DISS. ETH NO. 21789
Fungal lifestyle: analysis of the cell
wall and secreted antibacterial proteins
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
ANDREAS ESSIG
MSc ETH Biology
born on June 10, 1982
citizen of Mettauertal, Aargau
accepted on the recommendation of
Prof. Dr. Markus Aebi
Dr. Markus Künzler
Prof. Dr. Ruedi Aebersold
Prof. Dr. Gerald Hart
Prof. Dr. Hans-Georg Sahl
2014
Cover image
NMR structure of Copsin
Acknowledgments
First of all, I would like to thank my two supervisors Prof. Dr. Markus Aebi and Dr. Markus
Künzler for giving me the opportunity to perform my PhD thesis in their group. I greatly
appreciated their support and the freedom they gave me to design my projects. Fruitful
discussions and a stimulating working atmosphere they created had certainly a major impact on
the outcome of this thesis.
Many thanks go to my PhD committee members Prof. Gerald Hart, Prof. Hans-Georg Sahl, and
Prof. Ruedi Aebersold for their helpful inputs and feedbacks during my PhD.
Furthermore, I would like to acknowledge my scientific collaborators for their extensive
contributions to my projects, especially Paolo Nanni, Peter Gehrig, Bernd Roschitzki, and René
Brunisholz from the FGCZ, Prof. Gerhard Wider and Daniela Hofmann from the institute of
molecular biology and biophysics, and Tanja Schneider and Daniela Münch from the university
of Bonn.
I owe many thanks to my two Master students Savitha Gayathri and John Hintze for their
valuable support. It was a great pleasure to work with them.
I would like to thank members of the molecular life sciences PhD program, in particular, Susanna
Bachmann.
Many thanks go to past and present members of the Aebi Group and the institute of microbiology
for a very nice time inside and outside the lab. Especially, I want to thank Susanne, Andreas,
David, Martina, Stefanie, Niels, Ivan, Ramon, Jörg, Sonja, Palmira and Pauli.
Finally, I am deeply thankful to my parents, my brother and Alessia for their love and constant
support.
Table of Contents
Summary …………………………………………………………………………………. i
Zusammenfassung …………………………………………………………………… iii
Chapter 1 ………………………………………………………………………………… 1
Introduction - Antimicrobial peptides
Chapter 2 ……………………………………………………………………………….. 17
Copsin, a novel peptide-based fungal antibiotic interfering with the
peptidoglycan synthesis
Chapter 3 ……………………………………………………………………………….. 51
Studies towards the mode of action of copsin and its homologs
in C. cinerea
Chapter 4 ……………………………………………………………………………….. 75
Fungal lysozyme identified in the secretome of C. cinerea
Chapter 5 ……………………………………………………………………………..... 99
O-mannosylated cell wall proteins of S. cerevisiae
Chapter 6 ……………………………………………………………………………... 131
Discussion and future perspectives
Curriculum vitae ...…………………………………………………………………... 139
Summary
Fungi and bacteria coexist in a variety of biological niches, including soil, plants, and animals. In
these environments, the cell wall and secreted substances provide fungi with an effective
machinery to interact and to compete with bacteria. The discovery of penicillin by Alexander
Fleming, a fungal antibacterial metabolite, demonstrated the importance of understanding such
defense mechanisms.
Antimicrobial peptides (AMPs) are a highly diverse group of defense molecules identified in
prokaryotes and eukaryotes. As a part of the innate immune system, AMPs act microbicidal and
possess immunomodulatory properties. The first chapter provides an overview of the structure
and activity of AMPs including pharmaceutical applications.
The identification and characterization of a novel fungal AMP is described in chapter 2.
Coprinopsis cinerea is a basidiomycete that naturally occurs in herbivorous dung. Based on a
defined analytical setup, we studied the interactions of C. cinerea with different bacterial species
and analyzed secreted fungal proteins. A novel fungal defensin, copsin, was identified by
quantitative mass spectrometry (MS) and recombinantly produced in Pichia pastoris. The
structure of copsin was solved by NMR, which revealed an α/β-fold stabilized by six disulfide
bonds. Both the structural compactness and its terminal modifications render copsin extremely
heat stable and insensitive towards proteases. Characterization of the antibacterial activity
showed that copsin acts bactericidal by binding to the cell wall precursor lipid II predominately in
Gram positive species. Its high stability and potent activity against bacteria resistant to
conventional antibiotics make copsin to a valuable candidate for a novel antibacterial drug.
Further characterization of copsin and its homologs is reported in chapter 3. A sequence
comparison of copsin exhibited several homologous AMPs encoded in the C. cinerea genome.
Copsin and the homologous defensin CC82 were successfully produced in a bioreactor using P.
pastoris as the heterologous host. The distinct antibacterial profiles determined for both
polypeptides indicate that C. cinerea expresses a diversified group of AMPs against a multitude
of microbial competitors found in herbivorous dung.
Saprophytic fungi such as C. cinerea secrete an arsenal of enzymes to digest dead organic
matter. The secretome of C. cinerea was analyzed by MS, as described in chapter 4. Besides
numerous proteases and glycosidases, we discovered a novel lysozyme, for which we optimized
the heterologous expression in P. pastoris. This fungal lysozyme has a low sequence identity to
known peptidoglycan cleaving enzymes and possesses a very specific antibacterial profile.
i
The cell wall of fungi is a unique and complex network of polysaccharides and glycoproteins.
Besides the function as a determinant of the cell shape, it acts as a protective line for invading
and competing microbes. In order to gain more insights in the cell wall structure, we developed a
MS based workflow for the analysis of O-mannose (O-Man) glycans, a structurally and
functionally important modification of cell wall proteins. In chapter 5, a comprehensive yeast cell
wall O-Man glycoproteome is described with an in-depth analysis of the enormous heterogeneity
of this type of O-glycosylation. Furthermore, we implemented SILAC (stable isotope labeling by
amino acids in cell culture), which is a valuable tool for quantitative MS measurements of O-Man
glycans in yeast and other organisms.
ii
Zusammenfassung
Pilze und Bakterien interagieren in einer Vielzahl von biologischen Nischen; einschliesslich dem
Erdreich, Pflanzen und Tieren. Dabei dienen die Zellwand und sekretierte Substanzen den
Pilzen unter anderem zur Abwehr von Bakterien. Die Entdeckung des pilzlichen Metaboliten
Penicillin durch Alexander Fleming und dessen medizinische Anwendung hat gezeigt, wie
wichtig das Verständnis solcher Abwehrmechanismen ist.
Antimikrobielle Peptide (AMPs) sind eine vielfältige Gruppe von Molekülen, die von Prokaryoten
und Eukaryoten exprimiert werden. Als Teil der angeborenen Immunabwehr besitzen AMPs
antimikrobielle und immunomodulatorische Eigenschaften. Das erste Kapitel gibt einen
Überblick über die Struktur und Aktivität von bekannten AMPs und deren pharmazeutische
Anwendungen.
Die Identifizierung und Charakterisierung eines neuen pilzlichen AMP wird in Kapitel 2
beschrieben. Coprinopsis cinerea ist ein Ständerpilz, der natürlicherweise auf Dung von
pflanzenfressenden Tieren vorkommt. Die Interaktion von C. cinerea mit verschiedenen
Bakterienarten wurde in einem Modellsystem studiert, welches es ermöglichte sekretierte
Pilzproteine zu analysieren. Dabei wurde das Defensin Copsin mittels quantitativer
Massenspektrometrie identifiziert und in Pichia pastoris rekombinant produziert. Die durch NMR
gelöste 3D Struktur ergab eine α/β-Faltung, die durch sechs Disulfidbrücken stabilisiert ist. Die
strukturelle Kompaktheit und die terminalen Modifikationen machen Copsin extrem
hitzebeständig und unempfindlich gegenüber Proteasen. Die Charakterisierung der
antibakteriellen Aktivität zeigte, dass Copsin an den Peptidoglykan-Vorläufer Lipid II bindet und
dabei die Zellwandsynthese von vorwiegend Gram positiven Bakterien hemmt. Die enorme
Stabilität und eine starke Aktivität gegen Bakterien, die gegenüber herkömmlichen Antibiotika
resistent geworden sind, machen Copsin zu einem vielversprechenden Kandidaten für ein neues
Antibiotikum.
Die weitere Charakterisierung von Copsin und dessen homologen AMPs wird in Kapitel 3
diskutiert. Ein Sequenzvergleich von Copsin zeigte, dass im Genom von C. cinerea mehrere
homologe AMPs kodiert sind. Copsin und das homologe Defensin CC82 wurden erfolgreich in
einem Bioreaktor in P. pastoris produziert. Die unterschiedlichen antibakteriellen Profile von
Copsin und CC82 deuten darauf hin, dass C. cinerea eine diversifizierte Gruppe von AMPs
exprimiert, die gegen spezifische Mikroorganismen aktiv sind.
Saprophytisch lebende Pilze wie C. cinerea sekretieren ein Arsenal von Enzymen die dem
Abbau von totem organischem Material dienen. Das Sekretom von C. cinerea wurde mittels
iii
Massenspektrometrie analysiert und ist in Kapitel 4 beschrieben. Unter zahlreichen Proteasen
und Glykosidasen identifizierten wir ein neuartiges Lysozym, das wir in P. pastoris heterolog
exprimierten. Dieses pilzliche Lysozym zeigt eine niedrige Sequenzidentität mit bereits
bekannten Peptidoglykan-spaltenden Enzymen und besitzt ein sehr spezifisches antibakterielles
Profil.
Die Zellwand von Pilzen ist ein einzigartiges und komplexes Netzwerk von Polysacchariden und
Glykoproteinen. Sie dient nicht nur zur Stabilisierung der Zelle, sondern auch zum Schutz vor
invasiven und konkurrierenden Mikroben. Basierend auf einem massenspektrometrischen
Ansatz haben wir eine Methode zur Analyse von O-Mannose (O-Man) Glykanen entwickelt.
Diese O-Glykane stellen eine strukturell und funktionell wichtige Modifikation von
Zellwandproteinen dar und ermöglichten uns weitere Einblicke in die Zellwandstruktur. In Kapitel
5 beschreiben wir ein umfassendes Hefe-Zellwand O-Man Glykoproteom und analysieren
eingehend die enorme Heterogenität dieser Art von O-Glykosylierung. Zur quantitativen
Messung von O-Man Glykanen in Hefe integrierten wir SILAC (stable isotope labeling by amino
acids in cell culture) in die Analyse.
iv
Chapter 1
Introduction
Antimicrobial peptides
1
Chapter 1
Introduction
More than 2300 antimicrobial peptides (AMPs) are described and are cataloged in the AMP
database (http://aps.unmc.edu/AP/main.php). AMPs are expressed by all organism
examined and they play a key role in defense strategies against bacteria, fungi, and viruses
. Especially organisms that lack an adaptive immune system rely heavily on the action of
1,2
AMPs as first line of defense. In addition to directly killing microbes, AMPs are frequently
involved in modulation of the immune system, for example, by attracting and activating
immune cells, by supporting wound repair and angiogenesis, or by neutralizing bacterial
endotoxins 3,4.
The diversity of AMPs with regard to primary sequences and structural elements makes a
proper classification difficult. Nevertheless, there are some general features that count for
most AMPs. AMPs are gene encoded and are often synthesized as prepro-proteins that are
proteolytically processed to release an active AMP. They are short with a length of
approximately 6 to 80 amino acids and are rich in arginine and lysine residues and thus,
have an overall positive charge. However, there are anionic peptides active against
microbes. But only little is known about their targets and mechanism of action 5. They are not
further covered in this review. Hancock and Sahl classified AMPs according to their primary
amino acid sequence and structural features into four classes: (i) β-sheet peptides stabilized
by two to four disulfide bonds; (ii) α-helical peptides; (iii) extended structures enriched in
certain amino acids; (iv) loop peptides with one or more disulfide bond 2.
The three biggest classes of AMPs, bacteriocins, cathelicidins and defensins, are described
in more detail in the following sections with a special emphasize on defensins.
2
Chapter 1
Bacteriocins
Bacteriocins are a heterogeneous subgroup of AMPs exclusively produced by bacteria. They
mainly act as growth inhibitors of other microorganisms competing for nutrients 6.
Bacteriocins expressed by Gram positive bacteria are commonly categorizes in modified
(class I) and unmodified peptides (class II). Both classes are further divided according to
their primary sequences and structural features 7.
Amino acids side chains of class I peptides undergo extensive post translational
modifications. An example is the group of lantibiotics, characterized by the dehydration of
serine and threonine residues and subsequent formation of lanthionine thioether bonds 8.
Nisin and mersacidin are two very prominent members of lantibiotics. Both are synthesized
as an inactive precursor containing a leader peptide that directs them to the transport and
modification machinery 9. Nisin is produced by strains of Lactococcus lactis and exhibits
potent activity against different Gram positive bacteria
10.
For the mode of action, different
models are proposed. In the lipid II independent mechanism, nisin aggregates in the outer
leaflet of the membrane, which leads to the formation of short-lived pore-like structures. In
the second model, nisin integrates into the membrane and forms a complex with lipid II,
which leads to an oligomerization and subsequent pore formation
11.
The lipid II dependent
pores exhibit a much higher stability, increasing the activity of nisin approximately by three
orders of magnitude 9. Due to its low toxicity and high antibacterial activity, nisin is approved
as food additive since 1988. Mersacidin, a lantibiotic with globular structure, kills bacteria
also via interaction with lipid II
12.
However, in comparison to nisin, it does not lead to pore
formation.
Class II bacteriocins lack modified residues. They include the class IIa one-peptide pediocinlike bacteriocins and the class IIb two-peptide bacteriocins
13.
Pediocin PA-1 is a plasmid
encoded member of the class IIa peptides initially characterized from Pediococcus acidilactici
14.
As it shows activity in the nanomolar range against human pathogens like Listeria
monocytogenes, it is currently tested as food preservative. Lactococcin G was the first twopeptide class IIb bacteriocin identified in 1992
15.
It consists of the 39 residue α-peptide and
the 35 residue β-peptide encoded next to each other in the same operon. Both peptides
together form a membrane penetrating helix-helix structure that renders the membranes
permeable for small molecules, leading to cell death. An optimal activity requires the
presence of both peptides in equal amounts. Class IIb bacteriocins have to be further
characterized and optimized for making them valuable candidates for applications in clinics
and in food industry 13.
3
Chapter 1
In some classifications, proteins with an enzymatic activity are categorized as class III
bacteriocins (e.g. enterolysin A) and circular bacteriocins as class IV 6. These two classes
are not covered in this review.
AMPs that are ribosomally synthesized by Gram negative bacteria can be divided in larger
proteins such as colicins and smaller peptides such as microcins 6.
Colicins are organized in a C-terminal catalytic domain, an N-terminal translocation domain
and a central receptor binding domain
. They are able to cross the outer membrane of
16
Gram negative bacteria by interacting with specific receptors and finally execute their
antibacterial activity on a periplasmic or intracellular compound. However, it is not fully
understood how colicins cross the membrane and finally kill the cells. Due to their high
molecular weight, they are not considered suitable for drug development and are often not
assigned to the group of AMPs similar to class III bacteriocins.
Microcins are peptides with a mass below 10 kDa secreted by enterobacteria. They are
synthesized as precursor peptides similar to bacteriocins of Gram positive bacteria and they
act mainly in regulation of the intestinal microflora
17.
Their killing mechanism is based on
pore formation or interaction with periplasmic or intracellular compounds such as the RNA
polymerase. Microcins are extremely heat and pH stable and insensitive towards proteases.
Cathelicidins
Cathelicidins are ubiquitously expressed in vertebrates, such as the fowlicidins in chicken
protegrins in pigs
19,
or cathelicidins identified in snake venoms
20.
18,
One of the first
cathelicidins described was a dodecapeptide isolated from bovine neutrophils, where it is
stored in secretory granules and released upon inflammatory stimuli 21. So far, no cathelicidin
has been identified in an invertebrate.
Cathelicidins are synthesized as precursor proteins that are characterized by a highly
conserved pro-sequence linked to a heterogenic C-terminal domain corresponding to the
mature antimicrobial peptide. The pro-sequence (~100 amino acids) shows a high similarity
to cathelin, an inhibitor of cathepsin L that gave the name to this group of AMPs
22,23.
The C-
terminal antimicrobial domain (~10-80 amino acids) comprises, in comparison to the cathelin
domain, a wide repertoire of structures including linear and cyclic molecules (Fig. 1).
4
Chapter 1
Fig. 1. Gene and protein structures of cathelicidins. Cathelicidin genes are composed of four
exons and three introns. The exons 1-3 specify the prepro-region including the cathelin pro-sequence.
The antimicrobial domain is encoded in exon 4. Mature AMPs include α-helical (a, LL-37), cysteinerich (b, protegrin-1), and tryptophan-rich (c, indolicidin) peptides and proline-rich peptides (prophenin).
Structures obtained from www.rcsb.org (adapted from Zanetti, M., 2005) 22.
In higher concentrations, cathelicidins are detected at sites of inflammation, where they act
as a part of the innate immune system to kill bacteria, fungi or viruses
24.
In addition to the
direct antimicrobial activity, cathelicidins possess different immunomodulatory properties, for
example, the chemoattractive action on neutrophils and monocytes or binding and
inactivation of LPS and thus, preventing an endotoxic shock
25,26.
In humans, one cathelicidin gene has been identified, which encodes a precursor protein
called human cationic antimicrobial peptide-18 (hCAP18) with in total 170 amino acid
residues
27.
After removal of the signal peptide, hCAP18 is stored as inactive precursor in
secondary granules. The pro-peptide is cleaved off by the serine proteinase 3 after secretion
and the active AMP LL-37 is released. The hCAP18/LL37 protein is widely expressed in
humans, for example, in mucous epithelia, sweat, skin, or salivary glands. It is also secreted
into wound fluid and airway surface fluid
22.
LL-37 adopts a cationic amphipathic helix-helix structure, which is composed of an N- and Cterminal α-helix and a C-terminal tail. The positively charged residues allow the peptide to
interact with negatively charged head groups of phospholipids of the bacterial membrane.
The subsequent pore forming activity is based on a toroidal pore carpet-like mechanism
5
Chapter 1
proposed
. LL-37 is active against a wide range of bacteria such as Escherichia coli,
28,29
Staphylococcus aureus, and Mycobacterium tuberculosis. LL-37 is involved in a multitude of
other functions of the immune system, such as an anti-endotoxic activity, immunostimulation,
or wound repair 24.
Several attempts were undertaken to develop cathelicidins and their derivatives in
antimicrobial or immunomodulatory drugs, but development was hindered by a high toxicity
and low solubility. One of the few promising candidates is omiganan, an indolicidin based
peptide variant. It is currently undergoing Phase III clinical trials mainly for topical applications
against acne and rosacea 6,30.
Defensins
More than 50 years ago, leukin, an extract of rabbit leukocytes, was shown to be able to kill
Gram positive bacteria
31.
Shortly after this discovery, another extract from rabbit leukocytes
exhibited activity against Gram positive and Gram negative bacteria. It was termed
phagocytin
32.
In the following decades, both extracts were further characterized and the
active component was called cationic antimicrobial proteins (CAPs). In 1985, the term
defensin was introduced for natural peptide antibiotics
33.
Nowadays, more than 250
defensins are described in the literature covering producing organisms from fungi, plants,
insects, to mammals (Fig. 2).
Defensins are synthesized as prepro-proteins, which are processed to release an active
AMP. In comparison to cathelicidins, the mature peptides are much more conserved
throughout evolution, which made a more accessible classification possible. Considering all
biological kingdoms defensins can be roughly divided in two big groups, fungal / plant /
invertebrate defensins and vertebrate defensins. This classification is mainly based on
structural motifs, which is explained in more detail in the following sections.
Vertebrate defensins
Vertebrate defensins are divided in linear α- and β-defensins and cyclic θ-defensins.
Predominantly expressed by epithelial cells, they often act as a first line of defense against
invading pathogens such as bacteria, fungi, or viruses. Similar to cathelicidins, vertebrate
defensins have a variety of other biological effects such as immunomodulation, neutralization
of endotoxins, or induction of angiogenesis and wound repair 4,34.
α- and β-defensins share the common structural motif of a triple stranded antiparallel β-sheet
structure stabilized by three disulfide bonds. A differentiation between these two groups is
based on their amino acid composition and the pattern of pairing between the cysteine
residues. α-defensins (cysteine pairing: 1-6, 2-4, 3-5) are relatively rare in lysine residues and
6
Chapter 1
are generally 5-10 amino acids shorter than β-defensins (cysteine pairing: 1-5, 2-4, 3-6),
which contain a high number of lysine and arginine residues.
In humans, six α-defensins have been identified, called HNP (human neutrophil peptides) 1-4
and HD (human defensin) 5-6 4. HNP 1-4 are fully processed to the active form before they
are stored in primary granules of leukocytes. HD 5-6 are expressed by Paneth cells of the
small intestine. Upon stimulation, α-defensins are released from granules into the
extracellular space together with other defense molecules such as lysozymes, tumor
necrosis factor-α (TNF-α), and immunoglobulin A 4.
Four β-defensins (HBD 1-4) have been described in more detail and more than 30 have
been predicted by bioinformatics tools in humans, but little is known about their biological
functions. HBDs are predominantly expressed in epithelial cells and differ substantially in
their activity profile against Gram negative and Gram positive bacteria
34.
In comparison to α-
defensins that have been identified in mammals only, AMPs with close homology to βdefensins could be tracked back to reptiles and birds such as the crotamine-myotoxin family
of snake venom or the avian β-defensin family (AvBD) 35,36,37.
The only cyclic peptides known from mammals are θ-defensins, first isolated from rhesus
macaque leukocytes and bone marrow
38.
They are produced through a head-to-tail ligation
of two truncated α-defensins, which results in a cyclic peptide stabilized by three disulfide
bonds. The antimicrobial spectrum is similar to the one of α-defensins with an enhanced
activity against viruses. Humans express mRNA encoding for θ-defensins, but premature
stop codons prevent the production of a functional peptide 39.
Due to the fact that most vertebrate defensins possess an amphiphilic structure with a high
net positive charge, mode of actions are proposed, where defensins accumulate and embed
into the lipid bilayer of bacterial membranes. Finally, this leads to membrane disruption and
cell death
28.
However, recent studies support a more targeted mode of action for different
AMPs. HBD-2 and crotamines are two examples that are able to bind to ion channels and
perturb the membrane potential similar to cobatoxins, a scorpion toxin family
36,40.
Furthermore, for several α-and θ-defensins it could be shown that they interact with specific
glycan structures, indicating that binding of carbohydrates mediated by a lectin domain could
play a central role in recognition and killing of microbes 4.
7
Chapter 1
Fig. 2. Major classes of defensins. Plectasin, Pseudoplectania nigrella; Rs-AFP1, Raphanus
sativus; MGD-1, Mytilus galloprovincialis; HNP-1, HBD-2, RTD-1, Homo sapiens. Disulfide bonds
shown in red form the core structural motif CSαβ. All 3D structures were obtained from the protein
data bank (http://www.rcsb.org) 41,42,43,44,45,46.
8
Chapter 1
Invertebrate, plant, and fungal defensins
The majority of invertebrate, fungal, and plant defensins share a core structure that consists
of a cysteine stabilized α-helix β-strand motif (CSαβ)
. The α-helix is connected over two
47
disulfide bonds to the second β-strand (cysteine pairing: 2-5, 3-6) and a cysteine residue of
the N-terminal part forms a disulfide bond with a cysteine of the first beta-strand (1-4).
Plant defensins are a diverse group of AMPs with regard to their amino acid composition,
with the exception of eight conserved cysteine residues that form four disulfide bonds
. In
48,49
the Arabidopsis thaliana genome more than 300 defensin-like peptides could be identified, of
which 78% comprise the CSαβ fold
. The majority of plant defensins have been isolated
50
from seeds such as the radish Raphanus sativus defensins (Rs-AFPs)
. Rs-AFP1 and Rs-
51
AFP2 are composed of a CSαβ motif extended by an additional N-terminal β-strand and a
fourth disulfide bond. Upon disruption of the seed coat, Rs-AFPs are released and represent
up to 30% of secreted seed proteins. Unlike most vertebrate and invertebrate defensins that
have an antibacterial impact, the majority of plant defensins exert their antimicrobial action on
fungi, including plant and human pathogenic species. It is proposed that Rs-AFP2 interacts
with glucosylceramides in fungal membranes, inducing Ca2+ influx and thus inhibition of
hyphal growth
52,53.
In recent studies, several other biological activities of plant defensins
have been described such as the inhibition of α-amylases, inhibition of protein translation or
an influence on growth and development of plant roots. Due to their high stability and potent
activity against fungi, they are considered as valuable candidates for new antimycotic
drugs 54.
Defensins play a pivotal part in the innate immune system of all invertebrates investigated,
such as scorpions, spiders, mussels, and flies. Drosomycin is one of the best characterized
insect defensins isolated from Drosophila melanogaster
55.
Similar to the plant defensin Rs-
AFP, it consists of an extended CSαβ motif and is the major weapon of D. melanogaster
against fungi. However, a structure-function relationship could not be shown experimentally,
so far, mainly due to lack of an efficient expression system to produce an adequate amount
of peptide
56.
MGD-1 was originally isolated from the edible mediterranean mussel Mytilus
galloprovincialis and is composed of a CSαβ fold stabilized by four disulfide bonds
43.
The
activity of MGD-1 is directed against bacteria, mainly Gram positive species.
With the publication of plectasin in 2005, fungi were added to the list of defensin producing
organisms, as the last biological kingdom
41.
Plectasin is expressed by the saprophytic
ascomycete Pseudoplectania nigrella and is strongly active against Gram positive bacteria
such as Streptococcus spp. and Staphylococcus spp. Resembling a prototypic CSαβ fold
stabilized by three disulfide bonds, plectasin shows a high similarity to invertebrate and plant
defensins. This relation led to the assumption that there is a common ancestor of plant,
fungal, and invertebrate defensins, further supported by the recent finding of defensin-like
9
Chapter 1
peptides in bacteria
. In the following years, several other fungal defensins have been
57
identified, predominantly in the phyla of ascomycota and zygomycota
. These findings
58,59,60
indicate that AMPs are key components of the innate immune system of fungi active against
competing microbes and are very likely expressed by the majority of fungal species. Binding
studies of plectasin with different precursors of the peptidoglycan synthesis revealed that
plectasin binds to lipid II, a target of different antibiotics like nisin or vancomycin
.
9,61,62
However, in comparison to the bacteriocin nisin, it did not show any pore forming activity.
NMR based modeling exhibited that plectasin binds specifically to the pyrophosphate moiety
and to the D-γ-glutamate of the pentapeptide side chain of lipid II and interacts with the
membrane surface (Fig. 3). Due to a high efficacy in vivo against pneumococcal infections in
mice, efforts have been done to develop plectasin further to a commercial drug. Therefore,
different expression systems were successfully optimized to get a maximum yield of peptide,
such as in E. coli, Pichia pastoris, and Aspergillus oryzae
41,63,64.
Plectasin entered clinical
phase 1 trials and was considered as one of the most promising drug candidates tested.
Nonetheless, due to commercial and scientific reasons Novozymes and Sanofi-Aventis, both
companies involved in the development, decided to quite the studies on plectasin 65.
Fig. 3. Lipid II/plectasin/DPC complex based on NMR spectroscopy. (A) Yellow indicates residues
that interact with a dodecylphosphocholine (DPC) membrane. (B) Residues of plectasin (magenta)
that show substantial chemical shifts upon titration with lipid II. (C) Hydrogen bonds are formed
between the pyrophosphate and F2, G3, C4, and C37. The N-terminus of plectasin and the side chain
of His18 form a salt bridge with D-γ-glutamate of lipid II (from Schneider, T. et al., 2010) 61.
10
Chapter 1
Summary and concluding remarks
AMPs are produced by prokaryotes and eukaryotes, where they are an important part of the
innate immune system. The diversity at the sequence and structure level is immense from
highly derivatized peptides like the lantibiotics nisin and mersacidin to defensins with
secondary structural elements like MGD-1. Therefore, it is often rather difficult to categorize
and define groups of AMPs. In this regard, Yeaman and Yount discovered a structural motif,
which is preserved in all classes of cysteine stabilized AMPs
66
. The so-called γ-core is
defined by a hallmark Gly-X-Cys motif (X can be any amino acid), which is integrated in a
Cys-stabilized anti-parallel β-sheet structure (Fig. 4).
Fig. 4. γ-core structural motif. Shown is a Cys-stabilized anti-parallel β-sheet structure with the GlyX-Cys motif (X can be any amino acid). Positive charges are commonly found at the poles of the motif.
Peptides shown: mβD-8 (1), a murine β-defensin; BNBD-12 (2), bovine neutrophil β-defensin-12;
DEF3 (3), a human neutrophil defensin; protegrin (4), an antimicrobial peptide from porcine
neutrophils; plectasin (5); MGD-1 (6); and hPF-4 (7), human platelet factor-4 kinocidin (from Yeaman
and Yount, 2010) 67.
11
Chapter 1
The γ-core alone is sufficient for an antimicrobial activity (e.g. protegrin), but can also be
extended by additional α-helices and β-strands (e.g. plectasin). Recently, it has been shown
that the γ-core motif appears also in other classes of disulfide stabilized defense molecules
such as cytokines and chemokines with a direct antimicrobial activity. Interestingly, the γ-core
topologies show a clear pattern related to the complexity of the host immune system.
Invertebrates and fungi mainly produce molecules with a γ-α composition, often
characterized as the CSαβ fold. AMPs of higher organisms with an adaptive immune system
possess an altered structural composition of a γ-β fold, reflecting a co-evolution of AMPs with
the development of the immune system 67.
In contrast to the manifold appearance, most AMPs execute their antibacterial action in a
similar way of disrupting the bacterial cell membrane and forming pore-like structures. Only
for a few AMPs a more specific target has been identified, for example, plectasin and nisin
that both use lipid II as a specific anchoring point in the bacterial membrane. Nevertheless,
bacteria developed several strategies to get resistant against AMPs. For example,
esterification of teichoic acid phosphate groups with D-alanine is one way to reduce the net
negative charge of the cell wall and thus, repelling the overall positively charged AMPs
9,28.
Antimicrobial applications discussed in this review are manifold, directed against bacteria,
fungi, and viruses. We did not address the immunomodulatory properties of AMPs, which
could provide an additional pharmaceutical benefit in comparison to conventional antibiotics.
Altogether, AMPs are promising candidates for medical applications in animals and humans.
12
Chapter 1
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16
Chapter 2
Copsin, a novel peptide-based
fungal antibiotic interfering with the
peptidoglycan synthesis
Andreas Essiga, Daniela Hofmannb, Daniela Münchc, Savitha Gayathria, Hans-Georg
Sahlc, Gerhard Widerb, Tanja Schneiderc, Markus Aebia
a: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland
b: Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland
c: Institute of Medical Microbiology, Immunology, and Parasitology, Pharmaceutical Microbiology Section,
University of Bonn, Bonn 53105, Germany
Patent application (21.10.2013)
Essig A et al. (2013) Antimicrobial polypeptides, EP13005013 Patent pending.
Contributions
Co-cultivation of C. cinerea and bacteria
Discovery and purification of copsin
Recombinant expression in P. pastoris
MS based N-terminal sequencing
Determination of MICs and kill curve
TLC assays for binding to lipid I and lipid II
17
Chapter 2
Abstract
Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche.
This repertoire represents a rich and inexhaustible source for antibiotics and fungicides.
Antimicrobial peptides (AMPs) are an emerging class of fungal defense molecules that are
promising candidates for pharmaceutical applications. Based on a co-cultivation system, we
studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different
bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly
expressed in Pichia pastoris and the 3D structure was solved by NMR. The cysteine stabilized
α/β-fold (CSαβ) with a unique disulfide connectivity and an N-terminal pyroglutamate rendered
copsin extremely stable against high temperatures and protease digestion. Copsin was
bactericidal against a diversity of Gram positive bacteria, including human pathogens such as
Enterococcus faecalis and Listeria monocytogenes. Characterization of the antibacterial activity
revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore
interfered with the cell wall biosynthesis. In particular, the third position of the lipid II pentapeptide
was identified as a binding site for copsin. The unique structural properties of copsin make it a
possible scaffold for new antibiotics.
18
Chapter 2
Introduction
Fungi and bacteria coexist in a variety of environments, where fungi directly compete with
heterotrophic bacteria. However, bacterial-fungal interactions (BFIs) can also be commensal or
symbiotic associations. One of the best studied types of BFIs is the antibiosis, where secreted
substances play a key role in combating other microorganisms to defend a nutritional niche (1).
Secondary metabolites are the best characterized group of defense molecules, with their most
prominent member penicillin from the Penicillium chrysogenum mold (2). Numerous other
metabolites discovered in studies of BFIs have pharmaceutical applications, emphasizing the
importance of gaining knowledge on molecular mechanisms of BFIs.
Antimicrobial peptides (AMP) are a class of defense molecules that participate in competing
bacteria and other microbes as a part of an innate immune system. AMPs are a large and highly
diverse group of low molecular mass proteins (<10 kDa), expressed by both prokaryotes and
eukaryotes. They are broadly classified according to their structure and amino acid sequence.
Often, they are characterized by an amphipathic composition resulting in hydrophobic and
cationic clusters (3). Plectasin was the first fungal defensin identified in the secretome of the
ascomycete Pseudoplectania nigrella (4). It shows high similarity to plant and insect defensins
with a core structural motif of a cysteine stabilized α/β-fold (CSαβ). Plectasin acts bactericidal by
binding to the lipid II precursor and by inhibiting the peptidoglycan synthesis of predominantly
Gram positive bacteria (5). The cell wall biosynthetic pathways are a very effective target for
antibacterial substances, as shown for a number of clinically applied antibiotics. The far biggest
group of these drugs are the β-lactams, which inhibit the transpeptidation step of the
peptidoglycan layer (6). In an era of increasing bacterial resistance against many commercially
available antibiotics, AMPs are considered promising candidates for a new generation of
antibiotics.
Here, we studied the interaction of the basidiomycete C. cinerea with different bacterial species.
To model the lifestyle of this coprophilous fungus, a system was developed, where the fungus
was grown on medium-submerged glass beads. This method allowed for a co-cultivation with
different bacterial species and made it possible to analyze the secretome of both fungus and
bacteria.
The analysis of secreted proteins revealed that C. cinerea secreted AMPs that acted as a
defense line against bacteria. One of these peptides, hereafter called copsin, was characterized
further at a molecular and structural level and its mode of action was determined both in vivo and
in vitro.
19
Chapter 2
Results
Interaction of C. cinerea and bacteria
Dung of herbivores, the natural substrate of C. cinerea, is a rather complex environment. The
fungus grows on a solid substrate in high humidity and is confronted with a complex diversity of
bacteria and other microbes (7). To mimic this substrate and to study the interactions of C.
cinerea with bacteria, an artificial system was applied, in which the fungus was grown on glass
beads submerged in liquid medium (8). Because the fungal mycelium was kept in place by the
beads layer, the medium supporting fungal growth could be replaced and also allowed for the
co-cultivation with either Bacillus subtilis (strain 168), Pseudomonas aeruginosa (strain PA01), or
Escherichia coli (strain BL21). In addition, the growth of both organisms in competition could be
monitored easily (Fig. 1).
The Gram negative bacterium P. aeruginosa and E. coli had an inhibitory effect on the growth of
C. cinerea (Fig. 1A). B. subtilis, the Gram positive species tested, did not affect the expansion of
the fungal mycelium. But C. cinerea had an inhibitory impact on B. subtilis (Fig. 1B). P.
aeruginosa was not affected at the beginning of the co-cultivation, but the culture reached the
stationary phase much earlier than the fungal-free control. The growth of E. coli did not display
any dependency on C. cinerea. Our results showed that C. cinerea was indeed interacting in a
species-specific way with bacteria. In particular, we noticed an antagonistic behavior in
competition with B. subtilis and P. aeruginosa.
20
Chapter 2
Fig. 1. Interactions between C. cinerea and bacteria. (A) Vegetative mycelium of C. cinerea was grown
on submerged glass beads in minimal medium. After 60 h, 4 ml medium was replaced by a suspension of
Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. 0 h and 48 h after addition of the bacteria,
the plates were photographed. Fungal growth is indicated by the white mycelium. (B) Bacterial growth was
monitored by measuring OD600 over 48 h. As controls, bacteria and fungus were grown independently in
the beads system. All data points were acquired in three biological replicates and are displayed with the
standard deviation. C. cinerea exhibited a bactericidal effect in competition with B. subtilis and was
strongly inhibited by P. aeruginosa.
21
Chapter 2
Identification and recombinant expression of secreted AMPs
The co-cultivation experiments suggested a secreted fungal substance with a negative effect on
B. subtilis growth (Fig. 1B). To characterize this fungal activity, it was purified from conditioned
medium: Unchallenged C. cinerea was grown in the glass beads system in minimal medium.
After five days, the medium was collected, concentrated and the proteins precipitated with
ammonium sulfate. After a proteinase K treatment at 60 °C, the remaining proteins were
separated on a cation exchange column and evaluated according to their activity against B.
subtilis in a standard disk diffusion assay (Fig. S1 A and B). Fractions that displayed an
inhibition zone against B. subtilis were treated with reducing agent followed by a tryptic digest
and subjected to a mass spectrometry (MS) measurement. Five C. cinerea-derived proteins
were identified in all of the active fractions. These proteins were quantified by spectral counting
and the relative amount of each protein was correlated to the activity in the disk diffusion assay
(Fig. S1C). The relative concentration of the protein CC1G_13813 (copsin) was congruent to the
activity profile.
The transcript of the copsin locus was analyzed by reverse transcriptase-initiated PCR and the
results revealed an open reading frame encoding a protein with in total 184 amino acids. In silico
analysis suggested a signal peptide (position 1-23), a pro-peptide (position 24-127) and a
carboxy-terminal domain of 57 amino acids corresponding to the mature AMP (Fig. S2).
To obtain pure copsin, the cDNA encoding the native signal sequence was cloned in a pPICZA
expression vector and transformed in Pichia pastoris. The secreted recombinant product was
purified by cation-exchange chromatography and yielded a mature polypeptide with a
monoisotopic mass of 6059.5 Da, determined by electrospray ionization (ESI)-MS (theoretical
monoisotopic mass: 6059.5 Da).
Sequencing of the N-terminus by ESI-MS/MS of recombinant copsin and of the native
polypeptide from C. cinerea revealed the same peptide, both with an N-terminal glutamine
converted to a pyroglutamate (Fig. S3). These findings showed that the recombinant preproprotein was processed in P. pastoris in the same way as in the host fungus C. cinerea. Mature
copsin contained 12 cysteine (Cys) residues, all involved in a disulfide bond as displayed by a
mass shift of 12 Da after a reduction with dithiothreitol. This was further confirmed by typical 13C
NMR chemical shifts, which differ from reduced Cys residues. [ 1H,
15N]-HMQC
NMR spectra
recorded at pH 6.8 and 7.4 exhibited a stable positive charge on the His26 side chain and a
delta protonated, uncharged His21. Thus, a net positive charge of +7 was assigned to the
polypeptide.
A sequence comparison on the AMP database and by the Blastp algorithm exhibited maximum
identities of 20-27% of copsin with defensins from invertebrates, fungi, and plants, such as
plectasin from P. nigrella or eurocin from Eurotium amstelodami, both assigned to the fungal
phylum of ascomycota (Fig. 2A) (4, 9, 10).
22
Chapter 2
Fig. 2. 3D structure of copsin and similarity to other AMPs. (A) A sequence alignment of copsin with
AMPs exhibited the highest sequence identities with defensins from plants, fungi, and invertebrates (4, 1012). Disulfide bonds shown in solid lines form the core structural motif CSαβ. Disulfide bonds in dashed
lines were additionally detected in copsin. Regions of the α-helix and the two β-strands as well as the
positions of the Cys residues are indicated according to the sequence of copsin. The alignment was
performed with the ClustalW algorithm and visualized with the Jalview software (13, 14). (B) Cartoon
representation of the structure of copsin determined by NMR spectroscopy. Left: ribbon diagram of the
structure with the lowest energy after energy refinement with AMBER (15), highlighting secondary
structure elements; α-helical region (red) and the β-sheet (green). N and C terminus are denoted by N and
C, respectively, and selected residues are indicated with the residue type and sequence number; middle:
bundle of 20 conformers after energy refinement; right: Cys residues are denoted by the sequence
position and are colored in yellow for the conserved disulfide bonds and in orange for the three additional
disulfide bonds of copsin.
23
Chapter 2
Structural features of copsin
The three dimensional structure was determined using nuclear magnetic resonance (NMR)
spectroscopy. An almost complete assignment of resonances was obtained (99.1% backbone
resonances, 85.7% sidechain resonances). The structure calculation was performed as
described in the Supporting Information (SI) Materials and Methods. The input data for the
structure calculation and the characterization of the NMR structure are summarized in Table S1.
The structure of copsin contains one α-helix (residues 15-23) followed by two β-strands and is
stabilized by disulfide bonds (CSαβ) (Fig. 2B). The two β-strands (residues 36-40; 46-50) form a
small antiparallel sheet structure. These secondary structural elements exhibited a high
sequence identity to known CSαβ defensins, whereas the loop regions and the termini were very
unique in their length and composition. Standard identification of disulfide bonds following
proteolytic digest could not be applied, due to lack of single disulfide bond cleavage products.
Therefore, the disulfide bond pattern was investigated based on NMR data using target function
analysis (16), ambiguous disulfide restraint (17), and cysteine-cysteine distance measurements
(18), described in SI Materials and Methods. A comparison of an initial bundle of structures
calculated without disulfide constraints (Fig. S4A) with the three dimensional structures of other
defensins strongly supports the assumption of three conserved disulfide bonds. These bonds
connect the α-helix to the second β-strand (C18-C48; C22-C50) and the N-terminal region to the
first β-strand (C10-C40). Also, these disulfide bonding connectivities converged to a slightly
better target function, than structures obtained for other possible disulfide patterns (Table S2).
Three additional disulfides stabilize copsin, linking the termini to the loop between the α-helix
and the β-sheet. Given the conserved disulfide bonds, it follows that the two cysteines C35 and
C54 are connected. Calculating bonding probabilities for the remaining cysteines, based on
cysteine Cβ-Cβ distances (Table S2), the disulfides C3-C32 and C25-C57 seem most probable.
This assignment of all six disulfide bonds was further validated using ambiguous disulfide
restraints, where the same connectivity was obtained for all calculated structures (Fig. S4B and
C).
The compactness of the structure with its six disulfide bonds and the modifications at the termini
are mainly responsible for the very high stability of copsin. The activity was completely retained
after heat treatment or incubation at different pHs shown in a disk diffusion assay (Fig. S5A and
B). Copsin exhibited a strong resistance towards different proteases, such as proteinase K,
trypsin, or pepsin (Fig. S5C). However, antibacterial activity was lost when treated with a
reducing agent, supporting the pivotal role of disulfide bonds for the structural integrity of copsin.
24
Chapter 2
Antibacterial profile of copsin
The antibacterial activity of recombinant copsin was tested in disk diffusion assays against a
variety of Gram positive and Gram negative bacteria. For selected species the minimal inhibitory
concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the
standard microdilution broth method in Mueller Hinton Broth (MHB) at pH 7.3 (Table S3). Copsin
exhibited MIC values in the low microgram per milliliter range for Gram pos. bacteria, such as B.
subtilis, Listeria spp., and Enterococcus spp., including a vanA type vancomycin resistant E.
faecium strain. Most potent activity was detected against Listeria monocytogenes with MIC
values of 0.25-0.5 µg/ml. A change in pH to 6 in MHB did not affect the activity of copsin,
determined against B. subtilis and S. carnosus. Gram negative species such as E. coli were not
affected in viability when exposed to copsin.
MICs and MBCs did not differ more than twofold in their value indicating a bactericidal effect of
copsin on Gram pos. bacteria. To verify this finding, a kill curve assay using B. subtilis was
performed in MHB at pH 6 and 7.4 (Fig. 3). Independent of the pH, a clear reduction of the
viable count after 30 min of incubation was observed, supporting a bactericidal activity of copsin.
Fig. 3. Killing kinetics of copsin. B. subtilis grown in MHB at pH 6 and 7.3 and incubated with and
without (Ctrl) 4 µg/ml copsin (4xMIC). The OD600 is shown above the spectrum, measured at 0, 2 and 5 h.
25
Chapter 2
Molecular target of copsin in bacteria
The antibacterial activity and specificity of copsin was comparable to cell wall biosynthesis
interfering antibiotics such as plectasin or vancomycin (5). We, therefore, determined the cellular
localization of copsin on B. subtilis and S. carnosus cells during exponential growth phase, using
TAMRA labeled copsin and BODIPY-FL labeled vancomycin as a control. Fig. S6 shows a dual
staining of BODIPY-vancomycin and TAMRA labeled copsin. Vancomycin is known to localize
specifically to sites of active cell wall synthesis (19). Copsin exhibited binding at the cell surface,
preferably in curved regions of the rod-shaped bacterium B. subtilis and similarly distributed on
the spherical-shaped S. carnosus cells. A co-localization with vancomycin was found at the cell
septa.
To gain further insights on the molecular targets, we studied the binding affinity of copsin to
essential precursors of bacterial cell wall synthesis in vitro. Lipid I was synthesized as Nacetylmuramic
acid-pentapeptide
(MurNAc-pentapeptide)
linked
to
the
bactoprenolpyrophosphate lipid carrier (C55-PP). The pentapeptide was composed of L-alanyl-γD-glutamyl-L-lysyl-D-alanyl-D-alanine, a composition found in Staphylococcus spp. For lipid II, an
N-acetylglucosamine (GlcNAc) was attached to the MurNAc moiety of lipid I (Fig. 4A). Binding
studies included bactoprenolphosphate (C55-P) and lipid III, a teichoic acid synthesis precursor
consisting of GlcNAc linked to C 55-PP (20). Copsin was incubated with the synthesized
compounds in a defined molar ratio for 20 min and the free substrate was analyzed by thin layer
chromatography (TLC) (Fig. 4B). It was found that copsin bound in a 1:1 molar ratio to lipid I and
II, but had no affinity for C55-P and lipid III.
This findings suggested specific interaction of copsin with lipid I and lipid II and indicated that
MurNAc-pentapeptide might be pivotal for a stable complex. To examine whether the peptide
chain was necessary for binding to copsin, truncated versions of the pentapeptide were
synthesized and the binding affinity to copsin was analyzed (Fig. 4C). Copsin showed a strongly
reduced affinity for the lipid I-dipeptide (L-Ala-D-Glu) in comparison to the lipid I-tripeptide (L-AlaD-Glu-L-Lys). This result demonstrated that the third position of the pentapeptide is essential for
stable binding of copsin to the peptidoglycan precursor.
26
Chapter 2
Fig. 4. Binding of copsin to cell wall precursors. (A) Schematic of the peptidoglycan precursor lipid II
and the teichoic acid precursor lipid III (M: MurNAc; G: GlcNAc; P: Phosphate). (B) Purified cell wall
precursors were incubated with increasing molar ratios of copsin. After extraction with n-butanol / pyridine
acetate (pH 4.2), samples were analyzed by TLC, which displayed a binding of copsin to lipid I and lipid II.
(C) Truncated versions of lipid I (lipid I-dipeptide, lipid I-tripeptide) as well as lipid I-pentapeptide were
synthesized, using purified enzymes from S. aureus. Copsin was added in increasing molar ratios to the
lipid I versions. After incubation for 20 min at room temperature, samples were extracted as described and
analyzed by TLC. The result showed that copsin is binding to the third position of the pentapeptide.
Based on the hypothesis that copsin bound to lipid II, we wanted to know, whether copsin was
able to permeabilize the bacterial membrane, similar to the mode of action of nisin, a pore
forming lantibiotic (21, 22). Two assays were performed: The first relied on Carboxyflourescein
(CF) efflux from lipid II containing liposomes (Fig. 5A), the second on the potassium efflux of B.
subtilis cells (Fig. S7). In both assays, no pore formation was detected. Importantly, preincubation of the lipid II containing liposomes with copsin inhibited the action of nisin, which was
used as a positive control (Fig. 5B). These results showed that copsin interacted specifically with
lipid II at the extracellular site of the bacterial membrane, prevented the binding of nisin, but did
not lead to pore formation.
27
Chapter 2
Fig. 5. Carboxyfluorescein efflux from lipid II containing liposomes. Activity of copsin and nisin
against unilamellar liposomes made of DOPC supplemented with 0.1 mol% lipid II. Peptide-induced
marker release from liposomes with entrapped CF was measured. The 100% leakage level was
determined by addition of Triton X-100 after 300 sec. (A) 1 µM copsin (solid line) or nisin (dashed line)
were added after 100 sec. (B) First, 1 µM copsin was added (100 sec) following the addition of 1 µM nisin
(200 sec) to the same sample. Copsin did not permeabilize the membrane, but it blocked the action of
nisin.
28
Chapter 2
Discussion
Co-cultivation studies of C. cinerea with bacteria led to the identification of the peptide-based
antibiotic copsin, to our knowledge the first defensin identified in the fungal phylum of
basidiomycota.
Growing fungi on the surface of inert glass beads submerged in liquid medium provides a
reproducible and easy to handle model system for studying the interaction with bacteria. In
comparison to a confrontation assay on an agar plate or in a pure liquid culture, this setup has
the big advantage of combining a solid surface and a humid environment, two pivotal factors for
growth of bacteria and fungi in nature. Furthermore, it allows for a repeatable extraction of
secreted fungal and bacterial substances to perform an appropriate analysis at the metabolomic
and proteomic level. For our assays, B. subtilis, E. coli, and P. aeruginosa were selected as
competitors, which are well characterized and are known to interact with fungi (1, 23, 24). Under
the conditions tested, we observed an antagonistic behavior of the fungus with B. subtilis and P.
aeruginosa and a more commensal association with E. coli. These differentiated interactions
suggest an interdependent community of C. cinerea and bacteria on herbivorous dung to
preserve their nutritional niche. Different studies demonstrated that AMPs act as regulators of
microbial diversity, for example, α-defensins in mice or arminin peptides in Hydra species (25,
26). Based on the inhibition found against B. subtilis we developed a novel workflow for the
purification and identification of highly stable and active antibacterial peptides and proteins. This
analytical method involved a quantitative MS measurement, where we finally selected copsin for
further investigations.
Pichia pastoris, chosen for the production of copsin, is a highly efficient system for the
heterologous expression of secreted proteins at high yields in shake flasks and bioreactors (27).
It ensured also a correct processing of copsin, as P. pastoris expresses a Kex2 protease that
cleaves the pro-region at the lysine-arginine site, frequently identified in defensins (28). The 3D
structure of copsin was solved by NMR and revealed a core CSαβ fold with three disulfide
bonds. The CSαβ structural motif is a major characteristic of defensins of plants, invertebrates,
and fungi and is differentiating them from vertebrate defensins with a common motif of a triplestranded β-sheet structure (29). Copsin contains three additional disulfide connectivities not
described in a defensin, so far. The structural compactness with an N-terminal pyroglutamate
and a C-terminal cysteine involved in a disulfide bond render copsin extremely stable in a wide
pH and temperature range and insensitive towards proteases. Apart from the conserved α-helix
and β-strand regions, sequence comparisons exhibited no significant alignments with known
defensins.
Microscopy studies and binding assays with cell wall precursors revealed lipid II as the molecular
target of copsin. Lipid II is highly conserved molecule throughout the bacterial kingdom. Binding
29
Chapter 2
to this essential building block is an effective way to interfere with a proper cell wall synthesis
and consequently to kill a bacterium (30). Furthermore, it is unlikely that toxic effects on, for
example, mammalian cells would occur due to the unique structural composition of this
peptidoglycan precursor and specific binding pattern of copsin. Antibiotics found to interact with
lipid II exert their actions over defined binding sites, such as vancomycin, which interacts with the
D-Ala residues of the pentapeptide (19). In depth studies of plectasin exhibited that the Nterminal GFGC part is essential for a correct binding to pyrophosphate and glutamate of lipid II
(5). This motif is found in many other defensins such as MGD-1 or eurocin, but is absent in
copsin (10, 11). Binding studies of copsin with truncated versions of lipid I revealed that the third
amino acid in the pentapeptide side chain of lipid II is crucial for binding to copsin, independently
of whether lysine or diaminopimelic acid are located at this position. This finding is consistent
with the strong activity of copsin against a vancomycin resistant Enterococcus faecium strain,
where D-lactate is located at the C-terminal site of lipid II instead of D-Ala (31). Similar to other
fungal defensins as plectasin or eurocin, copsin exhibited a distinct antibacterial profile
predominantly active against Gram positive bacteria. Besides Enterococcus spp., the most
potent activity was determined against L. monocytogenes, a food-borne pathogen causing
severe forms of listeriosis in animals and humans (32).
The exceptional stability of copsin together and potent activity against bacteria are important
features for further applications in clinics or food industry.
30
Chapter 2
Materials and Methods
Chemicals and fungal strains
The C. cinerea strain AmutBmut (A43mut B43mut pab1.2) was used for all experiments involving
the fungus. All chemicals, if not otherwise mentioned, were bought at the highest available purity
from Sigma-Aldrich.
C. cinerea in competition with bacteria on glass beads
Preparation of the glass bead plates was adapted from van Schöll L et al. 2006 (8). In brief, 40 g
of sterile borosilicate glass beads (5 mm) were poured into a Petri dish and 15 ml of C. cinerea
minimal medium (CCMM) was added (composition per liter medium: 5 g glucose, 2 g
asparagine, 50 mg adenine sulfate, 1 g KH 2PO4, 2.3 g Na2HPO4 (anhydrous), 0.3 g Na2SO4, 0.5
g C4H12N2O6, 40 µg thiamine-HCl, 0.25 g MgSO4 x 7H2O, and 5 mg p-aminobenzoic acid).
C. cinerea was grown on 1.5% (w/v) agar plates containing YMG (0.4% (w/v) yeast extract
(Oxoid AG, England), 1% (w/v) malt extract (Oxoid AG, England), 0.4% (w/v) glucose) for 4 days
in the dark at 37 °C. Afterwards, two mycelial plugs were cut from the margin of the mycelium
and transferred two the center of a glass bead plate in a distance of 1 cm. After 60 h of letting
the fungus grow on the beads (37 °C, dark), 4 ml of the medium underneath the fungus was
replaced by 4 ml of a bacterial suspension (Bacillus subtilis 168, Pseudomonas aeruginosa 01,
or Escherichia coli BL21) grown previously in CCMM to an optical density (OD600) of 0.2. Both
organisms were grown in competition for 48 h (37 °C, dark). For the fungal growth control, the
medium was replaced by the same amount of CCMM (4 ml). As control for the bacterial growth,
the three bacterial strains were grown in the beads system independently for 48 h. The growth of
the bacteria was monitored by measuring the OD600 of the medium.
Purification of AMPs from fungal secretome
C. cinerea was grown in a glass bead plate (19 cm diameter) with 200 g glass beads and 80 ml
CCMM for 5 days in the dark. Subsequently, the medium was extracted and concentrated to 4 ml
by lyophilization. The proteins were then precipitated with 2.2 M ammonium sulfate at 4 °C for 30
min. After centrifugation at 20000 x g for 15 min, the pellet was dissolved in phosphate buffered
saline (PBS) pH 7.4 and the proteins digested with 100 ng/ul proteinase K (Roche Applied
Science, Germany) at 60 °C for 2 h. The solution was loaded in a 10 kDa MWCO dialysis
cassette (Thermo Scientific, USA) and dialyzed against PBS pH 7.4 at 4 °C for 24 h. The
remaining proteins were applied to a Resource S cation exchange column (GE Healthcare,
England) and eluted with a 100-600 mM NaCl gradient in 50 mM Na-phosphate buffer pH 7.4.
0.5 ml fractions were collected and the effluent monitored by absorbance at 280 nm. The flow
through and fractions showing activity against B. subtilis in a standard disk diffusion assay were
31
Chapter 2
subjected to a reduction with 10 mM DTT at 37 °C for 45 min and alkylation with 40 mM
iodoacetamide at 25 °C for 30 min in the dark. A proteolytic digest was performed with 0.25
µg/ml trypsin (Promega AG, USA) for 16 h at 37 °C and the peptides were desalted on C18
ZipTip columns (Millipore, Germany). The MS analyses were performed on a hybrid Velos LTQ
Orbitrap mass spectrometer (Thermo Scientific, USA) coupled to an Eksigent-nano-HPLC
system (Eksigent Technologies, USA). Separation of peptides was done on a self-made column
(75 µm x 80 mm) packed with C18 AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were
eluted with a linear gradient from 2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250
nl/min. MS and MS/MS spectra were acquired in the data dependent mode with up to 20 collision
induced dissociation (CID) spectra recorded in the ion trap using the most intense ions. All
MS/MS spectra were searched against the p354_filteredMod_d C. cinerea database using the
Mascot search algorithm v2.3 (Matrix Science Inc. , USA) with oxidation (methionine) as variable
modification and carbamidomethyl (cysteine) as fixed modification. Further statistical validation
was performed with Scaffold 4.0 (Proteome Software, USA) with a minimum protein probability of
90% and a minimum peptide probability of 50%. This program was also used for determining the
total non-normalized spectral counts for a protein identified in the fractions active against B.
subtilis and in the flow through.
cDNA synthesis and cloning of the copsin precursor
To extract RNA, 20 mg of lyophilized mycelium was lysed with 25 mg of 0.5 mm glass beads in
three FastPrep steps of 45 s at 4.5, 5.5 and 6.5, cooling the sample for 5 min on ice between
each step. RNA was extracted with 1 ml Qiazol (Qiagen, Germany) and 0.2 ml chloroform. After
a centrifugation at 12000 x g at 4 °C for 15 min, RNA was recovered in the aqueous phase,
washed on-column using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) and eluted in
RNase-free water. cDNA was synthesized from 2 µg of extracted RNA using the Transcriptor
Universal cDNA Master kit (Roche Applied Science, Germany) following the manufacturer
instructions.
The coding sequence of the copsin precursor protein was amplified from cDNA by PCR with the
Phusion high-fidelity DNA polymerase (Thermo Scientific, USA) according to standard protocols
(Sambrook J, Russell D, 2001, Molecular cloning, 3 rd edition).
FP: 5’-CCGGAATTCATGAAACTTTCTACTTCTTTGCTCG-3’;
RP: 5’-ACGCGTCGACTTAACAACGAGGGCAGGGG-3’;
The PCR product was cloned into the pPICZA expression plasmid (Life Technologies, USA)
containing a zeocin resistance gene using the EcoRI and SalI (Fermentas GmbH, Switzerland)
restriction sites. The resulting plasmid was linearized by the SacI restriction enzyme and
transformed into the P. pastoris strain NRRLY11430 by electroporation with 1.2 kV of charging
voltage, 25 µF of capacitance and 129 Ω resistance (33). Positive clones were selected on YPD
32
Chapter 2
plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar) containing
100 µg/ml zeocin (LabForce, Switzerland).
Expression and purification of recombinant copsin
P. pastoris transformants were inoculated in BMGY medium (1% (w/v) yeast extract, 2% (w/v)
peptone, 1.3% (w/v) YNB w/o amino acids (Becton Dickinson, USA), 100 mM K-phosphate
buffer pH 6, 1% (v/v) glycerol) and cultured at 30 °C for 48 h. The cells were harvested by
centrifugation at 3000 x g for 10 min, resuspended in P. pastoris minimal medium (1.3% (w/v)
YNB w/o amino acids, 100 mM K-phosphate buffer pH 6, 0.4 µg/ml biotin, 0.5% (w/v) NH4Cl, 1%
(v/v) MeOH) and cultured at 30 °C for 72 h. Methanol was added to 1% (v/v) in a time interval of
12 h and NH4Cl was added to 0.5% (w/v) in a time interval of 24 h.
The culture broth was centrifuged at 3000 x g for 10 min and the supernatant concentrated in a
3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc., USA) by a treatment with
polyethylene glycol 6000 at 4 °C. The concentrated supernatant was dialyzed against 20 mM Naphosphate, 50 mM NaCl buffer pH 7 (buffer A) at 4 °C for 24 h. The protein solution was sterilefiltered and loaded on a self-made SP sephadex cation exchange column equilibrated with buffer
A. The column was washed with 180 mM NaCl and bound proteins were eluted with 400 mM
NaCl in 20 mM Na-phosphate buffer pH 7. The eluent was subjected to a size exclusion
chromatography for further polishing. The separation was performed on a Superdex75 column
(HiLoad 16/60; GE Healthcare, USA) equilibrated with 20 mM Na-phosphate, 50 mM NaCl buffer
pH 6. The effluent was monitored by absorbance at 210 nm. The fractions containing copsin
were combined and the molecular mass was determined by ESI-MS.
Expression of 15N/13C labeled copsin in Pichia pastoris
The expression of labeled copsin was adapted from a previously described protocol (34). In
brief, P. pastoris transformants were inoculated in minimal medium supplemented with 0.5%
(w/v) 15NH4Cl (98 atom% 15N) and 13C3-glycerol (99 atom% 13C), respectively, and cultivated in
shake flasks at 30 °C to an OD 600 of approximately 35. After centrifugation at 3000 x g for 10
min, the cell pellet was dissolved in P. pastoris minimal medium containing 0.5% (w/v) 15NH4Cl
(98 atom% 15N) and 13C-methanol (99 atom% 13C). The culturing was performed at 30 °C for 3 d.
13C-methanol
was added to 1% (v/v) in a time interval of 12 h and 15NH4Cl was added to 0.5%
(w/v) in a time interval of 24 h. Purification of 15N/13C copsin was performed as described for the
unlabeled product.
NMR
Preparation of NMR Samples. Uniformly 15N/13C isotope-labeled copsin in 20 mM Na-phosphate,
50 mM NaCl buffer was prepared as described above.
33
Chapter 2
NMR Spectroscopy. Sequence specific resonance assignments were obtained using a set of
two-dimensional (2D) [15N,1H], and [13C,1H] heteronuclear single quantum coherence (HSQC),
constant-time [13C,1H] HSQC, [1H,1H] total correlated spectroscopy (TOCSY) (mixing time
τm=80ms), exclusive correlation spectroscopy (ECOSY), [1H,1H] ] nuclear Overhauser
enhancement spectroscopy (NOESY) (τm=40ms), three-dimensional (3D) HN(CO)CA and 15Nresolved [1H,1H]-TOCSY (τm=40ms) spectra recorded on Bruker Avance III 500, 600, 700 and
900 MHz at a temperature of 293K. Data was processed with TopSpin 3.0 (Bruker, Germany)
and analyzed with CARA 1.8.4 (cara.nmr.ch). The protonation state of histidine residues was
determined by recording a [15N,1H]-HMQC spectrum with a long (22 ms) INEPT transfer period
(35). Proline residues were assigned to cis and trans conformation based on chemical shifts of
13Cβ and 13Cγ
nuclei (36).
Structure calculation. Structure calculation was performed using 2D H 20 and D2O [1H,1H]NOESY and 3D
13C-, 15N-resolved
[1H,1H]-NOESY spectra (τm=40 and 60ms), recorded on
Bruker Avance 700 and 900 MHz spectrometers. Eight hydrogen bonds, identified based on
slow exchange of amide protons in D2O and based on correlations in long-range HNCO spectra
(37), were included as restraints. The NOEs were manually picked; the resulting peak list, the
amino acid sequence and the NOESY spectra were used as input for a structure calculation with
the software Cyana 3.0 (38), using the simulated annealing protocol. In each of the seven cycles,
800 structures were calculated. The 40 structures with the lowest target function were then
selected and used to calculate the final structure. At the outset of the structure calculation, no
assumptions on disulfide bond topology were used and the cysteines defined as negative
charged without a H atom. After mapping of the disulfide bonds, a last round of structure
calculation was performed and the best calculated structures were subjected to constrained
energy minimization using the software AMBER (15). The non-standard N-terminal amino acid
pyroglutamate was specially included into the library of AMBER.
Identification of disulfide bonds. Three methods for mapping of the disulfide bonds were used:
(A) ambiguous intersulfur restraint (17), (B) probability of a certain disulfide bond ensemble (18),
and (C) the averaged target function (16). (A) The cysteines were forced to form disulfide bonds
by the Cyana algorithm using ambiguous distance restraints, for every cysteine, between one
specific cysteine and all other cysteines (38). Clustering of at least two sulfur atoms together
satisfies the restraint. Standard distances between the sulfur and carbon atoms Sγ-Sγ (2.1/2.0 Å),
Sγ-Cβ (3.1/3.0Å) and Cβ-Sγ (3.1/3.0Å) were used for upper and lower limit, respectively. The
topology of the disulfide bond of the best 40 structures selected on the basis of their energy was
analyzed and visualized by PyMOL (www.pymol.org) (Fig. S4B-C). (B) An initial set of 800
conformers was calculated with no explicit constraint for disulfide bonds and the sulfur Hγ atoms
removed (Fig. S4A), subsequently the interatomic distances between all cysteine Cβ atoms were
extracted and averaged over the 40 final lowest energy structures. Based on the initial structure
34
Chapter 2
ensemble, all conceivable disulfide patterns were formed and a weighting factor representing the
probability of a specific disulfide bond topology was then calculated for all these possibilities
(Tab. S2). (C) The averaged target function of the 40 best Cyana structures was compared for
different fixed disulfide bond connectivities and violation were analyzed.
Effect of temperature, pH, and proteases on copsin activity
The pH stability of copsin was determined after incubation for 1 h at room temperature in a pH
gradient of buffered solutions, including 250 mM KCl/HCl buffer (pH 2), 100 mM Na-acetate
buffer (pH 4 and 5), 250 mM Na-phosphate buffer (pH 6) and 250 mM HEPES buffer (pH 8). As
a control, copsin form stock solution in 20 mM Na-phosphate,50 mM NaCl buffer pH 6 was
loaded on one. Thermal stability was tested in PBS buffer pH 7.4 at 4, 25, 50, 70, and 90 °C for
1 h incubation.
The effect of proteases as pepsin, trypsin, and proteinase K on the activity of copsin was
investigated by incubation with respective proteases at 37 °C for 3 h in a reaction mixture
containing a ratio of 1:10 (w/w) of protease to copsin. For pepsin the reaction was performed in
250 mM KCl/HCl buffer pH 2 and for trypsin, proteinase K, and for the control without any
protease in 100 mM Tris-HCl buffer pH 8. Copsin was also incubated in 5 mM DTT at pH 8. In
order to ensure that the proteases and buffers themselves do not contribute to any inhibition,
each of the proteases alone in the corresponding buffer was used a control.
After incubation, the samples were centrifuged at 12000 x g and the antibacterial activity of the
supernatant was tested by a disk diffusion assay on B. subtilis.
Antimicrobial activity
The MIC/MBCs were determined by the microdilution broth method (39). In brief, bacteria were
grown to an OD of 0.1-0.2 in Mueller-Hinton broth (MHB; Becton Dickinson, USA). The bacterial
suspension was diluted to 105-106 cfu/ml and added to a two-fold dilution series (0.25-64 µg/ml)
of copsin in a 96-well microtiter plate (Enzyscreen B.V., Netherlands). The plates were incubated
at 37 °C for 20-24 h. For all bacteria, the assays were performed in MHB pH 7.3. For Listeria
spp. and C. diphtheria, MHB was supplemented with 3% laked horse blood (Oxoid AG,
England). For B. subtilis and S. carnosus, the assay was additionally performed in MHB buffered
with 50 mM Na-phosphate pH 6. The cfu/ml was determined by plating serial dilutions on LBagar plates. The MIC is defined as the lowest concentration of copsin where no visible growth
was observed. The MBC is defined as the concentration of polypeptide that killed 99.9% of
bacteria after 20 – 24 h. All determinations were performed at least in duplicates.
35
Chapter 2
Killing kinetics
B. subtilis was grown in MHB to an OD600 of 0.2 and diluted to an OD600 of 0.1 in MHB
supplemented with 50 mM Na-phosphate buffer adjusted to pH 6 and 7.3. Copsin was added to
4 µg/ml (4xMIC) and the cultures incubated at 37 °C for 5 h. The cfu/ml were determined in
intervals of 30 min by plating serial dilutions on LB-agar plates. The OD600 was measured at 0, 2
and 5 h.
Light microscopy
For TAMRA labeling, copsin was dissolved in 1 M NaHCO 3 buffer pH 8.0 to 1.6 mg/ml. TAMRA
(Life Technologies, USA) was added to a final concentration of 1 mg/ml and incubated at room
temperature for 1 h. The excess dye was removed by passing the solution through a pre-packed
Sephadex G-25M PD-10 desalting column (GE Healthcare, USA) in 20 mM Na-phosphate, 50
mM NaCl buffer pH 6.0.
Bacteria were grown in LB medium to OD600 of 0.3 and incubated with TAMRA-copsin at 0.5xMIC
(0.5 µg/ml) for Bacillus subtilis and 2xMIC (16 µg/ml) for S. carnosus for 10 min. The cells were
washed twice with PBS and then immobilized on a poly-L-lysine coated cover slip. After washing
away the unbound cells with sterile water, BODIPY-vancomycin (Life Technologies, USA) was
used at 1 µg/ml concentration for staining on the cover slip. The cells were observed under a
100x oil-immersion objective (Zeiss, Germany) on a spinning-disk confocal microscope (Visitron,
Germany) and imaged with Evolve 512 EMCCD camera (Photometrics, USA) using standard
filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine (540/575 nm). Images were
processed using ImageJ v1.46.
Binding of copsin to cell wall precursors
2 nmol of each purified C55P, lipid I, lipid II (40) or lipid III (41) were incubated in the presence of
increasing molar copsin concentrations from 0.5:1 - 4:1 (copsin:lipid precursor) in a total volume
of 30 μl. After incubation for 20 min at 25 °C, the mixture was extracted with 2:1 (v/v) n-butanol/
pyridine acetate (pH 4.2), analyzed by thin layer chromatography (TLC) as described earlier (42,
43). Analysis was carried out by PMA staining.
Synthesis of UDP-MurNAc-peptides by S. aureus MurA-F enzymes
UDP-MurNAc-pp was synthesized as described (5) with modifications. UDP-GlcNAc (100 nmol)
was incubated with 15 µg of each of the recombinant histidine-tagged muropeptide synthetases
MurA to MurF in 50 mM Bis-Tris propane, pH 8, 25 mM (NH4)2SO4, 5 mM MgCl2, 5 mM KCl, 0.5
mM DTT, 2 mM ATP, 2 mM phosphoenolpyruvate, 2 mM NADPH, 1 mM of each amino acid (LAla, D-Glu, L-Lys and D-Ala), 10% DMSO in a total volume of 100 µl for 120 min at 30 °C. 33 µl
of the reaction mixture, corresponding to 25 nmol of UDP-MurNAc-pp, were used in a MraY
36
Chapter 2
synthesis assay without further purification (42). UDP-MurNAc-peptide variants with shortened
stem peptide, i.e. UDP-MurNAc-dipeptide and -tripeptide, were synthesized in the presence of
the corresponding subset of muropeptide synthetases and were used for the synthesis of lipid I
(dipeptide) and lipid I (tripeptide), respectively. After synthesis, lipid intermediates were
incubated at room temperature with increasing molar ratios of copsin for 20 min. TLC analysis
was performed as described above.
CF-efflux from lipid II containing liposomes
Large unilamellar vesicles were prepared by the extrusion technique, essentially as described by
Wiedemann I et al. 2001 (22). Vesicles were made of DOPC supplemented with 0.5 mol% Lipid
II (referring to the total amount of phospholipid). Carboxyfluorescein (CF)-loaded vesicles were
prepared with 50 mM CF and then diluted in 1.5 ml of buffer (50 mM MES-KOH, 100 mM K2SO4,
pH 6.0) at a final concentration of 25 μM phospholipid on a phosphorous base. After addition of
1 µM peptide (nisin or copsin), the increase of fluorescence intensity was measured at 520 nm
(excitation at 492 nm) on an RF-5301 spectrophotometer (Shimadzu, Japan) at room
temperature. Leakage was documented relative to the total amount of marker release after
solubilization of the vesicles by addition of 10 μl of 20% Triton X-100.
Potassium efflux from whole cells
For potassium efflux experiments a microprocessor pH meter (pH 213; Hanna Instruments,
Germany) with a MI-442 potassium electrode and MI-409F reference electrode was used. In
order to obtain stable results, the electrodes were pre-conditioned by immersing both the
potassium selective and the reference electrodes in choline-buffer (300 mM choline chloride, 30
mM MES, 20 mM Tris, pH 6.5) for at least 1 hour before starting calibration or measurements.
Calibration was carried out before each determination by immersing the electrodes in fresh
standard solutions containing 0.01, 0.1 or 1 mM KCl in choline buffer. Cells of B. subtilis were
grown in MHB and harvested at an OD600 of 1.0 to 1.5 (3300 x g, 4 °C, 3 min), washed with 50 ml
cold choline buffer, and resuspended in the same buffer to an OD 600 of 30. The concentrated cell
suspension was kept on ice and used within 30 min. For each measurement the cells were
diluted in choline buffer (25°C) to an OD 600 of about 3. Calculations of potassium-efflux in
percent were performed according to the equations established by Orlov D. S. et al. 2002 (44).
Peptide-induced leakage was monitored for 5 min, with values taken every 10 sec, and was
expressed relative to the total amount of potassium release induced by addition of 1 µM nisin.
Copsin was added at 10xMIC.
37
Chapter 2
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Güntert P (2004) Automated NMR structure calculation with CYANA. Methods Mol Biol
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Brotz H, Bierbaum G, Reynolds PE, & Sahl HG (1997) The lantibiotic mersacidin inhibits
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Rick PD, et al. (1998) Characterization of the lipid-carrier involved in the synthesis of
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Orlov DS, Nguyen T, & Lehrer RI (2002) Potassium release, a useful tool for studying
antimicrobial peptides. J Microbiol Meth 49(3):325-328.
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Petersen TN, Brunak S, von Heijne G, & Nielsen H (2011) SignalP 4.0: discriminating signal
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Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, & Thornton JM (1996) AQUA and
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40
Chapter 2
Supplementary Information
Fig. S1. Identification of an AMP in the secretome of C. cinerea. (A) Proteins were extracted from the
unchallenged C. cinerea secretome, digested with proteinase K, and the remaining proteins fractionated
on a cation exchange column. The effluent was monitored at 280 nm (solid line) and the conductivity was
measured (dashed line). (B) Fractions collected (0.5 ml) during the run were spotted in a disk diffusion
assay against B. subtilis. Activity was detected from 7 to 8.5 ml elution volume. (C) Proteins in the active
fractions and flow through (FT) were identified by an electrospray ionization tandem mass spectrometry
(ESI-MS/MS) measurement and quantified by spectral counting. The relative counts corresponding to the
protein CC1G_13813 (copsin) were best correlating to the diameter of the inhibition zones displayed
against B. subtilis.
41
Chapter 2
1
ATG AAA CTT TCT ACT TCT TTG CTC GCT ATC GTC GCT GTG GCG TCT ACC TTC ATT GGG AAC
M
K
L
S
T
S
L
L
A
I
V
A
V
A
S
T
F
I
G
N
>-------------------------------- Signal peptide ------------------------------
61
GCC CTC TCA GCC ACC ACC GTC CCC GGA TGC TTC GCT GAG TGC ATT GAC AAG GCT GCC GTA
A
L
S
A
T
T
V
P
G
C
F
A
E
C
I
D
K
A
A
V
----------< >-------------------- Pro peptide ---------------------------------
121
GCC GTC AAT TGC GCC GCG GGG GAT ATC GAC TGC CTC CAG GCT TCC TCG CAG TTC GCT ACT
A
V
N
C
A
A
G
D
I
D
C
L
Q
A
S
S
Q
F
A
T
-------------------------------------------------------------------------------
181
ATC GTT AGT GAA TGC GTC GCT ACC AGC GAC TGC ACT GCA CTT TCT CCT GGC TCG GCT TCT
I
V
S
E
C
V
A
T
S
D
C
T
A
L
S
P
G
S
A
S
-------------------------------------------------------------------------------
241
GAC GCG GAC TCC ATC AAC AAG ACC TTC AAC ATT CTC TCG GGT CTT GGT TTC ATT GAC GAA
D
A
D
S
I
N
K
T
F
N
I
L
S
G
L
G
F
I
D
E
-------------------------------------------------------------------------------
301
GCC GAC GCC TTC AGC GCC GCC GAT GTT CCC GAA GAG CGC GAT CTC ACT GGG TTG GGC CGT
A
D
A
F
S
A
A
D
V
P
E
E
R
D
L
T
G
L
G
R
-------------------------------------------------------------------------------
361
GTT TTG CCC GTT GAA AAG CGC CAG AAC TGC CCT ACC CGT CGT GGT TTG TGT GTC ACC TCA
V
L
P
V
E
K
R
Q
N
C
P
T
R
R
G
L
C
V
T
S
--------------------------< >--------------------------------------------------
421
GGC TTG ACG GCG TGT CGA AAC CAC TGT CGC TCA TGC CAC CGG GGA GAT GTA GGT TGT GTT
G
L
T
A
C
R
N
H
C
R
S
C
H
R
G
D
V
G
C
V
--------------------------------- Mature copsin --------------------------------
481
AGG TGC AGT AAT GCA CAG TGC ACG GGC TTT TTG GGC ACC ACA TGC ACC TGC ATT AAC CCC
R
C
S
N
A
Q
C
T
G
F
L
G
T
T
C
T
C
I
N
P
-------------------------------------------------------------------------------
541
TGC CCT CGT TGT TAA
C
P
R
C
--------------<
Fig. S2. Prepro-protein of copsin. Prediction of the signal peptide was performed with SignalP 4.0 (45).
42
Chapter 2
Fig. S3. Pyroglutamate at the N-terminus of copsin. A tryptic digest of purified recombinant copsin and
the extracted defense peptide of C. cinerea (CC1G_13813) was measured by ESI-MS/MS and the
detected fragments analyzed by the Xcalibur software. The recombinant and natural version of copsin
exhibited both the same N-terminal peptide with a glutamine converted to a pyroglutamate (pE). The
peptide with an N-terminal glutamine was not detectable. y* is a y ion with a loss of an ammonia molecule.
43
Chapter 2
Fig. S4. Structure calculations for the delineation of the six disulfide bonds. Cartoon representation
of the structure of copsin. Cysteine residues are marked with their residue number and colored in orange
for the new and in yellow for the conserved disulfides (see main text). N and C indicate N and C termini,
respectively. (A) Structure calculated with cysteine residues defined as negative charged without any use
of disulfide constraints. (B) Bundle of 40 best structures calculated by Cyana using ambiguous disulfide
constraints (38). Each individual cysteine was forced to build a disulfide bond to at least one of the
remaining eleven cysteines by an upper and lower distance restraint. For all calculated structures the
same disulfide connectivity was obtained. (C) Same as (B) but 90 degrees rotated into the plain of the
paper showing the terminal disulfides.
44
Chapter 2
Fig. S5. Temperature, pH, and protease stability of copsin. (A and B) Shown are disk diffusion assays
on B. subtilis with copsin that was exposed to different temperatures (4, 25, 50, 70, 90 °C) and in a pH
range of 2 to 8 for 1 h. (C) To evaluate the protease resistance, copsin/protease mixtures in a ratio of 10:1
(w/w) were incubated at pH 8 for trypsin and proteinase K and at pH 2 for pepsin for 3 h. Copsin was also
subjected to a treatment with 5 mM DTT. Additionally, the reaction mixtures were spotted without copsin.
As control (Ctrl), untreated copsin was loaded on a disc. The activity of copsin was retained in the whole
temperature and pH range tested and it showed no sensitivity in a treatment with proteases. The only way
to delete the activity of copsin was by disrupting the disulfide bonds.
45
Chapter 2
Fig. S6. Co-localization of copsin and vancomycin. B. subtilis and S. carnosus cells in the exponential
growth phase were stained with TAMRA-copsin (red) and subsequently stained with BODIPY-vancomycin
(green). Cells showed a co-localization of copsin with vancomycin at the cell septa (scale bar: 2 µm).
Fig. S7. Potassium efflux from living cells. Potassium efflux from B. subtilis cells was monitored with a
potassium-sensitive electrode. Ion leakage is expressed relative to the total amount of potassium released
after addition of 1 µM pore-forming lantibiotic nisin (100%, circles). Copsin was added at 10xMIC
(squares). Controls were without peptide antibiotics (triangles). Peptides were added after 60 sec. Copsin
was unable to form pores in the cytoplasmic membrane of B. subtilis cells.
46
Chapter 2
Supplementary Tables
Table S1. Structural statistics of the 20 best NMR structures of copsin
A: calculated without disulfide constraints, B: with defined ensemble of disulfide bonds, C: after energy refinement
A(A)
B(B)
C(C)
NMR Restraints
Distance Restraints
809823
823823
823
intraresidual
462
223
223
sequential (|i-j|=1)
115224
236237
236
medium range (1<|i-j|<5)
232129
126
126
long range (|i-j|>=5)
8 225
230
230
hydrogen bondsa
8 8
8
8
Torsion Anglesb
0 0
0
0
Disulfide bonds
0
6
6
Energy Statisticsc
Average distance constraint violations
0.1-0.2 Å
0.2-0.3 Å
0.3-0.4 Å
>0.4 Å
Maximal (Å)
0
Average angle constraint violations
<5 degree
>5 degree
Maximal (degree)
1
0
8.9 +/- 2.5
3.1 +/- 1.5
0.7 +/- 0.6
0.1 +/- 0.4
0.33 +/- 0.07
Mean AMBER Violation Energy
Constraint (kcal mol-1)
Distance (kcal mol-1)
Torsion (kcal mol-1)
8.8 +/- 2.0
2.2 +/- 1.2
0.2 +/- 0.4
0.0 +/- 0.0
0.28 +/- 0.03
1
0.0 +/- 0.0
0.0 +/- 0.0
0.0 +/- 0.0
16.0 +/- 0.0
0.0 +/- 0.0
96.57 +/- 0.20 (needs to be improved)
Mean AMBER Energy (kcal mol-1)
9.1 +/-1.2
1470.8 +/-3.3
9.1 +/-1.2
11.3 +/-3.0
0.0 +/- 0.0
1459.6 +/-0.8 (needs to be improved)
-2331.2 +/- 5.2
-2380.0 +/- 8.8
Mean Deviation from ideal covalent geometry
Bond Length (Å)
Bond Angle (degrees)
0.0043 +/- 0.0001
1.790 +/- 0.014
0.0043 +/- 0.0001
1.728 +/- 0.009
Ramachandran plot Statisticsc,d,e
Residues in most favored regions (%)
Residues in additionally allowed regions (%)
Residues in generously allowed regions (%)
Residues in disallowed regions (%)
70.483.6 +/- 2.5
29.615.7 +/- 2.5
0.1 0.7 +/- 1.2
0.0 0.0 +/- 0.0
64.2
35.8
0.0
0.0
79.2 +/- 1.9
20.7 +/- 2.1
0.1 +/- 0.5
0.0 +/- 0.0
RMSD to mean structure Statisticsc,d
Backbone atoms (Å)
Heavy atoms (Å)
0.500.42
+/- 0.09
+/- 0.09
0.980.86
+/- 0.16
+/- 0.11
0.43 +/- 0.09
0.87 +/- 0.11
0.34 +/- 0.09
0.78 +/- 0.08
Target function
0.21 +/-0.003
0.14 +/-0.003
Structure calculation by simulated annealing protocol of Cyana (38) (A), (B), 40 structures were selected on the basis of lowest target function and
energy minimized using Amber (15)(C).
(A)
No constraint for disulfide linkage was used
(B)
Each disulfide bond was explicitly defined by upper and lower distance limit restraints between the sulfur and carbon atoms of the two linked cysteins:
C3-C32, C10-C40, C18-C48, C22-C50, C25-C57, C35-C54
(C)
Energy refinement of the structure (B) using Amber (15).
a
H-bond constraints were identified from HNCO (37) experiments and slow exchanging amide protons in D 2O
c
Statistics computed for the best structures
d
Based on structured residue range, res 3-57
e
Ramachandran plot, as defined by the program Procheck (46)
47
Chapter 2
Table S2. Elucidation of disulfide network by target function a and intercysteine distances b analysis
Target function a
Violations a
Weight factor b
Variation of conserved disulfide c
10-40; 18-48; 22-50; 35-54; 3-32; 25-57 *
0.14
1 VdW e
0.0648
10-40; 18-48; 22-35; 50-54; 3-32; 25-57
0.18
1 VdW e
10-40; 18-48; 22-54; 35-50; 3-32; 25-57
0.60
5 distance / 1 VdW
10-48; 18-40; 22-50; 35-54; 3-32; 25-57
0.16
1 VdW e
10-48; 18-40; 22-35; 50-54; 3-32; 25-57
0.20
1 VdW
10-48; 18-40; 22-54; 35-50; 3-32; 25-57
0.95
6 distance / 2 VdW
10-18; 48-40; 22-50; 35-54; 3-32; 25-57
0.15
1 VdW e
10-18; 48-40; 22-35; 50-54; 3-32; 25-57
0.22
1 VdW
10-18; 48-40; 22-54; 35-50; 3-32; 25-57
0.82
8 distancef/ 1 VdW e
10-40; 18-22; 48-50; 35-54; 3-32; 25-57
0.54
3 distancef/ 1 VdW e
1.7643e-5
10-40; 18-50; 48-22; 35-54; 3-32; 25-57
1.29
9 distancef/ 2 VdW e
3.1976e-10
Variation of new disulfides d
10-40; 18-48; 22-50; 35-54; 3-32; 25-57 *
0.14
1 vdW e
0.0648
10-40; 18-48; 22-50; 35-54; 3-25; 32-57
0.15
1 vdW
10-40; 18-48; 22-50; 35-54; 3-57; 32-25
0.30
5 distance / 1 vdW
Disulfide bond pattern
0.0283
f
5.3654e-7
1.3644e-4
5.9653e-4
e
f
f
e
e
1.1294e-9
0.0044
0.0019
e
3.6487e-8
0.0091
e
e
7.5591e-5
Characterization of bundles of 40 conformers of copsin with different disulfide topologies. The cysteine bond pattern of the final structure (Fig 2B) is
represented in the top row of the first column and denoted by a star. Different other conceivable disulfide patterns are listed below and deviations in
cysteine connectivities from the favored one are indicated in bold. Target function (second row), amount of violations (third row) and weights based on
averaged Cβ-Cβ distances (forth row) are shown for the different cysteine pairings. Worse target function and weight factors were obtained for all
remaining combinations of cysteine pairing.
a
Target function and violation analysis. For every topology a structure calculation was performed using Cyana (38). The disulfides were given as fixed
constraints determined by the standard three upper and lower distance limits. Target function and violations are shown averaged over the 40 selected
best energy structures.
b
Characterization of disulfide bond pattern by cysteine Cβ-Cβ distances analysis as described by Klaus (18). Averaged distances were extracted from the
structure ensemble generated without any disulfide constraints. Subsequently weights were assigned to every disulfide pattern. The weight is directly
proportional to the likelihood of certain disulfide bond pattern to be realized in the final structure.
c
All possible combination of disulfide bond patterns for the conserved six cysteines are shown with the respective calculated target function, number of
violations and the weight factor.
d
Analysis of the disulfide bond connectivity of the new disulfides in copsin. The bond between C35-C54 is taken as fix, based on the initial structure and
the assigned conserved cysteines (see main text). All possible combinations of the remaining four cysteines (C3, C25, C32, C57) are listed and analyzed.
e
Van der Waal violations
f
Distance violations
* Disulfide bonding connectivity of the final structure as described in results (main text). Variations of single disulfide bonds from this assignment are
shown in bold.
48
Chapter 2
Table S3. Antibacterial profile of copsin
Bacterium
Copsin [µg/ml]
Source
Bacillus subtilis 168
MIC
MBC
1
1
Streptococcus
S. pneumoniae
DSM 20566
active
n.d.
S. pyogenes
DSM 20565
active
n.d.
Staphylococcus
S. carnosus
DSM 20501
8
8
S. epidermidis
DSM 20044
64
>64
DSM 4910
>64
>64
ATCC 29213
>40
>40
L. monocytogenes
WSLC 1042
0.5
1
L. monocytogenes
WSLC 1001
0.25
0.5
L. ivanovii
WSLC 3009
0.25
0.5
L. innocua
WSLC 2012
0.5
1
E. faecium
DSM 20477
4
8
E. faecium VRE
DSM 13590
2
2
E. faecalis
DSM 20478
8
16
S. aureus 113
S. aureus
Listeria
Enterococcus
Micrococcus luteus
DSM 1790
Escherichia coli BL21
Corynebacterium diphtheriae
DSM 44123
0.6
n.d
>64
>64
4
n.d
MICs and MBCs were determined with the microdilution broth method in MHB pH 7.3. The MBC is defined as the
concentration of copsin where 99.9% of bacteria are killed within 20-24 h. Active, displayed an inhibition zone in a
standard disk diffusion assay; n.d. not determined.
49
50
Chapter 3
Studies towards the mode of
action of copsin and its homologs
in C. cinerea
Contributions
Identification and characterization of homologous proteins of copsin
Further experiments were performed by Savitha Gayathri and John Hintze during their
Master studies at the ETH Zürich under the supervision of Andreas Essig and Pauli Kallio.
51
Chapter 3
Introduction
The following chapter is divided in three sections: 1. Homologous proteins of copsin encoded in
the genome of Coprinopsis cinerea. 2. The recombinant expression of copsin in a bioreactor.
3. Further characterization of the mode of action of copsin.
AMPs are an ancient part of the innate immune system known from all biological kingdoms.
Their evolution strongly contributed to immunological diversity and is essential for host survival 1.
Amplification of defensins is achieved by gene duplications, an event well described for
mammalian β-defensins 2,3. Followed by a positive selection, gene duplications can diversify and
specify the defense machinery, particularly important for invertebrates and fungi that lack an
adaptive immune system. However, little is known about defensins in fungi and their gene copy
numbers.
Pichia pastoris demonstrated to be an appropriate host for the heterologous production of
defensins such as plectasin 4. A big advantage of this expression system is the easy way of
upscaling from shake flask cultures to a fermenter culture, using defined and low cost basal salts
media. A bioreactor system provides a highly controlled environment that allows for growing
P. pastoris to cell densities of approximately 500 OD600 U/ml or 100 g/l cell dry weight 5. A high
biomass is especially important for secreted proteins, due to the fact that the cell density is
roughly proportional to amount of product in the culture supernatant 6. Furthermore, transcription
induced over the AOX1 promoter, is more efficient when methanol is continuously provided at
growth limiting rates, instead of methanol excess commonly added in shake flask cultures.
Deciphering the mode of action of an antibiotic is often difficult and includes a variety of
biochemical assays. A common first step is to investigate, which macromolecular biosynthesis
pathway is affected by an antibiotic, for example, through the incorporation of radioactively
labeled precursors (e.g. DNA replication, protein synthesis, cell wall (peptidoglycan)
synthesis) 7,8. Besides complementary approaches like genetic assays or transcriptional profiling,
recent publications showed that fluorescence microscopy is a powerful tool to get a first idea
how an antibiotic works 9,10. In combination with specific labeling strategies, microscopy allows
for a bacterial cytological profiling (BCP) and discrimination between different antibacterial
substances.
52
Chapter 3
Results and Discussions
1.
Homologous proteins of copsin
Sequence comparisons of copsin based on the Blastp algorithm revealed the highest identities
to proteins encoded by C. cinerea itself (Fig. 1). Comparing the homologs at the sequence level,
it was remarkable that the overall scaffold of the peptides was highly conserved with the cysteine
residues and the N-terminal pyroglutamate.
Fig. 1. Copsin and homologs. The highly conserved cysteine pattern is indicated in red. The mature
peptides are shown, including the Kex2 protease recognition site (KR) at the N-terminus. The alignment
was performed with the ClustalW algorithm and visualized with the Jalview software 11,12.
The homolog CC1G_08260, hereafter called CC82, was recombinantly expressed in P. pastoris
(Fig. S1). The antibacterial profiles of recombinant copsin and CC82 were similar with an overall
weaker activity of CC82, determined in a disk diffusion assay (Table 1). Copsin revealed its most
potent inhibition on L. monocytogenes and L. ivanovii, whereas CC82 was not at all active
against these two species. These findings indicated that C. cinerea encodes a diversified group
of AMPs, which are active against specific bacteria and potentially also against other microbes.
The expression of other homologs, such as CC1G_15644 and further investigations on the
antibacterial profiles is needed to confirm this hypothesis. In addition, the high similarity of copsin
and CC82 at the sequence level in contrast to a distinct activity against bacteria can give further
insights, how they interact with lipid II and other compounds of the bacterial cell wall.
53
Chapter 3
Table 1. Antibacterial profile of copsin and its homolog CC82
Bacterium
Copsin [µg/ml]
Source
Bacillus subtilis 168
CC82
MIC
MBC
1
1
active
Streptococcus
S. pneumoniae
DSM 20566
active
n.d.
active
S. pyogenes
DSM 20565
active
n.d.
active
Staphylococcus
S. carnosus
DSM 20501
8
8
active
S. epidermidis
DSM 20044
64
>64
n.a.
S. aureus 113
DSM 4910
>64
>64
n.a.
>40
>40
n.d.
S. aureus N315
>40
>40
n.d.
ATCC 29213
>40
>40
n.d.
L. monocytogenes
WSLC 1042
0.5
1
n.a.
L. monocytogenes
WSLC 1001
0.25
0.5
n.d.
L. ivanovii
WSLC 3009
0.25
0.5
n.a.
L. innocua
WSLC 2012
0.5
1
n.d.
E. faecium
DSM 20477
4
8
active
E. faecium VRE
DSM 13590
2
2
active
E. faecalis
DSM 20478
8
16
active
S. aureus HG003
S. aureus
Listeria
Enterococcus
Micrococcus luteus
DSM 1790
Escherichia coli BL21
Corynebacterium diphtheriae
DSM 44123
0.6
n.d
n.d.
>64
>64
n.a.
4
n.d
n.d
MICs and MBCs were determined with the standard microdilution broth method in MHB pH 7.3. The MBC is defined as the concentration of copsin where
99.9% of bacteria are killed within 20-24 h. Active, displayed an inhibition zone in a standard disk diffusion assay; n.a. no activity in disk diffusion assay; n.d.
not determined.
2.
Recombinant expression in a bioreactor
The expression of copsin was performed in a 3.6 l bioreactor with the open reading frame (ORF)
encoding the native signal sequence. In general, we followed standard procedures with an initial
biomass production step, using glycerol as the carbon source 13,14. After a transition phase, the
expression of copsin was initiated with methanol and continued over 90 h.
54
Chapter 3
Fig. 2. Copsin production in a bioreactor. Samples were taken from the fermenter culture at defined
time points after starting the induction with methanol. (A) After a centrifugation step, the supernatant was
directly loaded on a SDS-PAGE and a silver-staining was performed. (B) The antibacterial activity was
determined in a disk diffusion assay on Bacillus subtilis. The increase of copsin concentration detectable
at ~10 kDa is clearly correlating with the antibacterial activity.
After methanol induction, we reached a cell wet weight of 320 g/l and a final yield of purified
copsin of 20 mg/l, which is a tenfold increase compared to shake-flask cultures. A silver staining
displayed a very low amount of endogenous proteins secreted by Pichia and a continuously
increasing amount of copsin correlating with its antibacterial activity (Fig. 2).
Taken together, performing the Pichia expression in a bioreactor is absolutely crucial to achieve
high amounts of heterologous product. Based on the setup and protocol that we designed,
optimization is needed at different steps, for example, with a codon optimized version of copsin
(Fig. S2).
3.
Mode of action of copsin
Localization of copsin and induced morphological changes
In a first experiment, we studied the morphological effect of copsin on B. subtilis. Therefore, we
treated bacteria with sub-MIC concentrations of copsin and imaged them after 2 h. Differential
interference contrast (DIC) microscopy images revealed bacteria, which were severely swollen
and curved in comparison to untreated cells (Fig. 3). Furthermore, chains of non-separated and
shortened cells were visible with a higher frequency of septation. Some bacteria were
undergoing lysis, most likely due to a lethal concentration of copsin molecules encountered by
that particular cell. Since the cell wall is the major determinant of the bacterial cell shape, the
altered morphology indicated an indirect or direct impact of copsin on cell wall formation.
55
Chapter 3
A
B
C
D
E
Fig. 3. Morphological effect of copsin on B. subtilis. Cells at OD600 of 0.2 were exposed to sub-MIC
concentrations (0.5 µg/ml) of copsin in LB medium for 2 h and mounted on 1% agarose pads. (B - E)
Treated cells showed severe malformations compared to the untreated control sample (A). White arrows
point to swollen and shortened cells and the black arrows point to cells that have lyzed (scale bar: 4 µm).
Peptidoglycan, a polymer of alternating amino sugars of N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc), is the major constituent of the cell wall of Gram positive bacteria
15,16.
To visualize better the underlying features of the morphological effect, fluorescently labeled
BODIPY-FL vancomycin was used as a reporter for sites of active peptidoglycan synthesis on
sub-MIC treated B. subtilis cells. Vancomycin specifically binds to the D-Ala-D-Ala part of newly
synthesized lipid II precursor 17. Phase contrast and the corresponding fluorescence microscopy
images are shown in figure 4. In the control staining, vancomycin was specifically located at the
cell septa and only faintly at the sidewalls of B. subtilis cells (Fig. 4A). A dispersed binding
pattern of vancomycin was visible on copsin treated cells, correlating with the phenotype of bend
cells and multiple septation events in close proximity (Fig. 4B-F). These findings suggested that
copsin induced a disturbed synthesis of peptidoglycan that resulted in the display of vancomycin
targets at the cell surface.
56
Chapter 3
Fig. 4. Localization of vancomycin on sub-MIC treated B. subtilis. Cells at OD600 of 0.2 were
incubated with copsin at 0.5xMIC for 2 h and subsequently stained with BODIPY-vancomycin either in
culture (A - B) or after immobilization on a mounting cover slip (D - F). (A) Control cells that were not
treated with copsin showed an intense staining at the cell septa. (B - F) Copsin treated B. subtilis with an
abnormal septation displayed a localization of vancomycin at bend/swollen sites (scale bar: 5 µm).
Next, we studied the localization of TAMRA labeled copsin on B. subtilis cells. TAMRA is a red
fluorescent dye that specifically reacts with terminal alkyne groups. Importantly, the fluorescent
derivative of copsin retained its activity, as shown in a disk diffusion assay on B. subtilis (Fig.
5A). Additionally, we tested, whether the TAMRA dye itself is leading to an unspecific binding on
B. subtilis. Therefore, we treated cells with TAMRA labeled Marasmius oreades agglutinin
(MOA), a Galα1,3Gal/GalNAc-specific lectin that has no affinity for the cell wall of Gram positive
bacteria 18. As shown in figure 5B, there was no binding of TAMRA dye detectable. Incubation of
bacterial cells with TAMRA-copsin exhibited a staining exclusively at the cell wall. Copsin was
similarly distributed on the spherical-shaped S. carnosus cells (Fig. 5D). For the rod-shaped
bacterium B. subtilis, the fluorescence intensity was slightly increased at the septa and poles of
the cells, suggesting that copsin is preferably binding to curved membrane regions (Fig. 5C).
57
Chapter 3
Fig. 5. Localization of TAMRA-copsin on bacteria. (A) Copsin labeled with or without TAMRA displayed
the same inhibition zone in a disk diffusion assay on B. subtilis. (B) TAMRA attached to the lectin MOA
was used as a negative control and showed that there is no affinity of the dye itself for B. subtilis. At OD600
of 0.3, B. subtilis (C) and S. carnosus (D) cells were incubated with TAMRA-copsin at 2xMIC for 10min.
For both bacterial species, the fluorescence was located at the cell wall, with an accumulation at the poles
and septa of B. subtilis (scale bar: 8 µm).
C
A co-staining of B. subtilis and S. carnosus cells with BODIPY-vancomycin and TAMRA-copsin
revealed that both dyes indeed co-localize at the cell septa (Fig. 6). Differences were detectable
in the overall distribution of the fluorescence intensity, as copsin stained the cells more uniformly
than vancomycin that was focused at the cell septa and poles.
58
Chapter 3
Fig. 6. Co-localization of copsin and vancomycin. B. subtilis and S. carnosus cells in the exponential
growth phase were stained with TAMRA-copsin (red) and subsequently stained with BODIPY-vancomycin
(green). Cells showed a co-localization of copsin with vancomycin at the cell septa (scale bar: 6 µm).
To investigate, whether there is a correlation between binding sites of copsin and the
morphological phenotype, B. subtilis cells were incubated with sub-MIC concentrations of
TAMRA-copsin for 2 h. Subsequently, the cells were in addition stained with BODIPYvancomycin to compare the binding pattern. Interestingly, copsin was fully covering cells that
were undergoing lysis, where vancomycin was almost completely absent (Fig. 7). These cells did
not show the expected aberrant morphology. On the other hand, cells that received only a low
number of copsin molecules developed severe malformations with mislocalization of
vancomycin, as shown in figure 4. On particular cells, red fluorescence was clearly detectable at
bend regions, suggesting that copsin affects directly the cell wall synthesis.
59
Chapter 3
Fig. 7. Correlation between binding and effect of copsin on B. subtilis cells. Cells were incubated
with 0.5xMIC TAMRA-copsin for 2 h and subsequently stained with BODIPY-vancomycin. The white
arrows point to cells that encountered a lethal dose of copsin and are about to lyse.
Taken together, copsin had a strong impact on the morphology and viability of bacterial cells. For
localization studies, copsin was fluorescently labeled with the TAMRA dye. B. subtilis treated
with supra-MIC concentrations of TAMRA-copsin for 10 min, exhibited a binding at the
membrane and cell wall with a stronger intensity at the cell poles and septa, similar to
vancomycin. Reducing the concentration of copsin to sub-MIC values with a prolonged exposure
of about 2 h led to an aberrant morphology with severely bend, swollen and shortened cells.
Moreover, vancomycin exhibited a disturbed binding pattern, mainly detectable in regions of
malformations, correlating with multiple septa formations in close proximity. Interestingly, copsin
was then preferably binding to cells that lyzed. These observations indicated that cells, which
received a critical number of copsin molecules and did not further divide, were not able to
develop the aberrant phenotype, but lyzed rapidly. Furthermore, it suggested that copsin
interferes with the cell division machinery or with compounds of the cell membrane or wall. A kill
curve performed for B. subtilis exhibited that there is indeed a strong drop of the viable count
after one generation time (30 min) of growth.
The morphological changes are rather different to phenotypes seen for cell wall interfering
agents, such as vancomycin or penicillin, as we never observed blebbing or spheroblasts.
Similarities can be found with antibiotics that affect the cell membrane. One example is nisin, a
type A lantibiotic produced by Lactococcus lactis with a potent activity against Gram positive
species 19. Nisin exerts its antibacterial action by binding to the pyrophosphate moiety of lipid II
precursor and subsequently penetrating into the cytoplasmic membrane of a bacterium 20. This
60
Chapter 3
process is followed by pore formation and efflux of vital molecules such as ATP. A recent study
showed that B. subtilis treated with nisin displays multiple septations and septal malformations
with shortened minicells 21. They correlated these observations with a deregulation of the ATP
dependent Min system, which controls and suppresses the formation of new septa
. The
22
lipopeptide daptomycin is another example, which even closer resembles our findings.
Daptomycin is naturally produced by the soil bacterium Streptomyces roseosporus and is
approved for the treatment of infections caused by Gram positive pathogens
. Even though
23
clinically used for almost one decade, the mode of action is still heavily discussed. Known is that
daptomycin acts in a calcium dependent manner on the cell envelope through interference with
the cell wall biosynthesis and/or the cell membrane integrity
. Treatment of B. subtilis with
24,25
sub-MIC concentrations of daptomycin exhibited a phenotype as observed for copsin with bend,
shortened, and swollen cells 9. Fluorescent microscopy studies showed that daptomycin colocalizes with the cell division protein DivIVA at bend regions. DivIVA is an essential protein in
the cell division machinery of B. subtilis that localizes to curved regions of the cell membrane
26,27.
It is proposed that DivIVA causes local changes in peptidoglycan synthesis, leading to an
asymmetric extension of the cell wall and consequently to bending 9. Analog to copsin, B. subtilis
cells treated with a supra-MIC concentration of daptomycin did not develop the malformations,
but lyzed rapidly due to membrane damages.
Membrane permeabilization of copsin
The morphological malformations induced by copsin indicated a strong similarity to antibiotics
that permeabilize the cell membrane or even lead to pore formation, such as nisin. To examine,
whether copsin has an impact on the cell membrane integrity, we used Sytox Green, a
fluorescent dye of about 600 Da. Sytox Green can only enter bacteria with a compromised
plasma membrane, often used for Live/Dead staining
28.
Upon binding to nucleic acids, the
fluorescence emission of Sytox Green is approximately 500-fold enhanced.
First, we used B. subtilis cells in the early exponential growth phase to compare the influx of
Sytox Green upon treatment with copsin and nisin. Nisin is known to induce uptake of Sytox
Green, resulting from its pore forming activity. As a negative control, we exposed the cells to
vancomycin, which does not directly affect the plasma membrane. The three antibiotics were
applied in supra-MIC concentrations. We monitored an immediate increase in fluorescence
emission after adding copsin to a B. subtilis suspension pre-incubated with Sytox Green, similar
to nisin (Fig. 8). After treatment with vancomycin, there was no change in fluorescence
detectable.
61
Chapter 3
Fig. 8. Sytox Green uptake after antibiotic treatment. After pre-incubation of B. subtilis cells with Sytox
Green, the antibiotics were added at 4xMIC (arrow). The fluorescence emission measured at 520 nm
showed a comparable increase after treatment with nisin and copsin. Vancomycin and the medium control
had no impact on the Sytox Green uptake.
To confirm these results, the Sytox Green uptake was analyzed by microscopy (Fig. 9). The
percentage of B. subtilis cells emitting green fluorescence was calculated manually in the field of
view. After 10 min of treatment with the corresponding antibiotic, images obtained for nisin and
copsin showed a strong signal with 50% and 85%, respectively, of cells affected. Only a minor
fluorescence was detectable for the control (1.6%) and cells treated with vancomycin (1.5%).
The immediate influx of Sytox Green indicated that copsin permeabilized the cell membrane of
B. subtilis.
62
Chapter 3
Fig. 9. Visualization of Sytox Green uptake. B. subtilis cells in the exponential growth phase were
treated with antibiotic for 10 min and immobilized on a 1% agarose pads. Nisin and copsin induced an
influx of Sytox Green shown by cells with green fluorescence signal. The untreated control cells and cells
exposed to vancomycin were not permeabilized.
Next, we investigated whether the permeabilization of the bacterial membrane induced by copsin
depends on the growth stage of a bacterial culture. Therefore, B. subtilis cells in the early and
late exponential growth phase were treated with supra-MIC concentrations of copsin and nisin. A
4-fold decrease in fluorescence emission at higher optical density was monitored for both
antibiotics (Fig. 10).
63
Chapter 3
Fig. 10. Cell stage dependency of copsin and nisin. B. subtilis cell cultures at OD600 of 0.15 and 1.2
were diluted to the same OD600 (0.1) and pre-incubated with Sytox Green. Copsin and nisin were added to
4xMIC (arrow). At a later growth stage, a clear drop of fluorescence emission (520 nm) was visible.
We can summaries that copsin had a strong impact on the integrity of the bacterial cytoplasmic
membrane when applied in lethal concentrations. Together with the microscopy studies, it
indicated a pore forming mechanism similar to nisin, dependent on a cell wall precursor.
However, the killing kinetics of copsin is more similar to cell wall interfering agents than to rapidly
lytic antibiotics 8. Furthermore, our experiments exhibited that the permeabilization ability of
copsin is strongly dependent on fast dividing cells, which made an interaction solely with the cell
membrane more unlikely.
These results are not consistent with the findings for the carboxy-fluorescein efflux from lipid II
containing liposomes and the K + release from B. subtilis cells. In both assays, there was no
permeabilization of the bacterial membrane detectable within 3 min after exposure to copsin, in
comparison to nisin that was immediately active on the membrane. However, factors such as
buffer or medium conditions, properties of the reporter ion, or technical setups potentially had a
strong impact on the outcome of these assays. Further experiments are required to give a
conclusive reason for the differences in cell membrane permeabilization. Nevertheless, one fact
that was clearly demonstrated by these assays was that copsin acts differently on lipid II than
nisin, as indicated by the kinetic measurements.
64
Chapter 3
Synergy of copsin with lysozyme and EDTA
The synergistic effect of antibiotics together with other substances is widely known and exploited
to kill resistant pathogens 29. Microscopy studies and Sytox Green assays showed that copsin
interacts with a compound of the cytoplasmic membrane, leading to cell lysis. In Gram positive
bacteria, this membrane is covered and protected by a thick layer of murein and in Gram
negative species even by an additional outer membrane that hinders the penetration of many
antibiotics. Here we tested, whether copsin acts synergistically with lysozyme or
ethylenediaminetetra-acetic acid (EDTA). Lysozyme specifically hydrolyses the β1,4-linkages
between MurNAc and GlcNAc and thus, is degrading the peptidoglycan layer of mainly Gram
positive bacteria 30. EDTA is known to damage the outer membrane of Gram negative bacteria
making it permeable for extracellular molecules 31.
Fig. 11. Double disk diffusion synergy test. (A) Copsin (C; 23 µg) spotted on B. subtilis and two
adjacent disks with each 23 µg of hen egg white lysozyme HEWL (L). The inhibitory halo of copsin was not
affected by lysozyme. (B) Copsin together with EDTA (E; upper row: 0.5 M; lower row: 0.1 M) on E .coli
cells. A slight halo is visible around the disks loaded with copsin, showing that EDTA renders E. coli
susceptible for copsin.
A double disk synergy test showed that the antibacterial activity of copsin was not supported by
the action of lysozyme against B. subtilis (Fig. 11A) 32. EDTA in combination with copsin induced
an inhibitory halo on E. coli that we could not detect when copsin was applied independently
(Fig. 11B). This result showed that the target molecule of copsin is not specific for Gram positive
species.
65
Chapter 3
Materials and Methods
All chemicals, if not otherwise mentioned, were bought at the highest available purity from
Sigma-Aldrich.
Antimicrobial activity
The MIC/MBCs for copsin were determined by the microdilution broth method and as described
in chapter 2 33. For disk diffusion assays, 15 µg of purified CC82 and copsin was spotted on an
individual disk and plates incubated at appropriate growth temperatures for 24 h.
Expression in bioreactor
Bioreactor and control software
A Labfors 5 bioreactor with a 3.6 l working volume (WV) (Infors AG, Switzerland) was used for P.
pastoris fermentations. The bioreactor was equipped with a jacketed glass vessel, pH probe
(Mettler-Toledo, Switzerland), dissolved oxygen probe (Mettler-Toledo, Switzerland), foam probe,
air-flow meter, 2x Rushton impellers, baffles, sparger, pumps for acid, base, feed and anti-foam.
An auxiliary peristaltic pump was used for the initial glycerol feed. PC running IRIS NT version 5
software (Infors AG, Switzerland) was used for implementation of fermentation control
sequences.
Bioreactor operation
Expression was performed following the P. pastoris fermentation process guidelines of
Invitrogen 13 with slight modifications according to Diethard Mattanovich, BOKU, Vienna: Instead
of a direct change from glycerol to methanol feed, a pulse of 5 ml/l methanol is added before the
start of the methanol feed. This is to avoid accumulation of methanol in the medium as the cells
change their carbon metabolism. Initial volume of fermentation was 1.2 l expecting a final volume
of 1.5-2 l depending on length of the methanol fed-batch.
The fermentation process has two distinct phases. The first phase consists of a 24 h batch and 4
h fed-batch using glycerol as a carbon source with the purpose of generating a high cell density
without any recombinant protein production. The second phase is a 100 h methanol fed-batch,
where production of recombinant protein is induced under the control of the AOX1 promoter.
1.2 l of basal salts medium (per liter: 26.7 ml H3PO4, 0.93 g CaSO4, 18.2 g K2SO4, 14.9 g
MgSO4*7H20, 4.13 g KOH, 40 g glycerol) was added to the reactor, the pH probe calibrated and
the vessel with probes and medium autoclaved at 121 °C for 20 min. The medium was
maintained at the desired temperature (30°C) and the pH was set to 5 by sterile addition of 25%
NH4OH followed by addition of 4.35 ml/l of PTM1 trace salts solution (PTM1 trace salts per liter:
6.0 g CuSO4*7H20, 0.08 g NaI, 3.36 g MnSO4*H20, 0.2 g Na2MoO4*2H20, 0.02 g boric acid,
66
Chapter 3
0.82 g CoCl2*6H20, 20 g ZnCl2, 65 g FeSO4, 5 ml 95% H2SO4) and 0.87 mg/l biotin of a fresh
0.2 g/l biotin stock. The vessel was flushed with N 2 allowing the calibration of the dissolved
oxygen probe. Stirrer speed and airflow rate was set to 750 rpm and 1.2 l/min.
50 ml BMGY (1% (w/v) yeast extract, 2% (w/v) peptone, 1.3% (w/v) YNB w/o amino acids
(Becton Dickinson, USA), 100 mM potassium phosphate buffer pH 5, 1% (v/v) glycerol)
supplemented with 100 µg/ml zeocin was inoculated with P. pastoris NRRLY11430 transformed
with native copsin. The culture was grown overnight at 30°C to an OD600 of 6-10 and 40 ml of the
culture was used to inoculate the bioreactor.
The fermentation was run in batch mode for approximately 24 h. We attempted to maintain the
dissolved oxygen above 20 % of air saturation by regulating the stirrer speed and air flow rate
using a PID cascade loop. When all glycerol was consumed, indicated by a rise in dissolved
oxygen levels to > 90%, a limiting glycerol feed was started at a rate of 15.5 ml/h*l. After 4 h, a
total of 62 ml of feed was added and the pump was stopped. Depletion of glycerol in the reactor
was indicated by an increase of dissolved oxygen to > 90%. Methanol is toxic to P. pastoris at
too high concentrations. To avoid methanol accumulation while cells changed their metabolism
from glycerol to methanol a pulse of 5 ml/l of methanol was added to the culture. The methanol
was allowed to be consumed before the methanol feed pump was started approximately 4 h
later. Using a control sequence written in the IRIS software (Infors AG, Switzerland), the addition
of the methanol pulse and subsequent start of the methanol feed were automated. The methanol
feed was also regulated by the software to maintain the dissolved oxygen level above 10%, while
the stirrer speed and air flow rate were kept at 1170 rpm and 1.2 to 2 l/min, respectively. If the
oxygen level could not be maintained above 10% the feed was regularly turned off to see, if
methanol was accumulating in the reactor. After 90 h of methanol feed and addition of
approximately 700 ml of methanol the culture was harvested. An overview of the process is
shown in table 2.
Table 2: Pichia pastoris fermentation process scheme
Step
Carbon source
Duration h
Stirrer (rpm)
Aeration (l/min)
Batch
Glycerol
~24
750 - 1170
1.2
Fed-batch 1
Glycerol
≥4
1170
1.2
Transition
Methanol
~4
750 - 1170
1.2
Fed-batch 2
Methanol
90
1170
1.2-2
67
Chapter 3
Purification of copsin and CC82
The post-fermentation broth was centrifuged at 3000 x g for 20 min and the supernatant
concentrated in a 3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc., USA) by
a treatment with polyethylene glycol 6000 at 4 °C. The concentrated supernatant was dialyzed
against 20 mM Na-phosphate, 50mM NaCl buffer pH 7 (buffer A) at 4 °C for 24 h. The protein
solution was sterile-filtered and loaded on a self-made SP sephadex cation exchange column
equilibrated with buffer A. The column was washed with 180 mM NaCl and bound proteins were
eluted with 400 mM NaCl in 20 mM Na-phosphate buffer pH 7. The eluent was subjected to a
size exclusion chromatography for further polishing. The separation was performed on a
Superdex75 column (HiLoad 16/60, GE Healthcare, USA) equilibrated with 20 mM Naphosphate, 50 mM NaCl buffer pH 6. The effluent was monitored by absorbance at 210 nm. The
fractions containing the active peptide were combined.
Copsin induced morphological changes of B. subtilis
B. subtilis cells were grown in LB medium to an OD600 of 0.2 and treated with 0.5 µg/ml copsin
for 2 h. Untreated B. subtilis cells were used as control. For microscopy, 10 µl of the cultures
were immobilized on 1% agarose slabs. DIC images were acquired with an Axioscope 2
Apotome microscope with a 100x/1.4 Zeiss objective using an AxioCam MR (Zeiss, Germany).
Poly-L-lysine coating of coverslips
20 x 20 mm cover slips were washed thoroughly with ethanol for 5 min and air dried. The sterile
cover slips were then incubated in 0.01% poly-L-lysine solution for 5 min at RT. The cover slips
were washed with sterile water and air dried for 1 h.
Confocal microscopy
Bacterial cells were observed under a 100x oil-immersion objective (Zeiss, Germany) on a
spinning-disk confocal microscope (Visitron, Germany) and imaged with Evolve 512 EMCCD
camera (Photometrics, USA) using standard filter sets for GFP (excitation/emission:
488/525 nm) and rhodamine (540/575 nm). Images were processed using ImageJ v1.46.
Localization of TAMRA-copsin on bacteria
Bacteria were grown in LB medium to an OD 600 of 0.3 and incubated with TAMRA-copsin at
2xMIC (B. subtilis: 2 µg/ml; S. carnosus: 16 µg/ml) for 10 min. The cells were washed twice with
phosphate buffered saline (PBS) at pH 7.4 and immobilized on a poly-L-lysine coated cover slip.
Unbound cells were washed away with sterile water. The cover slip was then mounted on a
microscopic glass slide with 90% (v/v) glycerol. The cells were observed as described. The red
68
Chapter 3
fluorescence signal from the TAMRA dye was visualized by an excitation/emission wavelength of
540/575 nm with a standard filter set for rhodamine.
Co-localization of copsin and vancomycin
Bacteria were grown in LB medium to OD600 of 0.3 and incubated with TAMRA-copsin at 0.5xMIC
(0.5 µg/ml) for Bacillus subtilis and 2xMIC (16 µg/ml) for S. carnosus for 10 min. The cells were
washed twice with PBS and then immobilized on a poly-L-lysine coated cover slip. After washing
away the unbound cells with sterile water, BODIPY-vancomycin (Life Technologies, USA) was
used at 1 µg/ml concentration for staining on the cover slip. The cells were observed as
described, using standard filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine
(540/575 nm).
Localization of copsin and vancomycin on sub-MIC treated B. subtilis cells
B. subtilis cells were grown in LB medium to an OD600 of 0.2 and treated with 0.5 µg/ml of
unlabeled copsin or TAMRA-copsin for 2 h. Untreated B. subtilis cells were used as control. The
cells were then washed with 0.2% (v/v) saline, diluted to an OD600 of 0.2 and immobilized on a
poly-L-lysine coated coverslip. The unbound cells were washed with sterile water and stained
with 1 µg/ml BODIPY-vancomycin for 5 min on the coverslip. The cells were observed as
described, using standard filter sets for GFP (excitation/emission: 488/525 nm) and rhodamine
(540/575 nm).
Sytox Green assays
B. subtilis cells were grown to an OD600 of 0.2 and washed twice with 20 mM Na-phosphate,
30 mM NaCl buffer at pH 6.0 and centrifuged at 7000 x g for 1.5 min. The cells were
resuspended and diluted to an OD600 of 0.1 in 20 mM Na-phosphate, 30 mM buffer at pH 6.0
containing 1 µM Sytox Green dye (Life Technologies, USA). After incubation in the dark at RT for
15 min, 100 µl of the cell suspension was filled in wells of a 96-well plate and fluorescence
measurements were made using the Victor Wallac 1420 Spectrofluorimeter (PerkinElmer, USA).
At approximately 7 min after initiating and stabilizing fluorescence signal reading, 4xMIC
concentrations of antibiotic was added to the cell suspension, and the increase in Sytox Green
fluorescence was measured every 40 seconds for 45 min (excitation wavelength at 485 nm and
emission at 520 nm). Permeabilization of the cells with nisin (MIC: 40 ng/ml) was used as a
positive control. Vancomycin (MIC: 50 ng/ml) was used as a negative control. The MIC for nisin
and vancomycin against B. subtilis was determined in the lab by the microdilution broth method
as described
33.
A blank control with cells incubated with Sytox Green but no antibiotic added
was also included in the study.
69
Chapter 3
For the cell stage dependency assay, a bacterial culture was divided in two sub-cultures and
grown independently to an OD600 of 0.15 and 1.2, respectively. Both culture were then diluted to
an OD600 of 0.1 and processed as described.
For microscopy, 100 µl of the cells incubated with Sytox Green were treated separately with
4xMIC of the corresponding antibiotic. At 10 min after treatment, cells were immobilized on a 1%
agarose pad for viewing under the microscope. The cells were observed under a 20x Zeiss
objective of an Axioscope2 Apotome microscope (Zeiss, Germany) and imaged using AxioCam
MR (Zeiss, Germany).
Double disk synergy test
A disk diffusion assay was performed by placing 2 sterile blank paper discs about 1 cm apart
and loading one disc with 30 µl of 750 µg/ml copsin and the other with 30 µl of 780 µg/ml of
lysozyme (HEWL). This synergy assay was also performed on an E. coli plate with 30 µl of a 0.1
and 0.5 M EDTA solution.
70
Chapter 3
References
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Patil, A. A., Cai, Y., Sang, Y., Blecha, F. & Zhang, G. Cross-species analysis of the mammalian
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Cregg, J. M., Cereghino, J. L., Shi, J. & Higgins, D. R. Recombinant Protein Expression in Pichia
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Cereghino, J. L. & Cregg, J. M. Heterologous protein expression in the methylotrophic yeast
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Cotsonas King, A. & Wu, L. Macromolecular synthesis and membrane perturbation assays for
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Schneider, T. et al. Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II.
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Pogliano, J., Pogliano, N. & Silverman, J. A. Daptomycin-mediated reorganization of membrane
architecture causes mislocalization of essential cell division proteins. J. Bacteriol. 194, 4494–504
(2012).
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Nonejuie, P., Burkart, M., Pogliano, K. & Pogliano, J. Bacterial cytological profiling rapidly
identifies the cellular pathways targeted by antibacterial molecules. Proc. Natl. Acad. Sci. U. S. A.
110, 16169–74 (2013).
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Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–8 (2007).
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Jahic, M., Veide, A., Charoenrat, T., Teeri, T. & Enfors, S.-O. Process technology for production
and recovery of heterologous proteins with Pichia pastoris. Biotechnol. Prog. 22, 1465–73 (2006).
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Schleifer, K. H. & Kandler, O. Peptidoglycan types of bacterial cell walls and their taxonomic
implications. Bacteriol. Rev. 36, 407–77 (1972).
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Harz, H., Burgdorf, K. & Höltje, J. V. Isolation and separation of the glycan strands from murein of
Escherichia coli by reversed-phase high-performance liquid chromatography. Anal. Biochem. 190,
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Sheldrick, G. M., Jones, P. G., Kennard, O., Williams, D. H. & Smith, G. A. Structure of
vancomycin and its complex with acetyl-D-alanyl-D-alanine. Nature 271, 223–225 (1978).
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Wohlschlager, T. et al. Nematotoxicity of Marasmius oreades Agglutinin (MOA) Depends on
Glycolipid Binding and Cysteine Protease Activity. J. Biol. Chem. 286, 30337–30343 (2011).
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Rogers, L. A. The inhibiting effect of Streptococcus lactis on Lactobacillus bulgaricus. J. Bacteriol.
16, 321–325 (1928).
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Breukink, E. & de Kruijff, B. Lipid II as a target for antibiotics. Nat. Rev. Drug Discov. 5, 321–32
(2006).
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Hyde, A. J., Parisot, J., McNichol, A. & Bonev, B. B. Nisin-induced changes in Bacillus
morphology suggest a paradigm of antibiotic action. Proc. Natl. Acad. Sci. U. S. A. 103, 19896–901
(2006).
22.
Hu, Z., Saez, C. & Lutkenhaus, J. Recruitment of MinC , an Inhibitor of Z-Ring Formation , to the
Membrane in Escherichia coli : Role of MinD and MinE. J. Bacteriol. 185, 196–203 (2003).
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Eisenstein, B. I., Oleson, F. B. & Baltz, R. H. Daptomycin: from the mountain to the clinic, with
essential help from Francis Tally, MD. Clin. Infect. Dis. 50, S10–5 (2010).
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Eliopoulos, G. M., Thauvin, C., Gerson, B. & Moellering, R. C. In Vitro Activity and Mechanism of
Action of A21978C1 , a Novel Cyclic Lipopeptide Antibiotic. 27, (1985).
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Schneider, T. & Sahl, H. G. An oldie but a goodie - cell wall biosynthesis as antibiotic target
pathway. Int. J. Med. Microbiol. 300, 161–9 (2010).
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Bramkamp, M. & van Baarle, S. Division site selection in rod-shaped bacteria. Curr. Opin.
Microbiol. 12, 683–8 (2009).
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Lenarcic, R. et al. Localisation of DivIVA by targeting to negatively curved membranes. EMBO J.
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antibiotics against infectious diseases. Phytomedicine 15, 639–52 (2008).
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Callewaert, L. & Michiels, C. W. Lysozymes in the animal kingdom. J. Biosci. 35, 127–160 (2010).
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Hocquet, D., Dehecq, B., Bertrand, X. & Plésiat, P. Strain-tailored double-disk synergy test
detects extended-spectrum oxacillinases in Pseudomonas aeruginosa. J. Clin. Microbiol. 49,
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Motyl, M., Dorso, K., Barrett, J. & Giacobbe, R. Basic microbiological techniques used in
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Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal
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72
Chapter 3
Supplementary Information
1
ATG GAA CTC ACT GCA TCC TTC CTC TCT GCA GTA GCG GTC GCC TCC ACC TTC GTC GGA ACC
F
V
G
T
M
E
L
T
A
S
F
L
S
A
V
A
V
A
S
T
>-------------------------------- Signal peptide ------------------------------
61
GCC CTC TCA GCG ACC ACC GTC CCC GGA TGC TAC GCC GAG TGC ATC GAG AAG GGT GCT GCA
A
L
S
A
T
T
V
P
G
C
Y
A
E
C
I
E
K
G
A
A
----------< >-------------------- Pro peptide ---------------------------------
121
GCC GTC AAC TGC GCC GTC GAC GAC ATC GAC TGC CTC AGA CTC GCT TCC TCG CAG TTC ACC
A
V
N
C
A
V
D
D
I
D
C
L
R
L
A
S
S
Q
F
T
-------------------------------------------------------------------------------
181
ACC ATC ACC CGC GAA TGC CTC GAC ACT AAC AAC TGT ACC AGT CTC ACC CCT GGT ACA CCC
T
I
T
R
E
C
L
D
T
N
N
C
T
S
L
T
P
G
T
P
-------------------------------------------------------------------------------
241
GCC GAC GAA GCC TCC ATC ACT ACA ACC TTC AAC ATC CTC TCA GGC CTC GGC CTC ATC GAC
G
L
I
D
A
D
E
A
S
I
T
T
T
F
N
I
L
S
G
L
-------------------------------------------------------------------------------
301
TCC TCC GAA GTC TTC AGC CTC GCC GAC GTC CTC CAA GTC CAC CAA CGC GAC CTC ACA GGC
S
S
E
V
F
S
L
A
D
V
L
Q
V
H
Q
R
D
L
T
G
-------------------------------------------------------------------------------
361
CTC AGC CGC ATC CTA CCT ATC GAC AAA CGC CAA AGG TGC ATC GTC CGT CGC GCC ACA TGC
L
S
R
I
L
P
I
D
K
R
Q
R
C
I
V
R
R
A
T
C
--------------------------------------< >--------------------------------------
421
GTC ACT TCA GGC TTG ACA GCA TGC GTA AAC CAC TGC ATC TCG TGC CAT CGC GGG AGT GGC
V
T
S
G
L
T
A
C
V
N
H
C
I
S
C
H
R
G
S
G
--------------------------------- Mature peptide ------------------------------
481
AGC AGC AAC TGT GTC GAG TGC AGC GGG GGT CGG TGC ACA GGC ACT CTG GGC ACG ACG TGC
S
S
N
C
V
E
C
S
G
G
R
C
T
G
T
L
G
T
T
C
-------------------------------------------------------------------------------
541
ACA TGT CAG AAC CCG TGT CGT AGT TGC TAA
T
C
Q
N
P
C
R
S
C
----------------------------------<
Fig. S1. Prepro-protein CC82 (CC1G_08260). Prediction of the signal peptide was performed with
SignalP 4.0 34.
73
Chapter 3
1
ATG AAA TTG TCA ACA TCC CTT CTT GCT ATC GTC GCA GTC GCT TCC ACC TTT ATT GGT AAC
M
K
L
S
T
S
L
L
A
I
V
A
V
A
S
T
F
I
G
N
>-------------------------------- Signal peptide ------------------------------
61
GCC TTG TCC GCT ACT ACA GTT CCA GGA TGT TTT GCT GAA TGC ATT GAT AAG GCT GCC GTT
A
L
S
A
T
T
V
P
G
C
F
A
E
C
I
D
K
A
A
V
----------< >-------------------- Pro peptide ---------------------------------
121 GCT GTC AAC TGT GCA GCT GGA GAT ATT GAC TGC TTG CAA GCC TCT TCC CAG TTC GCA ACT
A
V
N
C
A
A
G
D
I
D
C
L
Q
A
S
S
Q
F
A
T
------------------------------------------------------------------------------181 ATC GTT TCT GAG TGT GTC GCT ACT TCC GAC TGC ACA GCC CTT TCA CCA GGA TCA GCA AGT
I
V
S
E
C
V
A
T
S
D
C
T
A
L
S
P
G
S
A
S
------------------------------------------------------------------------------241 GAT GCT GAC AGT ATT AAC AAG ACT TTT AAC ATC TTG TCT GGT CTT GGT TTT ATT GAT GAA
D
A
D
S
I
N
K
T
F
N
I
L
S
G
L
G
F
I
D
E
------------------------------------------------------------------------------301 GCT GAC GCC TTC AGT GCC GCA GAT GTT CCT GAA GAG AGA GAC TTG ACA GGT CTT GGA AGA
A
D
A
F
S
A
A
D
V
P
E
E
R
D
L
T
G
L
G
R
------------------------------------------------------------------------------361 GTT TTG CCA GTC GAG AAA AGA CAA AAT TGT CCT ACC AGA AGA GGT TTG TGT GTT ACC TCC
V
L
P
V
E
K
R
Q
N
C
P
T
R
R
G
L
C
V
T
S
--------------------------< >-------------------------------------------------421 GGA CTT ACT GCT TGT AGA AAC CAT TGT AGA TCA TGC CAC AGA GGA GAT GTT GGA TGT GTC
G
L
T
A
C
R
N
H
C
R
S
C
H
R
G
D
V
G
C
V
--------------------------------- Mature copsin -------------------------------481 AGA TGC TCT AAT GCT CAA TGT ACT GGT TTT CTT GGA ACT ACC TGC ACT TGT ATC AAT CCT
R
C
S
N
A
Q
C
T
G
F
L
G
T
T
C
T
C
I
N
P
------------------------------------------------------------------------------541 TGT CCT AGA TGT TAG
C
P
R
C
--------------<
Fig. S2. Codon optimized prepro-protein of copsin.
74
Chapter 4
Fungal lysozyme identified
in the secretome of C. cinerea
Contributions
Analysis and description of the C. cinerea secretome by ESI-MS/MS
Identification and characterization of CC49 and CC92
The heterologous expressions of CC49 and CC92 were performed by John Hintze during his
Master studies at the ETH Zürich under the supervision of Andreas Essig and Pauli Kallio.
75
Chapter 4
Introduction
In 1909, P. Laschtschenko, a researcher of the University of Tomsk in Russia, first described
the inhibitory effect of hen egg white on bacteria 1. Thirteen years later, it was Sir Alexander
Fleming, known as the discoverer of penicillin (1928), who coined the word lysozyme, when
he discovered that nasal mucus of one of his patients strongly reduced the growth of
Micrococcus lysodeikticus 2. It took another forty years until the primary sequence of hen egg
white lysozyme (HEWL) was deciphered followed by the 3D structure
. HEWL is a
3,4
milestone in enzymology as it was the first enzyme for which an X-ray structure was solved
and for which the enzymatic mechanism was described in detail
. Lysozymes are
5,6
characterized by their ability to hydrolyze the β1,4-glycosidic bond between N-acetylmuramic
acid (MurNAc) and N-acetylglucosamine (GlcNAc) of peptidoglycan (PG), also called murein.
PG is a unique structural element of the bacterial cell wall, composed of linear chains of
alternating β1,4-linked MurNAc and GlcNAc residues interconnected by peptide bridges 7.
The disaccharide and the amino acid moieties are frequently modified and linked to other cell
wall compounds, leading to a high structural variation of PG in different bacterial species 8.
The cell wall and in particular PG provide the cell with stability and rigidity against turgor
pressure. Therefore, disruption of the integrity of the peptidoglycan layer through the action
of a lysozyme is leading to a rapid hypotonic lysis of a bacterial cell. Most lysozymes are
predominately active against Gram positive species, where the PG is freely accessible, in
comparison to Gram negative bacteria with a protective outer membrane.
Today, a wide variety of lysozymes is described in literature, originating from bacteria, plants,
animals, and viruses. Lysozymes are categorized according to their primary sequence and
biochemical properties, including the c-type (chicken-type) group in animals
9.
C-type
lysozymes are produced by most vertebrates, for example, human lysozyme and HEWL. In
mammals, they can be identified in various body fluids, such as saliva, tears, or breast milk,
but also in the lysosomal granules of neutrophils and macrophages. As a part of the innate
immune defense, lysozymes act against bacteria and may possess immunomodulatory
functions
10.
Additionally, some animals recruit lysozymes as digestive enzymes to gain
nutrients from bacteria 9. A different functionality of lysozymes is found in viruses and
bacteria. Bacteriophages exploit the action of lysozymes to partially digest the bacterial cell
wall before they enter the cell. At the end of the infection cycle, larger phages use lysozymes
and amidases, called endolysins, to induce the release from the host cell
11,12.
characterized examples are the member of the T4 phage group of endolysins
The best
13.
Bacteria
themselves express lysozymes or autolysins, which are required for an efficient degradation
and recycling of the cell wall during the cell division process
11.
Cellosyl, a bacterial
muramidase from Streptomyces coelicolor, shows a high similarity to the only fungal
76
Chapter 4
lysozyme identified, so far
. It was extracted from the secretome of Chalaropsis, a member
14
of the phylum of ascomycota and defines an own group of ch-type lysozymes
. They are
15,16
characterized by the ability to cleave 6-O-acetylated peptidoglycan, a modification that
renders the cell wall of, for example, Staphylococcus aureus insensitive towards most other
types of lysozymes 8. However, little is known about the structures and enzymatic properties
of ch-type lysozymes.
Nowadays, lysozymes and especially HEWL are used extensively in research and as food
preservatives. Treatments of systemic bacterial infections are restricted mainly due to
immunogenic reactions, a short plasma half-life, and an increased cytokine production upon
release of cell debris 17.
Here, we characterized a novel fungal lysozyme secreted by the basidiomycete Coprinopsis
cinerea. It showed a distinct antibacterial profile and revealed a highly conserved
autolysin/endolysin domain.
77
Chapter 4
Results
Lysozyme identified in the secretome of C. cinerea
A mass spectrometry based analysis of the secretome of C. cinerea grown on glass beads
revealed in total 182 proteins (Table S1). A majority of these proteins had a predicted
enzymatic or digestive function, such as glycosidases or peptidases, adapted to the
saprophytic lifestyle of this mushroom. The list included two lysozyme-like enzymes,
CC1G_03049 and CC1G_03076. CC1G_03049, hereafter called CC49, was further
characterized. The locus encoded a protein of 268 amino acids with a predicted signal
peptide (position: 1-20) (Fig. S1). A Blastp search with the amino acid sequence of CC49
showed a conserved autolysin/endolysin domain commonly identified in bacterial and phage
lysozymes (Fig. 1). No significant similarity was found to HEWL and human lysozymes.
Enterobacteria phage
C. cinerea CC49
Pseudomonas phage
C. crescentus
Xanthomonas phage
4
109
9
7
68
ISSNGITRLKREEG.[5].YSDSR
GIPTIGVGHTGK.[9].M
TITAEKSSELLKEDLQWV.[5].SLV
VNSRTVQEIKNSEG.[5].APDPI
GLPTVGYGHLCK.[3].C.[7].PLTEAQATSLLMTDLKTF.[5].DQI
ALAAALAGLVALEG.[5].YRDIA
GVPTICSGTTAG.[4].D
KATPEQCYQMTIKDFQRF.[5].DAI
VSRAAVDLIKRFEG.[5].AQLPD
GRWTVGYGHTLT.[4].A
SVSEKDAEALLLYDLISV.[5].EHT
LSAAGVVAISSHEG.[5].YPDPA.[3].APWTICYGHTGP.[5].L
VVTQSQCDKWLAQDLSKA.[5].AVV
75
181
75
73
138
Fig. 1. Alignment of CC49 with phage and bacterial lysozymes. The conserved catalytic
autolysin/endolysin domain is indicated within yellow borders (Enterobacteria phage, gi138699;
Pseudomonas phage, gi33300856; Caulobacter crescentus, gi16126412; Xanthomonas phage,
gi32128440). The alignment was performed with the BlastP algorithm
18.
Further analysis of the secretome revealed that the open reading frame of CC1G_06692,
hereafter called CC92, was highly similar to the N-terminal region of CC49 (Fig. 2). The
genetic locus of CC92 encoded 104 amino acids, consisting of a predicted signal peptide
(position: 1- 19) and a mature polypeptide (position: 20–104) (Fig. S2). Comparisons on the
AMP database exhibited sequence similarities of 25–30% to defensins and bacteriocins,
such as the human beta defensin 2 (AP00524) and salivaricin P produced by Lactobacillus
salivarius (AP01173)
19.
The result of an alignment of CC92 with the N-terminal region of
CC49 is illustrated in figure 2, which exhibited a highly conserved pattern of twelve cysteine
residues, also found in copsin.
78
Chapter 4
Fig. 2. Alignment of CC92, CC49, and copsin. A schematic alignment of CC92 and CC49 precursor
protein is illustrated. The corresponding amino acid positions are indicated. A sequence alignment is
shown for CC92, CC49, and copsin. The Kex2 recognition site is marked in green and the cysteine
residues are marked in red. The sequence alignment was performed with the ClustalW algorithm and
visualized by Jalview 20,21.
Recombinant expression of CC92 and CC49
CC92 and CC49 were recombinantly produced in Pichia pastoris, an efficient expression
system for secreted fungal proteins, as demonstrated for copsin. Both proteins potentially
undergo cleavage of a pro-peptide, due to a Kex2 recognition site identified (Fig. 2)
22.
Antibacterial activities of the culture supernatants were tested against B. subtilis (Fig. 3A).
CC49 exhibited a clear inhibition zone in a disk diffusion assay, in contrast to CC92 that was
weakly active when expressed with a C-terminal polyhistidine (His) tag and its native signal
sequence (CC92-His). This construct showed a molecular mass corresponding to the
uncleaved pro-protein in an immunoblot (Fig. 3B). When the native signal sequence of CC92
was replaced by the α-factor secretion signal of S. cerevisiae, the polypeptide exhibited an
increased molecular mass on the blot, most likely due to an uncleaved pro-peptide of the αfactor. CC49 displayed multiple bands including the molecular mass for the processed and
unprocessed pro-protein. Therefore, it could not be concluded, whether a pro-peptide is
cleaved off. The assignments of the protein bands were additionally confirmed by tandem
mass spectrometry measurements of in-gel proteolytic digests.
79
Chapter 4
Fig. 3. Antibacterial activity of recombinantly expressed CC92 and CC49. The predicted signal
peptide of CC92 and CC49 was replaced by the α-factor secretion signal of S. cerevisiae and a
polyhistidine tag was fused to the C-terminus (αCC92-His; αCC49-His). The protein production in P.
pastoris was performed in shake flask cultures. CC92 was additionally expressed without the His-tag
(αCC92) and with the native signal sequence (CC92-His). (A) The culture supernatant was
concentrated and spotted on B. subtilis. The disk assay showed a clear halo for the lysozyme-like
protein CC49 and a weak inhibition zone for CC92-His. Copsin was used as a positive control. (B) The
concentrated culture supernatant was separated on a SDS-PAGE followed by immunoblotting with a
histidine specific antibody. CC92-His displayed exclusively the molecular mass corresponding the proprotein at 8.5 kDa. CC49 showed multiple bands with the molecular masses of the processed
(21.7 kDa) and unprocessed (26.2 kDa) pro-protein indicated in red. M indicates the marker lane.
Expression and purification of CC49
The histidine tagged construct of CC49 was expressed in P. pastoris shake flask cultures.
After concentrating the culture supernatant, a one-step purification was performed on Ni-NTA
beads. The different fractions were analyzed on an SDS-PAGE and the antibacterial activity
determined in a disk assay on B. subtilis cells (Fig. 4). The elution step exhibited multiple
bands consistent with the immunodetection assay and it was the only fraction that showed an
antibacterial activity.
Both, the immunoblot and the Coomassie stained SDS-PAGE, displayed proteins with a
molecular mass higher than expected (Fig. 3B and 4A). CC49 harbors three potential Nglycan sites (Fig. S2; N85, N208, N544), which could result in this mass shift. To investigate,
whether CC49 is expressed as a glycoprotein, we treated purified CC49 with two specific
endoglycosidases
23.
The Peptide-N-Glycosidase F (PNGaseF) cleaves at the N-
acetylglucosamine (GlcNAc) and asparagine residue of high-mannose, hybrid, and complex
N-glycans. The Endoglycosidase H (EndoH) specifically cleaves between the two innermost
GlcNAc residues of high-mannose N-glycan structures. Additionally, we analyzed the thermal
stability of CC49 and the sensitivity towards proteinase K.
80
Chapter 4
Fig. 4. Recombinant expression and purification of CC49. CC49, including the α-factor secretion
signal of S. cerevisiae and a C-terminal polyhistidine tag, was expressed in P. pastoris. The culture
supernatant was concentrated and dialyzed against PBS. A separation was performed on Ni-NTA
beads with a one-step elution of 400 mM imidazole. (A and B) Flow through (FT), wash (W), and
elution fraction (E) were analyzed on a SDS-PAGE and spotted on a disk diffusion assay against
B. subtilis. The elution step was the only fraction that displayed Coomassie stainable bands and an
inhibition of bacterial growth.
CC49 was completely degraded when simultaneously exposed to heat and proteinase K
(Fig. 5). Protein corresponding to the potential glycoform (>26,2 kDa) was not detectable
anymore upon treatment with PNGaseF or EndoH. This result indicated that CC49 is indeed
carrying N-glycans, most likely of the high-mannose type, a structure often found on secreted
fungal proteins.
Fig. 5. Stability and glycosylation of CC49. Purified recombinant CC49 was subjected to a heat
treatment (60 °C) with and without proteinase K (100 ng/µl) for 2 h and it was exposed to PNGaseF
and EndoH according to the manufacturers protocol. The proteins were separated and visualized on a
Coomassie stained SDS-PAGE. Proteinase K degraded CC49 and the endoglycosidases led to a shift
of the upper bands (>26.2 kDa), assumed to be the glycoform.
81
Chapter 4
Optimization of CC49 expression in P. pastoris and purification
The degradation of CC49 most likely resulted of the action of endogenous proteases of P.
pastoris secreted during cultivation. To reduce the level of these proteases, we combined
three commonly known strategies
. First, the expression was performed in the protease A
22
deficient strain SMD 1168H (pep4). Second, we reduced the pH of the culture medium, as it
is known that P. pastoris has the ability to grow at lower pH, while the activity of secreted
proteases is strongly reduced. Third, protease inhibitor was added to the concentrated
culture supernatant before the pH was increased again. Additionally, we used a CC49
construct without a His-tag. The histidine residues potentially interfere with the antibacterial
activity. Due to the high number of arginine and lysine residues identified in CC49, we
performed the purification on a cation exchange column (Fig. 6). The combined strategies
strongly reduced degradation of CC49 and its glycoform. The antibacterial activity was
retained without the His-tag, verified in a disk diffusion assay on B. subtilis.
Lysozyme activity of CC49 and the antibacterial profile
CC49 possessed a predicted endolysin/autolysin domain, as shown in the initial alignments
with known hydrolases (Fig. 2). Therefore, we compared the enzymatic activity of CC49 with
HEWL and bovine serum albumin (BSA), using a commercial lysozyme assay kit. Increasing
amounts of CC49, HEWL, and BSA were incubated with fluorescein labeled Micrococcus
lysodeikticus cell wall and fluorescence was measured after 30 and 60 min. Both, CC49 and
the HEWL positive control, showed a clear increase in fluorescence with an increasing
concentration of protein (Fig. 7). These results confirmed that CC49 exerts an enzymatic
action on peptidoglycan, hydrolyzing the β1,4-glycosidic linkage between MurNAc and
GlcNAc.
82
Chapter 4
Fig. 6. Optimized expression of CC49 and purification on an ion exchange column. The
expression of CC49 was performed in medium buffered at pH 4 with a protease A deficient P. pastoris
strain. The culture supernatant was concentrated, dialyzed, and loaded on a 5 ml self-made cation
exchange sephadex column. (A) The elution was done at 200 mM NaCl over 5 column volumes and
the eluent was monitored at 280 nm. (B) Collected fractions were concentrated and analyzed by SDSPAGE and silver-staining. The major fraction of CC49 (26.2 kDa) eluted at 200 mM NaCl (96 ml
elution volume) and showed a clearly reduced degradation.
83
Chapter 4
Fig. 7. Lysozyme activity of CC49. The EnzCheck lysozyme assay kit (Life technologies) was used
for determining the enzymatic activity of CC49. The assay measures lysozyme activity on Micrococcus
lysodeikticus cell wall, which is labeled with fluorescein. The initially quenched fluorescence is
released upon enzymatic treatment proportional to lysozyme activity. The final concentration of CC49
and BSA in the reactions were 3.9, 7.9, 15.8, 31.3, 63, 125, 250 µg/ml. HEWL was prepared according
to the manufacturers protocol. The excitation and emission wavelength was 485 and 535 nm,
respectively, and was measured after 30 and 60 min. Mean and standard deviation was calculated
from three replicates performed for HEWL and CC49. Two replicates were made for the negative
control BSA.
84
Chapter 4
For determining the antibacterial profile of CC49, it was spotted on B. subtilis, S. carnosus,
E. coli, and S. aureus cells and the activity was compared to HEWL (Fig. 8). CC49 and
HEWL displayed a comparable activity against B. subtilis and E. coli and no inhibitory effect
on S. aureus cells. HEWL was additionally active on S. carnosus, in comparison to CC49
that did not exhibit an inhibition against this bacterial species.
Fig. 8. Antibacterial activity of CC49. 130 µg of purified CC49 and HEWL dissolved in sodium
phosphate buffer pH 6.5 supplemented with 50 mM NaCl were spotted on Bacillus subtilis (strain 168),
Staphylococcus carnosus (strain 361), Escherichia coli (strain BL21), and Staphylococcus aureus
(strain 113) cells.
85
Chapter 4
Discussion
Analysis of the C. cinerea secretome revealed that this basidiomycete encodes a lysozyme,
CC49, active against Gram positive and Gram negative bacteria.
CC49 and CC92 were recombinantly produced in Pichia pastoris and an inhibition zone was
shown on B. subtilis for both proteins. It was a unique finding that a fungus encodes the Nterminal region of a lysozyme in an independent genetic locus with an antimicrobial function.
Known is that cleavage of lysozymes by specific proteases can lead to a release of AMPs.
One example is processing of the N-terminal region of human breast milk lysozymes by
pepsin and subsequent release of AMPs. It is proposed that this process is relevant in
stomach of newborns, protecting the gastrointestinal tract against bacterial infections
24,25.
However, the rather low expression levels of CC92 could not be reproduced and therefore,
further optimization of the expression system is needed to conclusively demonstrate that
CC92 acts antibacterial.
The expression of CC49 exemplarily exhibited the advantages and disadvantages of the
Pichia system
22.
P. pastoris can easily be genetically modified and yields high expression
levels of heterologous proteins in shake flasks and fermenters with an inexpensive basal
salts medium. Furthermore, P. pastoris is capable of efficiently secrete recombinant proteins
with disulfide connectivities and modifications as N- an O-glycosylations, with a very low level
of endogenously produced and secreted proteins. We identified initially an N-glycosylated
version of recombinant CC49, verified by an endoglycosidase treatment. However,
hyperglycosylation by N-linked high-mannose structures and O-mannoses is a known
problem of secreted proteins in yeast
26.
Therefore, further investigations are required to
show, whether the protein of C. cinerea is indeed glycosylated and what the impact of these
modifications are on the structure and activity. Unexpectedly, this glycoform disappeared
upon pH reduction of the culture medium, a phenomenon that we could not confirm in
literature. Another disadvantage of Pichia is the endogenous expression of intra- and extracellular proteases, which led to a degradation of CC49. A reduction of this proteolytic
cleavage was achieved by using a protease deficient strain in combination with a lowered pH
of the culture medium. However, the impact of the protease deficient strain was negligible, as
determined in an independent expression. Because these mutant strains often show reduced
viability and expression levels, an important fact for further optimization of the expression
system
22.
CC49 harbors a Kex cleavage site, which could potentially lead to processing of a
pro-peptide. Based on the molecular masses detected and the variability of Kex recognition
sites in different organisms, it was not possible to conclude, whether CC49 is indeed
encoding a pro-sequence 27.
86
Chapter 4
Comparisons at the sequence level revealed that CC49 has a highly conserved cysteine
pattern identified in several homologous proteins in C. cinerea (e.g. CC1G_03042, 03040,
03076) and other fungi, such as Laccaria bicolor (S238N-H82). Similar to copsin, the
potentially high number of disulfide bonds could serve as a scaffold to stabilize secreted
lysozymes and other proteins in the harsh environment of dung, as the natural habitat of C.
cinerea. Furthermore, sequence alignments demonstrated a similarity to autolysins and
endolysins with a highly conserved catalytic domain. Based on these findings, it can be
speculated about the biological function of CC49. Besides directly killing and potentially
acquiring nutrients from bacteria, it is possible that CC49 is involved in remodeling of the
fungal cell wall. Especially invertebrate type (i-type) lysozymes often possess a muramidase
and chitinase activity, cleaving the β1,4-linked backbone of PG and of the GlcNAc
homopolymer chitin 28,29.
Different modifications of the glycan strand of PG can contribute to a lysozyme resistance 8.
The O-acetylation at the C6 position of MurNAc, linkage of PG to teichoic acid, or a high
degree of cross-linking are three examples, which render PG of S. aureus insensitive to
HEWL
30,31.
In contrast, S. carnosus that lacks O-acetylated MurNAc is killed upon exposure
to HEWL. CC49 did not display an inhibition zone on S. carnosus and S. aureus cells, which
indicated that binding properties and enzymatic activity are different to c-type and ch-type
lysozymes.
Due to its low similarity to other lysozymes and the specific antibacterial profile, CC49 can be
considered as a novel class of lysozymes. However, further investigations at the structural
level and biochemical assays are needed to accurately categorize this enzyme.
87
Chapter 4
Materials and Methods
Secretome analysis of C. cinerea
The cultivation and extraction was performed twice (A and B) with two technical replicates
each time. C. cinerea was grown on 40 g glass beads in CCMM (A: 10 ml; B: 15 ml) for 4 (A)
and 5 (B) days, respectively. Afterwards, the medium was extracted, centrifuged (3800 x g,
15 min), and the supernatant concentrated to 1 ml by lyophilization. 200 µl of a 100% TCA
solution was added and the proteins precipitated at 4 °C for 15 min. After centrifugation
(16000 x g, 15 min), the pellet was washed twice with 100% acetone and dissolved in
denaturing buffer (8 M Urea, 100 mM ammonium bicarbonate buffer pH 8). After 30 min (60
°C, 900 rpm), DTT was added to 10 mM and incubated at 37 °C for 45 min. Then, the
proteins were incubated with 30 mM IAM for 45 min at 30 °C in the dark. The proteins were
digested by 500 ng trypsin in 250 µl ammonium bicarbonate buffer (100 mM, pH 8) at 37 °C
for 16 h. The peptides were desalted on C18 ZipTip columns (Millipore, USA). The MS
analyses were performed on a hybrid Velos LTQ Orbitrap mass spectrometer (Thermo
Scientific, USA) coupled to an Eksigent-nano-HPLC system (Eksigent Technologies, USA).
Separation of peptides was done on a self-made column (75 µm x 80 mm) packed with C18
AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were eluted with a linear gradient from
2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250 nl/min. MS and MS/MS spectra
were acquired in the data dependent mode with up to 20 collision induced dissociation (CID)
spectra recorded in the ion trap using the most intense ions. All MS/MS spectra were
searched against the p354_filteredMod_d C. cinerea database using the Mascot search
algorithm v2.3 (Matrix Science Inc. , USA) with oxidation (M) as variable modification and
carbamidomethyl as fixed modification. Further statistical validation was performed with
Scaffold 4.0 (Proteome Software, USA) with a minimum protein probability of 90% and a
minimum peptide probability of 50%. This program was also used for determining the total
non-normalized spectral counts for each protein identified in the fungal secretome.
Cloning of CC92 and CC49
The coding sequence of CC92 and CC49 precursor protein was amplified from C. cinerea
(strain AmutBmut (A43mut B43mut pab1.2)) vegetative mycelium cDNA library by PCR with
the Phusion high-fidelity DNA polymerase (Thermo Scientific, USA) according to standard
protocols (Sambrook J, Russell D, 2001, Molecular cloning, 3 rd edition) using the following
primers:
88
Chapter 4
Table 1. Primers, restriction enzymes and plasmids used for cloning in E. coli DH5α.
Construct
Primer (5' to 3')
Restriction enzyme
Plasmid
αCC92-His
FP: ATTTGAATTCGCCCTCAACGGCCCCTG
EcoRI
pPICZαA
RP: ATTTGTCGACTGATGGCAAGCAGCACCTAA
SalI
FP: ATTTGAATTCATGAAGTTTACCACATCCCTCTT
EcoRI
RP: ATTTGTCGACTGATGGCAAGCAGCACCTAA
SalI
FP: ATTTGAATTCGCCCTCAACGGCCCCTG
EcoRI
RP: ATTTTCTAGATCTAGATTATGATGGCAAGCAGC
Xbal
FP: ATTTGAATTCGCCATCAACGATCCCTGCTC
EcoRI
RP: ATTTGTCGACGCACCTGGGAGGATGCC
SalI
FP: ATTTGAATTCGCCATCAACGATCCCTGCTC
EcoRI
RP: ATTTGTCGACCTAGCACCTGGGAGGATGCC
SalI
CC92-His
αCC92
αCC49-His
αCC49
pPICZA
pPICZαA
pPICZαA
pPICZαA
The PCR product was cloned into the corresponding expression plasmid (Life Technologies,
USA) containing a zeocin resistance gene using the corresponding restriction enzymes
(Fermentas GmbH, Switzerland) (Table 1). The affinity tag was composed of six histidine
residues encoded on the plasmid. The resulting plasmid was linearized by the SacI restriction
enzyme (Thermo Scientific, USA) and transformed into the P. pastoris strain NRRLY11430
by electroporation with 1.2 kV of charging voltage, 25 µF of capacitance and 129 Ω
resistance
32.
Positive clones were selected on YPD plates (1% (w/v) yeast extract, 2% (w/v)
peptone, 2% (w/v) glucose, 2% (w/v) agar) containing 100 µg/ml zeocin (LabForce,
Switzerland).
Expression of CC92 and CC49 in P. pastoris
P. pastoris transformants were cultured in BMGY medium (1% (w/v) yeast extract, 2% (w/v)
peptone, 1.3% (w/v) YNB w/o amino acids (Becton Dickinson, USA), 100 mM K-phosphate
buffer pH 6, 1% (v/v) glycerol) supplemented with 100 µg/ml of zeocin at 30 °C, 200 rpm, for
48 h. Maximum 10 % of the baffled Erlenmeyer flask volume was used to provide sufficient
aeration for glycerol and methanol metabolism. Cells were harvested at 3800 x g for 5 min at
room temperature and resuspended in the same volume of minimal medium (1.3% (w/v) YNB
w/o amino acids, 100 mM K-phosphate buffer pH 6, 0.4 µg/ml biotin, 0.5% (w/v) NH4Cl, 1%
(v/v) MeOH) for production of recombinant protein using methanol induction. Expression
89
Chapter 4
cultures were incubated for 48 h at 30 °C, 200 rpm. Methanol was added to 1% (v/v) in a time
interval of 12 h and NH4Cl was added to 0.5% (w/v) in a time interval of 24 h.
For the optimized expression of CC49, the P. pastoris strain SMD 1168H (pep4; Life
technologies, USA) was used and the pH of the minimal medium was adjusted to 4.
Purification of CC49 on cation exchange column
The post-fermentation broth was centrifuged at 3000 x g for 10 min and the supernatant
concentrated in a 3.5 kDa Spectra/Por dialysis membrane (Spectrum Laboratories, Inc.,
USA) by a treatment with polyethylene glycol 6000 at 4 °C. For the optimized expression of
CC49, the concentrated supernatant was then incubated with 1 mM PMSF and protease
inhibitor cocktail (Roche, Germany) for 1 h at 4 °C. After dialysis against 20 mM Naphosphate, 50 mM NaCl buffer pH 6.5 (buffer A) at 4 °C for 24 h, the protein solution was
sterile-filtered and loaded on a self-made SP sephadex cation exchange column equilibrated
with buffer A. The column was washed with buffer A and CC49 eluted at 200 mM NaCl in
20 mM Na-phosphate buffer pH 6.5. The effluent was monitored by absorbance at 210, 254,
and 280 nm.
Ni-NTA purification
0.5 ml of Ni-NTA beads (Macherey-Nagel, Germany) were washed and equilibrated in a total
of 50 ml PBS pH 7.4 and transferred to a 10 ml drop column (Macherey-Nagel, Germany).
Conditioned expression supernatant was loaded, the column washed with 50 CV 50 mM
imidazole in PBS and the elution performed with 8 CV 400 mM imidazole in PBS. Elution
fractions were concentrated and dialyzed against PBS pH 7.4.
Protein separation on polyacrylamide gel and detection
The protein solution was mixed with Lämmli buffer and boiled at 95 °C for 20 min. The
denatured proteins were separated on a 15% polyacrylamide gel. Detection of proteins was
performed in three different ways:
Proteins were stained with Coomassie Brilliant Blue R.
Immunoblotting was performed with a primary mouse anti-His antibody (dilution 1:2000;
Qiagen, Germany) and with a goat anti-mouse secondary antibody (dilution 1:2000)
according to standard protocols.
Silver staining was performed according to a standard protocol (Blum et al, (1987),
Electrophoresis 8, 93)
90
Chapter 4
Lysozyme activity assay
The lysozyme assay kit EnzCheck (Life technologies, USA) was used according to the
suppliers protocol. The assay is based on Micrococcus luteus cell wall being labeled with
fluorescein in such a manner that fluorescence is quenched. When the cell wall is cleaved by
lysozyme the quenching is relieved and increased fluorescence can be detected. The
reactions were performed with a final concentration of 3.9, 7.9, 15.8, 31.3, 63, 125, and
250 µg/ml CC49. BSA was prepared in the same manner as negative control. Fluorescence
was measured using the Victor3 plate reader (PerkinElmer, USA) with an excitation
wavelength of 485 nm and emission wavelength of 535 nm. Reactions for the positive control
HEWL and CC49 were performed in triplicates and the negative control BSA in duplicates.
91
Chapter 4
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Supplementary Information
Table S1. Secretome of unchallenged C. cinerea. The cultivation and extraction was performed
twice (A and B) with two technical replicates. Signal peptides were predicted by the software SignalP
4.0
33.
Shown are the predicted biological function and the non-normalized spectral counts for each
protein and replicate.
Accession
CC1G_00158
CC1G_00306
CC1G_00332
CC1G_00344
CC1G_00471
CC1G_00563
CC1G_00916
CC1G_00929
CC1G_01102
CC1G_01107
CC1G_01219
CC1G_01248
CC1G_01253
CC1G_01315
CC1G_01443
CC1G_01543
CC1G_01658
CC1G_01797
CC1G_02104
CC1G_02174
CC1G_02182
CC1G_02286
CC1G_02351
CC1G_02845
CC1G_02862
CC1G_03046
CC1G_03049
CC1G_03076
CC1G_03149
CC1G_03154
CC1G_03158
CC1G_03180
CC1G_03223
CC1G_03329
CC1G_03339
CC1G_03340
CC1G_03354
CC1G_03407
CC1G_03420
CC1G_03425
CC1G_03442
CC1G_03477
CC1G_03541
CC1G_03653
CC1G_03680
CC1G_03940
CC1G_04051
CC1G_04162
CC1G_04169
CC1G_04257
CC1G_04305
CC1G_04336
CC1G_04337
CC1G_04470
CC1G_04562
CC1G_04712
CC1G_04843
CC1G_04844
CC1G_04876
CC1G_04923
CC1G_04928
CC1G_04948
CC1G_04997
Protein function
Hypothetical protein
Chitin deacetylase
Endopeptidase
Disulphide isomerase
Hypothetical protein
Glycosyl hydrolase 53 domain-containing protein
Predicted protein
Hypothetical protein
Crystal protein
Exocellobiohydrolase
Hypothetical protein
Peptidase
Hypothetical protein
Endochitinase
Hypothetical protein
Aminopeptidase
Aryl-alcohol oxidase
Hypothetical protein
Peroxidase
Hydrophobin
Hydrophobin
Peptidase
Hypothetical protein
Sphingomyelin phosphodiesterase
Predicted protein
Predicted protein
Lysozyme
Lysozyme
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Peptidylprolyl isomerase B
Copper radical oxidase
Hypothetical protein
Hypothetical protein
DJ-1/PfpI family protein
Trehalase
Beta-hexosaminidase
Beta-hexosaminidase
Endonuclease/exonuclease/phosphatase
Hypothetical protein
Predicted protein
Aminopeptidase
Predicted protein
Laccase
Glycosyl hydrolase family 16
Hypothetical protein
Hypothetical protein
Beta-mannase
Hypothetical protein
Predicted protein
Predicted protein
Serine-type endopeptidase
Serine protease
Copper radical oxidase
Hypothetical protein
5'-nucleotidase
alpha-glucosidase
FAD/FMN-containing protein
leucine aminopeptidase 1
Aminopeptidase
Glucoamylase precursor
94
A1
2
0
6
0
4
1
7
A2
2
1
8
5
4
1
8
0
0
1
3
3
1
6
2
1
14
4
2
1
13
1
14
1
15
1
1
1
0
1
1
0
0
1
1
1
2
4
1
5
9
5
0
6
3
2
1
3
2
11
4
1
1
1
1
4
2
21
64
3
5
22
70
3
31
7
38
10
1
6
10
2
8
B1
B2
19
3
11
21
2
7
9
2
5
14
13
2
4
12
2
2
0
0
21
3
3
113
6
4
32
2
1
3
1
2
25
6
3
92
5
3
29
1
0
3
6
5
4
4
2
3
5
2
2
4
4
3
3
4
2
5
10
19
1
3
12
2
3
14
0
7
5
5
4
0
4
0
10
10
41
2
1
11
2
1
10
3
7
1
1
2
3
8
13
213
487
0
14
60
3
5
1
33
0
1
6
12
20
256
600
0
19
77
3
8
2
32
Chapter 4
CC1G_05039
CC1G_05043
CC1G_05139
CC1G_05192
CC1G_05246
CC1G_05285
CC1G_05355
CC1G_05418
CC1G_05573
CC1G_05600
CC1G_05607
CC1G_05612
CC1G_05638
CC1G_05778
CC1G_05798
CC1G_05860
CC1G_05883
CC1G_05917
CC1G_05923
CC1G_05954
CC1G_06012
CC1G_06019
CC1G_06068
CC1G_06086
CC1G_06262
CC1G_06324
CC1G_06563
CC1G_06564
CC1G_06692
CC1G_06696
CC1G_06703
CC1G_06773
CC1G_06848
CC1G_06885
CC1G_06891
CC1G_06959
CC1G_07128
CC1G_07167
CC1G_07201
CC1G_07205
CC1G_07631
CC1G_07990
CC1G_08049
CC1G_08051
CC1G_08056
CC1G_08067
CC1G_08068
CC1G_08239
CC1G_08260
CC1G_08277
CC1G_08310
CC1G_08358
CC1G_08391
CC1G_08427
CC1G_08561
CC1G_08598
CC1G_08717
CC1G_08822
CC1G_08921
CC1G_08950
CC1G_09057
CC1G_09088
CC1G_09149
CC1G_09154
CC1G_09155
CC1G_09180
CC1G_09189
CC1G_09291
CC1G_09292
CC1G_09340
CC1G_09406
CC1G_09442
CC1G_09595
CC1G_09604
CC1G_09659
CC1G_09727
Predicted protein
Predicted protein
Predicted protein
Fasciclin domain family
Hypothetical protein
Endochitinase
Hypothetical protein
Aryl-alcohol oxidase
Hypothetical protein
Predicted protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
ycaC
Hypothetical protein
Hypothetical protein
Aminopeptidase
Aminopeptidase
Aminopeptidase
Hypothetical protein
Predicted protein
Laccase 5
Alginate lyase
Predicted protein
Hypothetical protein
Endoglucanase
Exo-beta-1,3-glucanase
Exo-beta-1,3-glucanase
Predicted protein
Predicted protein
Predicted protein
Vacuole protein
Hypothetical protein
Predicted protein
Metalloprotease
Thaumatin-like protein
Predicted protein
Cu/Zn superoxide dismutase
Glucoamylase
Predicted protein
Predicted protein
Elastinolytic metalloproteinase
Predicted protein
Predicted protein
Secreted protein
Macrophage activating glycoprotein
Predicted protein
Predicted protein
Predicted protein
Cellobiohydrolase II-I
Predicted protein
Metalloprotease
Predicted protein
Hypothetical protein
Predicted protein
G-X-X-X-Q-X-W domain-containing protein
Carboxylesterase
Putative fungistatic metabolite
Hypothetical protein
Metalloprotease
Hypothetical protein
Mannoprotein
Hypothetical protein
Snodprot1
Snodprot1
Carboxypeptidase C
Hydrophobin-315
Hypothetical protein
Glucooligosaccharide oxidase
CEL4b mannanase
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
95
5
1
1
4
1
0
4
7
3
11
1
2
12
1
3
3
3
2
0
1
3
1
3
1
2
0
1
1
0
1
1
3
3
6
2
5
1
1
1
1
3
3
3
3
3
3
10
3
2
7
2
1
2
1
2
1
4
3
1
0
12
2
1
11
2
4
2
2
7
5
4
2
6
6
4
9
3
2
1
13
0
15
2
0
2
1
5
3
2
3
7
5
6
0
22
1
1
25
5
2
6
0
22
0
5
21
2
7
2
5
3
0
2
1
1
3
5
2
2
2
7
3
6
5
2
1
0
3
11
2
9
30
18
2
0
3
4
6
2
6
23
15
5
9
6
4
0
4
10
0
9
4
2
4
20
7
15
2
7
0
2
10
4
13
10
0
2
8
3
90
5
4
14
7
0
2
7
0
0
23
1
1
3
10
4
5
0
4
8
0
6
2
2
3
15
5
13
3
5
2
2
8
4
9
7
0
2
6
3
105
1
3
11
6
0
3
5
2
1
24
1
1
0
13
4
3
0
0
11
3
4
1
Chapter 4
CC1G_09770
CC1G_09868
CC1G_09883
CC1G_09921
CC1G_09938
CC1G_09992
CC1G_10038
CC1G_10047
CC1G_10245
CC1G_10446
CC1G_10592
CC1G_10603
CC1G_10606
CC1G_10796
CC1G_10812
CC1G_10860
CC1G_10969
CC1G_11193
CC1G_11195
CC1G_11246
CC1G_11283
CC1G_11355
CC1G_11511
CC1G_11527
CC1G_11695
CC1G_11771
CC1G_12036
CC1G_12142
CC1G_12227
CC1G_12269
CC1G_12415
CC1G_12425
CC1G_12510
CC1G_13219
CC1G_13656
CC1G_13671
CC1G_13682
CC1G_13742
CC1G_13813
CC1G_14064
CC1G_15015
CC1G_15645
CC1G_15739
Mala s 12 allergen
Lipase
Lipase
Class V chitinase ChiB1
Hypothetical protein
Predicted protein
B-(1-6) glucan synthase
WSC domain-containing protein
Hypothetical protein
Mala s 12 allergen
Serine protease
G-X-X-X-Q-X-W domain-containing protein
Hypothetical protein
Hypothetical protein
B2-aldehyde-forming enzyme
Predicted protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Ricin B lectin
Predicted protein
FAD binding domain-containing protein
Glucuronyl hydrolase
Hypothetical protein
Glucoamylase
Metalloprotease
Predicted protein
Predicted protein
1,3-beta-glucanosyltransferase
Hypothetical protein
Hypothetical protein
Hypothetical protein
Carotenoid ester lipase
Predicted protein
Riboflavin aldehyde-forming enzyme
Cellulose-binding beta-glucosidase
1,3-beta-glucanosyltransferase
Calnexin
Copsin
Hypothetical protein
Hypothetical protein
Predicted protein
Macrophage activating glycoprotein
96
1
2
4
1
3
3
4
6
10
2
1
2
15
6
4
2
1
1
0
1
7
6
9
9
6
12
19
22
3
9
2
2
8
2
1
0
12
2
4
1
1
11
3
4
3
0
3
2
3
3
5
3
1
3
1
2
3
5
4
5
12
0
2
1
1
3
60
39
16
11
6
12
4
6
16
0
1
1
1
5
31
34
24
7
6
8
0
2
5
1
0
6
21
52
6
49
8
6
22
5
3
0
2
4
2
1
3
15
46
5
48
11
5
17
0
3
3
51
0
3
16
6
0
37
3
57
0
3
18
7
0
43
13
3
10
11
5
8
Chapter 4
1
ATG AAG CCC ATT GCT CTC CTC TCC ACC ATC GTC TTC GCC TTG ACC GTC TCG GTC CAA GGC
M
K
P
I
A
L
L
S
T
I
V
F
A
L
T
V
S
V
Q
G
>---------------------------- Signal peptide ---------------------------------<
61
GCC ATC AAC GAT CCC TGC TCA GTC AAC GGC ACG CCC GGT ATT TGC ATC ACC ACG ACG GCT
A
I
N
D
P
C
S
V
N
G
T
P
G
I
C
I
T
T
T
A
>-----------------------------------------------------------------------------121
TGT GCC AAC GCT GGT GGC ACC AAC GCT GTC GGT TTC TGC CCC AAC GAT CCT GCC AAC GTC
C
A
N
A
G
G
T
N
A
V
G
F
C
P
N
D
P
A
N
V
------------------------------------------------------------------------------181
CGT TGC TGC ACC AAG AAG TGC AGT ACC AAC GGC ACC TGC CGT TTC ACC AAC ACC TGT TCT
R
C
C
T
K
K
C
S
T
N
G
T
C
R
F
T
N
T
C
S
------------------------------------------------------------------------------241
AGC GGC AAC GTC CTC GTT GGC CTT TGC CCC GGT CCC TCC AAC TTC AGG TGT TGC ATC CCC
S
G
N
V
L
V
G
L
C
P
G
P
S
N
F
R
C
C
I
P
------------------------------------------------------------------------------301
TCT AGC AGC TGC GCC TAC AGC CCG GTC AAT TCC CGC ACC GTC CAG GAA ATC AAG AAC TCC
S
S
S
C
A
Y
S
P
V
N
S
R
T
V
Q
E
I
K
N
S
------------------------------------------------------------------------------361
GAA GGA TTC GTC AGG TCC CCT GCA CCC GAT CCA ATC GGT CTC CCC ACG GTC GGA TAC GGC
E
G
F
V
R
S
P
A
P
D
P
I
G
L
P
T
V
G
Y
G
---------------------------- Mature lysozyme ---------------------------------421
CAC CTC TGC AAG AAT AAG GGA TGC AGC GAG GTC CCA TAC AGT TTC CCC CTG ACC GAA GCG
H
L
C
K
N
K
G
C
S
E
V
P
Y
S
F
P
L
T
E
A
------------------------------------------------------------------------------481
CAG GCC ACC TCT CTC CTC ATG ACC GAC TTG AAG ACC TTC CAG AAG TGC ATC TCC GAC CAA
Q
A
T
S
L
L
M
T
D
L
K
T
F
Q
K
C
I
S
D
Q
------------------------------------------------------------------------------541
ATC AAC GAT TCC ATC AGG CTG AAC GAG AAC CAG TAC GGT GCC TTG GTT TCG TGG GCC TTC
I
N
D
S
I
R
L
N
E
N
Q
Y
G
A
L
V
S
W
A
F
------------------------------------------------------------------------------601
AAC GTT GGC TGC GGT AAC ACC GCC TCT TCT GCC TTG ATC TCG CGA CTC AAC AAG GGA GAG
N
V
G
C
G
N
T
A
S
S
A
L
I
S
R
L
N
K
G
E
------------------------------------------------------------------------------661
AGC CCG AAC AAG GTT GCG GAG GAG GAG CTT CCT CGA TGG AAG TAC GCC GGT GGC CAG GTT
S
P
N
K
V
A
E
E
E
L
P
R
W
K
Y
A
G
G
Q
V
------------------------------------------------------------------------------721
CTG CCC GGC TTG GTT GCT CGT AGG AAC AGG GAG ATC GCA TTG TTC AAG ACT GCT TCG AGC
L
P
G
L
V
A
R
R
N
R
E
I
A
L
F
K
T
A
S
S
------------------------------------------------------------------------------781
GTC GTC GGG CAT CCT CCC AGG TGC TAG
V
V
G
H
P
P
R
C
---
------------------------------<
Fig. S1. Precursor protein CC1G_03049 (CC49). Prediction of the signal peptide was performed with
SignalP 4.0 33. The potential Kex cleavage site and N-glycan sites are indicated in bold.
97
Chapter 4
1
ATG AAG TTT ACC ACA TCC CTC TTC GCC ATC TTC TTG ACC CTC GGT GTC GCC CAA GCC GCC
M
K
F
T
T
S
L
F
A
I
F
L
T
L
G
V
A
Q
A
A
>------------------------------ Signal peptide ---------------------------< >-61
CTC AAC GGC CCC TGC AAC ATC CCC GGC GTC GGT CCT GGT ACC TGC CTC CAC ACC TCC ACC
L
N
G
P
C
N
I
P
G
V
G
P
G
T
C
L
H
T
S
T
------------------------------------------------------------------------------121
TGC GCC AAC GGC GGT GGC GGT TCT TTC TCT GGC TAC TGC CCG AAC GAC CCC GCA GAC GTC
C
A
N
G
G
G
G
S
F
S
G
Y
C
P
N
D
P
A
D
V
------------------------------- Mature peptide -------------------------------181
CGG TGC TGC TTC AAG CGC TGC CCC ACA TCT CTT GGC AGT GGC AGA TGC CGC CCT GTT GCC
R
C
C
F
K
R
C
P
T
S
L
G
S
G
R
C
R
P
V
A
------------------------------------------------------------------------------241
TCG TGC CCC AGC GGC AGA ACC CTC ACA GGA TAC TGC CCT GGA CCC GCC ACG GTT AGG TGC
S
C
P
S
G
R
T
L
T
G
Y
C
P
G
P
A
T
V
R
C
------------------------------------------------------------------------------301
TGC TTG CCA TCA TAA
C
L
P
S
---
--------------<
Fig. S2. Precursor protein CC1G_06692 (CC92). Prediction of the signal peptide was performed with
SignalP 4.0 33. The potential Kex2 cleavage site is indicated in bold.
98
Chapter 5
O-mannosylated cell wall proteins
of S. cerevisiae
Andreas Essiga, Paolo Nannib, Markus Aebia
a: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland
b: Functional Genomics Center Zurich, CH-8057 Zurich, Switzerland
Contributions
Extraction of yeast cell wall proteins
MS sample preparation
MS measurements Orbitrap Velos
MS data analysis
99
Chapter 5
Abstract
The cell wall of fungi is composed of a unique polysaccharide scaffold interconnected to
structural and enzymatic proteins. The majority of cell wall proteins (CWPs) are extensively
decorated with N- and O-glycans influencing their stability, function, and localization. Omannosylation is one type of glycan found on CWPs, an ubiquitously known protein modification
up to humans. In yeast, O-mannose (O-Man) glycans are generated in the endoplasmic
reticulum by the transfer of a single mannose to serine and threonine residues catalyzed by
protein O-mannosyltransferases (PMTs). However, little is known about which proteins are
targeted by PMTs and about the actual site localization of O-Man glycans. Here, we present a
novel workflow for the detection of O-mannosylations on covalently linked cell wall proteins of
Saccharomyces cerevisiae, which combines an α-mannosidase treatment of extracted CWPs
with a liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement. Based on
this strategy, we described a comprehensive yeast cell wall O-Man glycoproteome with 24
unique O-mannosylated CWPs identified. Analysis of the acquired spectra revealed an
extremely high complexity and heterogeneity of this type of O-glycosylation. Further, we could
successfully implement SILAC (stable isotope labeling by amino acids in cell culture) for relative
quantification of O-mannosylated peptides, providing new opportunities for studying Omannosylated proteins in yeast and other organisms.
100
Chapter 5
Introduction
The cell wall of S. cerevisiae is a dynamic and unique network of proteins and polysaccharides,
which represents 15-30% of the dry weight of vegetative cells 1. This extracellular matrix
possesses important biological functions, such as maintenance of the cell shape, interaction with
the environment, or acting as barrier and protective line for biological molecules and competing
microbes
. The major compounds of the cell wall are β-glucans, chitin, and glycoproteins.
2,3,4
β1,3- and β1,6-linked glucose (Glc) polymers are the two predominant types of glucan found in
the cell wall of S. cerevisiae with more than 50% of the total cell wall dry weight. β1,3-glucan
chains are highly branched and can adopt helix-like structures, which provide the cell wall with
elasticity. β1,6-glucan chains are shorter than the ones with a β1,3 linkage and have a more
amorphous structure. Both types of glucan are essential for a proper cell wall assembly, as they
are highly interconnected to other cell wall components like chitin and glycoproteins 3. Chitin is a
linear polymer of β1,4-linked N-acetylglucosamine (GlcNAc), which occurs as free chitin or linked
to β-glucan 5. These polymer chains of more than 100 residues are able to form microfibrils with
an enormous tensile strength that confer a high rigidity to the cell wall. Major deposits of chitin
are found at the bud neck and septum of dividing cells and in a lesser extent also in the lateral
wall near the plasma membrane 1,4. In total, ~180 proteins are covalently linked or associated to
the polysaccharide scaffold 4. They execute a diversity of biological functions, such as enzymatic
activities like cross-linking and remodeling of cell wall components, mediating adhesion,
regulating permeability, or transmitting signals from environmental stimuli 6. Many of the
covalently linked cell wall proteins (CWPs) are synthesized with a glycosylphosphatidylinositol
(GPI) anchor that localizes the protein to the outer leaflet of the plasma membrane, for example,
the Tip1, Gas, and Sed1-Spi1 protein families
7,8.
GPI-CWPs can be further processed and
released from the lipid carrier by a cleavage between the glucosamine (GlcN) and Man residue
of the GPI anchor. The reducing end of the GPI remnant is then linked to a glucose residue of a
non-reducing end of β1,6-glucan 9. PIR (proteins with internal repeats) proteins are a group of
covalently attached CWPs that are characterized by the repeating sequence unit
SQ[I/V][S/T/G]DGQ[I/V]Q[A][S/T/A] and can be released from the cell wall by a mild alkali
treatment 4. The connection of PIR proteins to other cell wall components is mediated most likely
via linkages between their amino acids or glycan structures and the glucose residues of β1,3glucan 10. Treatment of the cell wall with reducing agents releases PIR proteins, indicating that
they can also be linked through a disulfide bond to other CWPs 11.
Most of these cell wall associated proteins are decorated with N- and O-linked glycans after
passing the secretory pathway. N-linked glycosylation is an essential protein modification in
eukaryotes, which is involved in numerous biological processes, such as protein folding and
quality control, solubility and activity of a protein, or mediating interactions with other biological
101
Chapter 5
molecules 12,13,14. N-glycans are initiated in the endoplasmic reticulum (ER) through the action of
an oligosaccharyltransferase complex (OST), which transfers the core oligosaccharide
Glc3Man9GlcNAc2 from a dolichylpyrophosphate lipid carrier to an asparagine site chain of a
nascent polypeptide chain 15,16,17. After trimming to a Man8GlcNAc2 structure, the glycoprotein is
moved to the Golgi complex, where the N-glycan structures are further processed. In baker's
yeast, a frequent elaboration of the core N-glycan is the addition of a mannose-rich
oligosaccharide with up to 150-200 mannoses (Man) residues. The so-called mannan structures
include the addition of chains of α1,6-linked Man, extensively branched with α1,2-Man
terminating in an α1,3-Man or a Man-1-P (mannose phosphate)
. The negatively charged
3,4
phosphate groups are most likely involved in regulation of the cell wall permeability and in
retaining water molecules 2,4.
In comparison to N-glycans, O-mannosylations in S. cerevisiae are short with a chain length of
up to five mannoses. The chain is α1-linked to hydroxyl-groups of serine and threonine residues
18.
The first mannose is attached in the luminal part of the ER by protein O-mannosyltransferases
(PMTs), which use dolichol phosphate β-D-mannose as their mannosyl donor
19.
PMTs are
evolutionarily conserved ER transmembrane proteins that are divided in three subfamilies
referred to as PMT1, PMT2, and PMT4
20,21.
In S. cerevisiae, six PMTs have been identified
assigned to the three subfamilies, PMT1 (Pmt1, Pmt5), PMT2 (Pmt2, Pmt3), and PMT4 (Pmt4).
To achieve highest activity, Pmts of the subfamilies PMT1 and PMT2 form heterodimers,
whereas Pmt1-Pmt2 and Pmt5-Pmt3 complexes are most prevalent 22. Pmt4, the only member of
subfamily PMT4, enhances its activity by forming homodimers. It was shown that PMTs act on
specific sets of proteins: the Pmt4 dimer exclusively O-mannosylates membrane associated
proteins 23. Substrates of the Pmt1-Pmt2 complex are soluble or membrane associated proteins
and also misfolded proteins in the ER. Different studies demonstrated that O-mannosylations are
involved in the unfolded protein response (UPR) and the ER-associated protein degradation
(ERAD) pathway
24,25,26.
Further processing of O-mannosylations takes place in the Golgi
apparatus by different mannosyltransferases using GDP-Man as sugar donor. The second and
third mannose is added in α1,2-linkage by three KTR family members (Kre2, Ktr1, Ktr3)
27.
Members of the MNN1 family (Mnn1, Mnt2, Mnt3) catalyze the attachment of the two terminal
α1,3-linked mannoses. The linear mannose chain can additionally be modified by Man-1-P, the
same structural element identified on N-linked mannans 28. The analysis of viable pmt∆ mutants
showed that O-mannosylations affect the stability, localization, and functionality of CWPs and
consequently also the cell wall integrity 18,29. Furthermore, inhibition of PMTs by the rhodanine-3acetic acid derivative OGT2468 revealed an activation of the cell wall integrity (CWI) pathway,
which is involved in maintenance and repair of the cell wall 30. The biological significance of Omannosylations could also be shown in humans, where the structural composition and diversity
of glycans is much more complex than in yeast 31. One of the few well studied O-mannosylated
102
Chapter 5
proteins is α-dystroglycan, a component of the dystrophin-glycoprotein complex (DGC). Within
the DGC complex, α-dystroglycan interacts with components of the extracellular matrix,
mediated among other glycan types by O-mannosylations. A non-functional O-Man structure
leads to a detachment of the extracellular matrix with a strong impact in muscular tissue, where it
can cause severe forms of congenital muscular dystrophy 31,32.
Despite efforts done in the past, the localization and stoichiometry of O-Man glycans on CWPs is
largely unknown. Reasons for this lack of knowledge are technical challenges to identify Omannosylated peptides, such as the instability of O-glycans in standard collision induced
dissociation (CID) MS/MS and the fact that there is no consensus sequence known for O-Man
glycan sites as for N-glycans. In these studies, we present a unique workflow for the detection
and quantification of O-mannosylations on S. cerevisiae CWPs based on electron transfer
dissociation (ETD) MS/MS 33,34. ETD has been proven to be a powerful tool for the identification
of labile post-translation modifications (PTMs), as it preserves PTMs during the fragmentation
process 35. In combination with higher energy collisional dissociation (HCD) MS/MS, 24 unique
O-mannosylated CWPs were identified and quantified by the implementation of SILAC (stable
isotope labeling by amino acids in cell culture)
36,37.
The detected O-mannosylated peptides
exhibited a highly complex pattern of O-Man modifications giving new insights in the diverse and
dynamic nature of this O-glycosylation.
103
Chapter 5
Results
Identification of O-mannosylated cell wall proteins of yeast
Previous studies indicated that O-Man glycans are highly clustered in serine and threonine rich
regions and appear in various lengths with up to five mannose residues connected. This
heterogeneity and the lability of O-glycosidic bonds were taken into account, when we developed
a LC-MS/MS based workflow for the identification of O-Man glycans on covalently linked CWPs
of yeast. An analytical method that had previously been established in our lab for the analysis of
N-linked glycans on CWPs, was further developed and adapted for ETD measurements of Omannosylated peptides 34. In brief, yeast cells grown to the midlog phase were lyzed and the cell
wall material separated and enriched. To achieve an optimal coverage of the amino acid
sequences, the proteolytic cleavages were performed with three different proteases ideal for
ETD-MS/MS measurements, including the lysyl-endopeptidase (LysC), the peptidyl-Lys
metalloendopeptidase (LysN), and the endoproteinase AspN 38,39. To simplify the data analysis,
proteolytic peptides released from the cell wall were incubated with the α-mannosidase from
Jack bean to trim down O-Man glycans to a single α1-linked mannose
40.
For each digest, an
ETD-MS/MS measurement was performed and the acquired data analyzed with a hexose as
variable modification on serine and threonine residues. To verify the ETD output and to cover
more optimally doubly charged peptides, an HCD-MS/MS measurement was executed
additionally for the LysC and LysN digest. As we determined a low stability of O-Man glycans in
HCD fragmentations, the subsequent data analysis was performed under the assumption of a
neutral loss of a single hexose from serine and threonine residues.
The ETD and HCD measurements resulted in the identification of in total 24 unique Omannosylated CWPs, most of them yet not experimentally shown to be modified by O-Man
glycans (Supplementary table 1). The majority of the identified proteins are connected to the
cell wall scaffold via a GPI anchor or remnant (e.g. Aga1, Ecm33, Gas1, Sag1) or by an alkalisensitive linkage (ASL), including PIR proteins (e.g. Hsp150, Cis3). Uth1, a SUN protein family
member, was shown to be released of the cell wall after a dithiothreitol (DTT) treatment
41.
Ym122 is uncharacterized concerning the type of linkage to the cell wall and was so far not
described as cell wall protein.
Considering the peptide level, 107 O-mannosylated peptides were identified by ETD- and HCDMS/MS with an overlap of 23 peptides between the two fragmentation techniques
(Supplementary table 1). Even though ETD was applied, only for a few peptides it was possible
to unambiguously localize the modifications (Table 1). In addition to an insufficient peptide
fragment coverage, the major reason for this failure was that often several isobaric versions of
an O-mannosylated peptide existed with the modifications located at different positions. The
peptide AAAVSQIGDGQIQAT*T*K containing a PIR repeat is illustrated as an example, where
104
Chapter 5
two hexoses could be localized by ETD and the peptide sequence be verified by HCD with a
neutral loss of two hexoses (Figure 1). ETD was highly efficient in sequencing of peptides with
multiple mannoses, for example, the triply charged peptide DGQIQAT*T*KT*KAAAVS*QIG with
four single mannoses attached and a mass of 2535.22 Da (Figure 2A). Another peptide is
shown in figure 2B with the amino acid sequence DSIKKIT*G originating from the CWP Ecm33.
Table 1. Peptides with localized O-Man glycans. O-mannosylated peptides, where the mannoses could
be localized on specific serine and threonine residues based on ETD-MS/MS spectra. Modified sites are in
bold and underlined. Peptides, which could not be assigned to a specific PIR protein are labeled with PIR
proteins.
105
Chapter 5
Fig. 1. ETD- and HCD-MS/MS of an O-mannosylated peptide. Spectra of the peptide
AAAVSQIGDGQIQAT*T*K containing a PIR repeat acquired by ETD with the precursor ion [M + 3H]3+ at
661.7 m/z (calculated: 661.7 m/z) and by HCD with the precursor ion [M + 2H]2+ at 991.99 m/z (calculated:
991.99 m/z). Two O-Man glycans were localized on threonine residues by ETD. The HCD analysis was
performed with a neutral loss of hexoses (162.0528 Da). Due to low peak intensities, certain areas were
multiplied indicated above the ETD spectrum.
106
Chapter 5
Fig. 2. Localization of single hexoses on serine and threonine residues by ETD-MS/MS. (A)
Fragmentation of the O-mannosylated peptide DGQIQAT*T*KT*KAAAVS*QIG originating from Pir1 with
the precursor ion [M + 3H]3+ at 846.5 m/z (calculated: 846.1 m/z). (B) Spectrum of the singly Omannosylated peptide DSIKKIT*G originating from the protein Ecm33 with the precursor ion [M + 2H]2+ at
512.5 m/z (calculated: 512.3 m/z). (C) The O-mannosylated peptide GT*T*T*KET*GVT*T*K originating
from the protein Sed1 with the precursor ion [M + 3H]3+ at 733.1 m/z (calculated: 732.7 m/z).
107
Chapter 5
Next, we analyzed peptides with different numbers of mannoses attached. Three examples are
described in table 2 (TSTNATSSSCATPSLK of Cis3, GTTTKETGVTTK of Sed1, and
ASSTSTSASASSSIK of Ym122).
Table 2. Heterogeneity of O-mannosylated peptides. For each peptide, all possible numbers of O-Man
sites (# Man) are shown originating from ETD-MS/MS measurements. As an indicator for a correct
assignment of a peptide version, the retention time (RT) is displayed. The area under the curve (AUC) was
determined manually from an extracted ion chromatogram (XIC) of the corresponding m/z. The relative
quantity of a peptide was calculated by dividing the AUC of the corresponding m/z by the sum of AUCs of
all peptide versions. The most abundant one is indicated in bold. No MS1 means that there was no MS1
trace detectable. No MS2 means that there was no MS/MS performed for the corresponding m/z.
108
Chapter 5
For each peptide only a certain range of number of modifications was detectable at the MS1
level. By determining manually the relative abundance of the different versions, it was shown that
within these ranges there is a maximum abundance as for the Cis3 peptide
TSTNATSSSCATPSLK at two amino acids modified. For none of the peptides it was possible to
clearly localize the modifications with the exception of the Sed1 peptide GT*T*T*KET*GVT*T*K,
where all threonine residues were modified (Figure 2C).
Relative quantification of O-mannosylated peptides
The site localization experiments revealed a high complexity of O-mannosylated peptides as
illustrated in table 2. To address this diversity in more detail and to get an idea how reproducible
these findings are, a quantitative assay was needed. Since a SILAC-MS/MS approach was
already implemented previously in the CWP extraction workflow, we adapted this strategy for the
relative quantification of O-mannosylated peptides by ETD- and HCD-MS/MS 34. A S. cerevisiae
wild type strain was grown independently in heavy and light labeled minimal medium to an
optical density (OD600) of 1.0. The extraction was performed as described above and in the
methods with a LysC proteolytic digest, applying the HCD and ETD fragmentation techniques for
the subsequent MS measurements.
Through this method, 14 O-mannosylated CWPs were identified combining the ETD and HCD
measurements (Supplementary table 2). All of them were reproducibly described in the site
localization experiments of the LysC digest (Supplementary table 1). A relative quantification
was performed for 36 O-mannosylated peptide pairs including the different versions of number of
sites modified. The light to heavy ratios were in a range of 0.7 to 1.5 with an average ratio of 1.1
for the ETD-MS/MS output and an average ratio of 1.0 for the HCD-MS/MS acquired spectra. An
exception was Hpf1 where the peptide ASSAISTYSK showed a ratio of 2.0 for the ETD
measurement and a ratio of 2.1 for the HCD measurement. In more detail, the result is shown for
the three peptides ISPTSANTK of Gas1, SEAPESSVPVTESKGTTTK of Sed1, and
ASSTSTSASASSSIK of Ym122 (Table 3). For each peptide a range of number of sites modified
was detected with a maximum of abundance. The peptide ASSTSTSASASSSIK of Ym122, also
shown in table 2, displayed reproducibly a detectable MS1 trace from 0 to 5 mannoses attached
with a slightly shifted maximum of abundance of 3 to 4 serine and threonine residues modified.
With the results obtained, it was shown that SILAC in combination with ETD- and HCD-MS/MS is
a valuable tool for the quantification of O-mannosylated peptides and proteins.
109
Chapter 5
Table 3. Relative quantification of O-mannosylated peptides. Wild type yeast cells were grown in light
and heavy medium to an OD600 of 1.0, combined, and the CWPs proteolytically digested with LysC. All
identifications shown here were acquired by HCD-MS/MS. The area under the curve (AUC) was
determined manually from a XIC chromatogram of the corresponding m/z. Light (L) to heavy (H) ratios
were calculated manually from the extracted AUCs. No MS1 means that there was no MS1 trace
detectable. No MS2 means that there was no MS/MS performed for the corresponding m/z.
110
Chapter 5
Discussion
The critical impact of O-mannosylations on yeast cell viability and especially on the cell wall
stability is demonstrated by the fact that Pmt double mutants exhibit an osmolabile phenotype
and the Pmt triple mutants pmt2∆ pmt3∆ pmt4∆ and pmt1∆ pmt2∆ pmt4∆ are even completely
inviable 4,42. To study this type of O-glycosylation in a high throughput manner, we developed a
novel analytical workflow for the identification of O-mannosylated peptides, based on an αmannosidase treatment of extracted and proteolytically digested CWPs followed by a LC-MS/MS
measurement. Using ETD and HCD as fragmentation techniques, this strategy revealed 24 Omannosylated and covalently linked CWPs, with a high reproducibility at the protein level.
However, O-mannosylated peptides identified in different runs exhibited a rather low overlap,
which is most likely due to experimental limitations or differences in the expression profile of OMan glycans on individual CWPs.
As expected, HCD preferably identified doubly charged peptides (87%) and ETD had a slight
bias toward charge states of three and higher. ETD preserved mannoses on serine and
threonine residues during the fragmentation procedure with up to eight single mannoses
identified on a peptide. However, based on ETD spectra it was often not possible to
unambiguously localize the modifications, because of several isobaric versions of a modified
peptide fragmenting together in the linear ion trap. Even though technically very challenging,
strategies originally developed for isobaric phosphopeptides could be implemented to separately
quantify these O-Man peptides 43,44. The α1 linkage of O-mannosylations to serine and threonine
residues was not stable in HCD fragmentations. Therefore, we applied a neutral loss scan for the
detection of O-mannosylated peptides by HCD, which revealed a set of peptides with a high
overlap to the ETD output. Taken together, both fragmentation techniques are capable of
conclusively identify O-mannosylated peptides.
The heterogeneity of O-mannosylated peptides turned out to be extremely high, due to the
existents of peptides that were modified with different numbers of mannoses. The number of
sites modified was restricted to a specific range with a maximum of attached mannoses.
However, differences in the ionization efficiency of glycopeptides can have an impact on these
distributions and they are difficult to predict. Further validation of these results was performed by
implementing SILAC in the CWP extraction workflow in combination with ETD- and HCDMS/MS. A relative quantification was done with a wild type yeast strain, which exhibited
differences of close to zero in quantities of O-mannosylated peptides. These findings
demonstrated that yeast cells in their exponential growth phase express a very similar profile of
O-Man glycans on their covalently linked cell wall proteins. The possibility of using SILAC for the
quantification of O-mannosylated peptides is opening new opportunities for studying differences
in O-Man glycoprofiles in yeast and other organisms, where SILAC is applicable.
111
Chapter 5
O-Man glycans are predominantly localized in protein regions with a high content of serine and
threonine residues, a feature of O-mannosylations described in literature, which we could also
confirm in our studies
. A prominent class of identified CWPs were the PIR proteins with the
18
internal repeat SQ[I/V][S/T/G]DGQ[I/V]Q[A][S/T/A], such as Pir1, Hsp150, Pir3, and Cis3 45. PIR
proteins are thought to be involved in maintenance of the cell wall integrity and the regulation of
permeability 3. How PIR proteins are interconnected with the other cell wall components is not
fully understood. Besides the two possibilities mentioned in the introduction, O-mannosylations
could play an important role in linking PIR proteins and other CWPs to the cell wall scaffold. GAS
protein family members and especially Gas1 were another group of CWPs, which was found to
be extensively modified by O-mannosylations. Gas1 is a putative β1,3-glucanosyltransferase
involved in the formation of β1,3-glucan 46. The functionality of most other O-mannosylated GPI
proteins identified is not fully understood. They are often described as structurally relevant for the
cell wall, such as the proteins Ecm33, Tip1, Ccw12, and Sed1 4.
The complex pattern of O-Man glycans described in our studies is pointing on the biological
function of this protein modification. A dynamic localization and stoichiometry of Omannosylations can be correlated to selective regulation of cell wall permeability or to CWP
linkages within the polysaccharide scaffold. Our workflow developed for quantitative studies of
O-mannosylated proteins, provides a powerful tool to uncover more details about how O-Man
glycans are involved in the formation and maintenance of the fungal cell wall.
112
Chapter 5
Materials and Methods
Chemicals and Yeast strains
All experiments were performed with the S. cerevisiae strain SS328 (ade2-101; his3Δ200; lys2801; ura3-52). For SILAC experiments arg4 deficient cells were used (ade2-101; his3Δ200; lys2801; ura3-52; delta arg4 : NAT).
All chemicals, if not otherwise mentioned, were bought at the highest available purity from
Sigma-Aldrich, USA.
S. cerevisiae cell wall protein preparation for MS analysis
The protocol is adapted from Schulz and Aebi 2009 and optimized for ETD-MS/MS
measurements of O-mannosylated cell wall proteins 34.
Yeast cells were grown in YPD medium (1% (w/v) yeast extract (Oxoid AG, England), 2% (w/v)
peptone (Oxoid AG, England), 2% (w/v) glucose) at 30 °C to an optical density (OD600) of 1 to 1.2
and 50 OD equivalent of cells (~5 x 10 8 cells). Cells were then harvested by centrifugation at
2500g at 4 °C for 10 min and resuspended in extraction buffer (10mM TrisHCl pH 7.5, 1x
protease inhibitor cocktail (Roche Applied Science, Germany), 2 mM PMSF). After lysis of the
cells using glass beads (0.5 mm; BioSpec, USA) at 4 °C, covalently linked cell wall material was
pelleted by centrifugation at 16000g for 1 min and resuspended in denaturing buffer (50 mM
TrisHCl pH 7.5, 2 M thiourea, 7 M urea, 2% (w/v) SDS). Cell wall proteins were reduced by the
addition of DTT (Axon Lab, Switzerland) to 10 mM and an incubation at 30 °C for 2 h. After an
alkylation with 25 mM acrylamide at 30 °C for 1 h in the dark, the cell wall pellet was five times
washed with denaturing buffer and subsequently six times with 2% (w/v) SDS. The pellet was
then dissolved in 2% SDS and the N-glycans trimmed down to a single HexNAc by the endo-βN-acetylglucosaminidase H for 16 h according to the manufacturer's protocol (EndoH; New
England Biolabs, USA). After washing and dissolving the pellet in 50 mM Na4HCO3 (pH 8), the
cell wall proteins were digested and released with either AspN (2 µg/ml; Merck, Germany), LysC
(20 µg/ml; Wako, Japan), or LysN (6 µg/ml; U-Protein Express BV, Netherlands) at 37 °C for 16
h. The remaining cell wall material was separated from soluble peptides by centrifugation at
16000 x g for 1 min and the supernatant reduced to complete dryness in a centrifugal
evaporator. The peptides were dissolved in 100 mM sodium acetate buffer (pH 4.6) and treated
with α-mannosidase from Jack beans at 37 °C for 16 h. Afterwards, the peptide solution was
acidified with formic acid to pH 2-3 and desalted using C18 ZipTip pipette tips (Merck Millipore,
Germany).
For both fragmentation techniques, the sample preparation was performed according to the
described procedure. For the LysN digest, HCD-MS/MS was performed with the same extract as
113
Chapter 5
prepared for ETD. For the LysC digest, the ETD and HCD measurement was performed with two
independently prepared extracts. ETD was solely used for the extract digested with AspN.
S. cerevisiae cell wall protein preparation for SILAC analysis
The lysine/arginine auxotrophic yeast strain was grown at 30 °C in minimal medium (0.67% (w/v)
Yeast Nitrogen Base w/o amino acids (Becton Dickinson, USA), 2% (w/v) glucose)
supplemented either with 20 mg/l of the light version of L-lysine [12C6/14N2] and L-arginine [12C6]
or the heavy version of L-lysine [13C6/15N2] and L-arginine [13C6] (Cambridge Isotope
Laboratories, USA). The cells of the light and heavy culture were grown both to an OD600 of 1.0,
mixed 1:1 (v:v), and harvested by centrifugation at 2500 x g at 4 °C. Afterwards, the cell wall
proteins were prepared as described with a LysC proteolytic digest using the same extract for
the ETD and HCD measurement.
MS analysis
All MS analyses were performed on a hybrid Velos LTQ Orbitrap mass spectrometer (Thermo
Scientific, USA) equipped with an ETD unit and coupled to an Eksigent-nano-HPLC system
(Eksigent Technologies, USA). Separation of peptides was done on a self-made column (75 µm
x 80 mm) packed with C18 AQ 3 µm resin (Bischoff GmbH, Germany). Peptides were eluted with
a linear gradient from 2% to 31% acetonitrile (ACN) in 53 min at a flow rate of 250 nl/min.
For the ETD measurements, full MS data were measured in the Orbitrap unit in a mass range of
300 – 1700 m/z, with an automatic gain control (AGC) setting of 1 x 106, at a resolution of 60000
at 400 m/z. MS/MS spectra were acquired in the data dependent mode with up to 10 ETD
spectra recorded in the linear ion trap using the most intense ions. Supplemental activation
energy was activated and the AGC value was set at 5 x 104. Fluoranthene was used as anion
with an AGC value of 1 x 105 and a reaction time of 100 ms.
For HCD measurements, full MS sans were done with a resolution of 30000 at 400 m/z. A
maximum of 10 HCD MS/MS scans were acquired with stepped normalized collision energy
starting at 15% using three steps with a collision energy width of 15. Fragment ions were
detected in the Orbitrap at a resolution of 7500.
Data analysis
For all measurements, MS/MS spectra were searched against the S. cerevisiae protein database
including common contaminants (fgcz_4932) using the Mascot search algorithm v2.4 (Matrix
Science Inc., USA) with the following parameters: Propionamide (C) as fixed modification;
oxidation (M), hexose (S), hexose (T) as variable modifications; 5 ppm peptide tolerance and
0.8 Da fragment ion tolerance; LysC, LysN, or AspN set as proteases with a maximum of 3
missed cleavages. For HCD, a neutral loss of hexose (162.0528 Da) from serine and threonine
114
Chapter 5
residues was set as variable modification in addition to oxidation of methionine. The maximum
false discovery rate was set at 1% and peptides with an e-value of below 0.05 were rejected. All
acquired data were manually verified using the Mascot search output and the Xcalibur software
(Thermo Scientific, USA). Peptide abundances were determined manually from extracted ion
chromatograms (XIC) by calculating the area under the curve (AUC) using the Xcalibur software.
For SILAC experiments, the fold abundance ratio was determined by dividing the “light” by the
“heavy” AUC of a XIC of the corresponding m/z.
115
Chapter 5
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Supplementary Information
119
120
Ccw14
Crh1
O13547
P53301
LysN
LysC
AspN
LysC
LysN
LysC
AspN
Ccw12
Q12127
Protease
LysC
Protein
GPI-CWPs
P32323
Aga1
Accession
DSTTAASSTASCNPLKTTGCTP
DSTTAASSTASCNPLKTTGCTP
GDTTTYDRGEFHGVDTPTDK
TLASSSVTTSSSISSFEK
TLASSSVTTSSSISSFEK
TPATVSSTTRSTVAPTTQQSSVSSDSPVQDK
TPATVSSTTRSTVAPTTQQSSVSSDSPVQDK
TTGCTPDTALATSFSEDFSSSSK
TTGCTPDTALATSFSEDFSSSSK
TVASSSTSESIISSTK
TVASSSTSESIISSTK
TVASSSTSESIISSTK
KTTGCTPDTALATSFSEDFSSSS
APSSEESSSTYVSSSK
ASSSSASSSTKASSSSAAPSSSK
ASSSTKASSSSASSSTK
DHVCSETVSPALVSTATVTV
DHVCSETVSPALVSTATVTVD
DHVCSETVSPALVSTATVTVD
DHVCSETVSPALVSTATVTVD
NGTSTAAPVTSTEAPK
NGTSTAAPVTSTEAPK
NGTSTAAPVTSTEAPK
KNGTSTAAPVTSTEAP
KNGTSTAAPVTSTEAP
SWVSSMTTSDEDFNK
SWVSSMTTSDEDFNK
Sequence
1
2
1
1
3
1
8
1
2
1
2
3
1
1
1
2
1
1
2
3
1
2
3
2
3
1
2
# Man
1209.5325
860.7094
594.2631
990.9767
1153.0299
1104.8675
1112.4947
858.3735
912.3911
873.9273
954.9537
1035.98
858.3732
897.8911
737.0028
628.9516
1125.0447
788.7083
842.7255
896.7426
847.4109
928.4368
1009.4618
948.3952
1029.4223
Observed
(m/z)
2
3
4
2
2
3
4
3
3
2
2
2
3
2
3
3
2
3
3
3
2
2
2
2
2
z
29.51
15.6
24.41
45.26
28.43
30.17
20.55
45.62
52.31
54.85
40.15
25.93
47.18
45.65
72.65
19.71
21.22
29.44
22.27
18.88
48.45
20.65
17.22
70.35
40.67
Score
ETD
0.0015
0.045
0.0036
8.2E-05
0.0038
0.0058
0.026
2.7E-05
5.9E-06
8.5E-06
0.00025
0.0049
1.9E-05
2.7E-05
8.1E-08
0.014
0.018
0.0029
0.018
0.041
2.1E-05
0.0099
0.023
9.2E-08
8.6E-05
Expect
1287.0546
1287.0561
912.3906
928.4352
1009.4601
1029.4217
Observed
(m/z)
by an M next to the number of mannoses (# Man) attached to the peptide. The score and expectation value are from the Mascot output.
2
2
3
2
2
2
z
41.83
118.18
55.69
35.54
54.39
98.38
Score
HCD
0.000066
1.5E-12
2.7E-06
0.00043
4.4E-06
1.5E-10
Expect
wall scaffold. Peptides originating from a PIR repeat, which could not be assigned to a specific protein are indicated. An oxidation of methionine is indicated
Table S1. O-mannosylated peptides and proteins identified by ETD- and HCD-MS/MS. Proteins were categorized according to their linkage to the cell
Chapter 5
121
Gas3
Q03655
Egt2
P42835
Gas1
Ecm33
P38248
P22146
Cwp1
P28319
LysC
LysN
LysC
AspN
AspN
LysC
AspN
LysC
LysC
AspN
ANSLNELDVTATTVAK
DDFNNYSSEINKISPTSANTKSYSATTS
DDFNNYSSEINKISPTSANTKSYSATTS
DDFNNYSSEINKISPTSANTKSYSATTS
ISPTSANTK
ISPTSANTK
ISPTSANTK
SDCSFSGSATLQTATTQASCSSALK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
TLDDFNNYSSEINK
KISPTSANT
KISPTSANT
KSYSATTSDVACPATG
KSYSATTSDVACPATG
KYGLVSIDGNDVKTLDDFNNYSSEIN
DSAQYAEHTNLVAI
LTEATATDK
TSLSTEESVVAGYSTTVGAAQYAQHTSLVPVSTIK
TSLSTEESVVAGYSTTVGAAQYAQHTSLVPVSTIK
TSLSTEESVVAGYSTTVGAAQYAQHTSLVPVSTIK
TSLSTEESVVAGYSTTVDSAQYAEHTNLVAIDTLK
TSTFQK
DSIKKITG
LSSTSTESSK
DATGVAIRPTSKSGSVAA
DDATGVAIRPTSKSGSVAA
DGSFKEGSES
DGSSYIFSSK
EGSESDAATGFSIK
LGSGSGSFEATITDDGK
SSSGFYAIK
YAVVNEDGSFK
QSDDATGVAIRPTSK
1
1
2
3
1
2
3
3
1
2
1
1
2
1
2
1
1
1
1
2
3
5
1
1
2
1
1
1
1
1
1
1
1
1
3
3
3
2
2
2
3
2
2
2
2
2
3
911.4039
540.7737
621.8
896.3998
1028.1483
2
2
3
3
3
3
2
2
2
3
3
2
2
2
2
2
2
1068.8149
1122.8321
1176.8501
540.7734
621.8008
702.8272
1027.447
896.4007
847.3984
556.2721
1248.9586
1302.9778
1356.9939
1504.3605
437.2136
512.2811
675.8035
617.3211
655.6635
602.7458
626.7811
780.8491
902.4094
561.2714
695.8215
63.65
43.72
36.48
45.61
63.09
45.46
32.17
25.99
23.78
15.12
21.62
18.48
34.68
41.02
53.2
33.15
33.82
34.81
33.3
21.36
17.74
33.32
69.19
67.7
25.88
37.26
35.79
31.11
17.04
22.2
1.9E-06
4.2E-05
0.00042
3.7E-05
4.9E-07
0.00012
0.0027
0.011
0.008
0.049
0.0069
0.014
0.00036
0.00014
4.8E-06
0.0021
0.0047
0.0041
0.0035
0.0088
0.017
0.00049
1.7E-07
3.2E-07
0.0026
0.00024
0.00026
0.00085
0.02
0.006
2
2
2
2
2
2
2
2
3
896.3995
977.4267
911.4031
540.7734
621.7999
896.4001
977.424
1028.1483
904.9591
2
2
3
540.7744
621.8006
569.9514
109.63
114.38
107.44
103.64
50.08
46.86
60.17
54.48
63.6
54.82
40.95
51.02
2.1E-11
3.6E-12
1.8E-11
4.3E-11
0.000018
0.000027
1.1E-06
3.6E-06
1.5E-06
6.1E-06
0.00013
0.000017
Chapter 5
122
Pry3
Pst1
Sag1
Q12355
P20840
Hpf1
P47033
Q05164
LysC
LysC
LysC
AspN
LysN
LysC
LysN
NTGYFEHTALTTSSVGLNSFSETAVSSQGTK
TLLSTSFTPSVPTSNTYIK
KFTSGDIK
DPTDNSASPTDNAKHTSTYGSSSTGASL
DPTDNSASPTDNAKHTSTYGSSSTGASL
DPTDNSASPTDNAKHTSTYGSSSTGASL
STTINPAK
ASSAISTYSK
ATSLTTAISK
ATSLTTAISK
ATSLTTAISK
ATSLTTAISK
ETSETSETSAAPK
ETSETSETSAAPK
ETSETSETSAAPK
ETSETSETSAAPK
IITSQIPEATSTVTATSASPK
IITSQIPEATSTVTATSASPK
IITSQIPEATSTVTATSASPK
IITSQIPEATSTVTATSASPK
SYTTVTSEGSK
SYTTVTSEGSK
SYTTVTSEGSK
SYTTVTSEGSK
TVTSEAPK
TVTSEAPKETSETSETSAAPK
KASSAISTYS
KETSETSETSAAP
KSYTTVTSEGSKATSLTTAIS
ANSLNELDVTATTVAK
ANSLNELDVTATTVAK
KANSLNELDVTATTVA
4
1
1
4
5
6
1
1
1
2
3
4
1
2
4
5
1
2
3
5
1
2
3
4
1
5
1
3
6
2
3
3
1290.5813
1110.0689
529.2738
1139.8101
1193.8276
1247.8447
588.785
577.8111
658.8373
739.8645
820.8894
750.3342
831.3591
993.4118
1074.4386
1133.0877
809.7456
863.7631
971.7981
661.304
742.33
823.3564
904.3834
497.7518
987.7708
588.7851
912.3842
1035.8065
985.9845
1067.0118
3
2
2
3
3
3
2
2
2
2
2
2
2
2
2
2
3
3
3
2
2
2
2
2
3
2
2
3
2
2
36.74
40.9
23.95
13.48
16.62
20.41
25.24
27.56
21.45
15.23
17.69
34.07
24.58
16.46
18.38
28.19
23.18
21.21
36
49.27
22.93
17.68
13.21
24.35
16.73
37.26
14.2
25.79
26.38
33.31
0.00087
0.00026
0.0066
0.047
0.022
0.0091
0.0037
0.0022
0.012
0.048
0.028
0.00039
0.0035
0.023
0.015
0.0036
0.015
0.033
0.0013
1.2E-05
0.0051
0.017
0.048
0.0073
0.044
0.00023
0.038
0.0094
0.0051
0.00098
1110.067
497.2585
2
2
2
2
2
742.3294
823.3555
588.784
2
2
2
2
2
2
2
2
588.7851
577.8115
658.8369
739.8634
820.8898
985.9842
1067.0113
1067.0112
35.33
38.12
47.12
50.82
43.54
69.4
64.69
53.98
47.25
39.68
66.53
100.17
34.74
0.00079
0.00026
0.000023
8.3E-06
0.000044
1.3E-07
3.9E-07
5.8E-06
0.000028
0.00016
4.8E-07
2.2E-10
0.00077
Chapter 5
123
Tir1
Yl042
Yl194
Q07990
Q05777
Tip1
P27654
P10863
Sed1
Q01589
LysC
AspN
LysN
LysC
LysC
AspN
LysN
LysC
EAQESASTVVSTGK
EAQESASTVVSTGK
DLNTALGQKVQYTFL
ITSAAPSSTGAK
ITSAAPSSTGAK
MLTMVPWYSSRLEPALK
SSSAAPSSTEAK
TSAISQITDGQIQATK
KITSAAPSSTGA
DAYTTLFSEL
DTSAAETAELQAIIG
DVLSVYQQVMTYTD
AASSSEATSSAAPSSSAAPSSSAAPSSSAESSSK
AASSSEATSSAAPSSSAAPSSSAAPSSSAESSSK
LPWYTTRLSSEIAAALASVSPASSEAASSSEAASSSK
LPWYTTRLSSEIAAALASVSPASSEAASSSEAASSSK
GTTTKETGVTTK
GTTTKETGVTTK
GTTTKETGVTTK
GTTTKETGVTTK
GTTTKETGVTTK
SEAPESSVPVTESK
SEAPESSVPVTESK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTKETGVTTK
SEAPESSVPVTESKGTTTKETGVTTK
SEAPESSVPVTESKGTTTKETGVTTK
SEAPESSVPVTESKGTTTKETGVTTK
KSEAPESSVPVTES
TLLSTSFTPSVPTSNTYIK
QPSSPSSYTSSPLVSSLSVSK
1
2
1
1
2
1M
1
1
1
1
1
1
1
3
1
2
2
3
4
5
6
1
2
2
3
4
5
6
8
1
2
1
778.3697
859.3971
936.9822
626.8174
707.8436
734.0385
642.7932
912.464
626.8166
661.3059
826.399
912.4163
1050.4615
1158.4963
1278.2898
999.4833
516.5888
570.6069
624.6242
678.6417
732.6589
804.8766
885.9037
753.692
807.7089
825.6383
866.1515
906.6654
987.6903
804.8769
1191.0949
2
2
2
2
2
3
2
2
2
2
2
2
3
3
3
4
3
3
3
3
3
2
2
3
3
4
4
4
4
2
2
29.39
17.87
37.46
28.54
44.65
29
16.52
28.69
42.51
40.83
28.33
31.91
39.29
25.57
50.97
64.87
42.11
21.97
53.18
37.28
27.86
30.51
14.26
43.45
52.44
45.94
29.74
28.93
24.12
33.99
19.32
0.0021
0.03
0.00018
0.0015
5.5E-05
0.005
0.022
0.0037
8.4E-05
8.3E-05
0.0023
0.00077
0.00019
0.0028
9.8E-05
4.3E-06
0.00012
0.01
7.9E-06
0.00034
0.0028
0.00089
0.037
0.00018
2.4E-05
0.00013
0.0048
0.0053
0.0066
0.0004
0.042
1278.2896
1332.3069
804.8773
804.8768
885.9033
1191.0942
1144.0619
3
3
2
2
2
2
2
77.77
86.6
49.96
73.26
54.86
54.23
87.28
1.7E-07
2.4E-08
0.00001
4.7E-08
3.3E-06
0.000012
5.8E-09
Chapter 5
LysC
AspN
P32478
Hsp150
AspN
PIR proteins
(ASL-CWPs)
Q03178
Pir1
DGQIQATTKTTSAKTTAAAVSQIS
DGQIQATTKTTSAKTTAAAVSQIS
DGQIQATTTTLAPKSTAAAVSQIG
DGQIQATTTTLAPKSTAAAVSQIG
DGQVQAATTTASVSTKSTAAAVSQIG
DGQVQAATTTASVSTKSTAAAVSQIG
DGQVQATTKTTAAAVSQIG
DGQVQATTKTTAAAVSQIG
DGQVQATTKTTAAAVSQIG
DGQVQATTKTTQAASQVS
DGQVQATTKTTQAASQVS
DGQVQATTKTTQAASQVS
DGQVQATTKTTQAASQVS
DGQVQATTTTLAPKSTAAAVSQIG
DGQVQATTTTLAPKSTAAAVSQIG
DGQVQATTTTLAPKSTAAAVSQIG
STAAAVSQIGDGQIQATTK
STAAAVSQIGDGQIQATTK
STAAAVSQIGDGQIQATTK
STAAAVSQIGDGQVQATTK
STAAAVSQIGDGQVQATTK
STAAAVSQIGDGQVQATTK
STAAAVSQIGDGQVQATTK
STAAAVSQIGDGQVQATTTTLAPK
STAAAVSQIGDGQVQATTTTLAPK
STAAAVSQIGDGQVQATTTTLAPK
STAAAVSQIGDGQVQATTTTLAPK
TSGTLEMNLK
TTAAAVSQIGDGQIQATTKTTSAK
TTAAAVSQIGDGQIQATTKTTSAK
TTAAAVSQIGDGQVQATTK
TTAAAVSQIGDGQVQATTK
DGQIQATTKTKAAAVSQIG
DGQIQATTKTKAAAVSQIG
DGQIQATTKTKAAAVSQIG
DGQIQATTKTKAAAVSQIG
5
6
2
3
2
3
2
3
4
1
2
3
4
1
3
4
1
3
4
1
2
3
4
1
3
4
5
1M
4
5
1
2
1
2
3
4
1063.8376
1117.8549
885.4459
939.4636
925.4494
979.468
724.3574
778.374
832.3911
991.9783
1073.0048
769.69
823.7077
826.7556
934.7915
988.8091
1005.0033
1167.0579
832.3919
997.9968
719.6865
773.7025
827.7195
826.7555
934.7897
988.8086
1042.8258
636.3047
999.8157
1053.8333
1005.0042
1086.0314
684.0261
738.0438
792.0615
846.0793
3
3
3
3
3
3
3
3
3
2
2
3
3
3
3
3
2
2
3
2
3
3
3
3
3
3
3
2
3
3
2
2
3
3
3
3
26.29
24.24
64.09
29.29
24.11
33.26
80.74
57.47
41.62
30.75
21.24
19.73
25.53
43.84
34.6
24.4
42.35
26.72
21.63
22.36
43.48
62.14
54.25
42.93
24.79
21.4
31.81
31.52
22.34
29.79
24.47
37.19
68.86
78.81
47.46
18.72
0.012
0.019
9E-07
0.0031
0.0093
0.0014
4E-08
1.1E-05
0.00044
0.0022
0.02
0.029
0.0069
7.4E-05
0.00073
0.0085
0.00015
0.0076
0.024
0.022
0.00016
1.8E-06
1.1E-05
0.00017
0.015
0.035
0.0029
0.0007
0.031
0.0054
0.0091
0.00066
2E-07
2.6E-08
4.4E-05
0.034
Chapter 5
124
Cis3
P47001
Not assigned PIR
sequences
Pir3
Q03180
125
LysC
AspN
AspN
LysC
AspN
LysC
LysN
DGQIQATTKTTAAAVSQIG
DGQIQATTKTTAAAVSQIG
DGQIQATTKTTSAKTTAAAVSQIG
DGQIQATTKTTSAKTTAAAVSQIG
DGQIQATTKTTSAKTTAAAVSQIG
DGQIQATTKTTSAKTTAAAVSQIG
DGKGRIGSIVANRQFQF
DGQIQATTKTTAAAVSQIG
DGQIQATTKTTAAAVSQIG
DGQIQATTKTTAAAVSQIG
AAAVSQIGDGQIQATTK
AAAVSQIGDGQIQATTKTTSAK
AAAVSQIGDGQIQATTKTTSAK
DAKGRIGSIVANRQFQF
ISSSASKTSTNATSSSCATPSLK
ISSSASKTSTNATSSSCATPSLK
ISSSASKTSTNATSSSCATPSLK
ISSSASKTSTNATSSSCATPSLK
ISSSASKTSTNATSSSCATPSLK
NSGTLELTLK
TSTNATSSSCATPSLK
TSTNATSSSCATPSLK
TSTNATSSSCATPSLK
TSTNATSSSCATPSLK
DGQVQATAEVK
AAASQITDGQIQASKTTSGASQVSDGQVQATAEVK
TTAAAVSQIGDGQVQATTK
TTAAAVSQISDGQIQATTTTLAPK
TTAAAVSQISDGQIQATTTTLAPK
KSTAAAVSQIGDGQVQATTTTLAP
KSTAAAVSQIGDGQVQATTTTLAP
KTSGTLEMNL
2
3
4
5
6
7
1
1
2
4
2
4
5
1
1
2
3
4
5
1
1
2
3
4
1
3
3
5
6
3
6
1
729.0282
783.0458
999.8157
1053.8339
1107.8516
1161.8677
685.6916
1012.012
729.0282
837.0635
661.6637
932.4511
986.4687
690.3625
817.0563
871.0743
925.0918
979.1102
1033.1272
619.3296
894.911
975.9376
1056.964
1137.9908
654.3193
1307.2903
778.3724
1062.1732
1116.1888
934.7907
1096.8432
628.3078
3
3
3
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
3
3
3
3
3
3
2
90.28
51.69
18.25
21.79
19.49
21.29
25.89
50.37
90.28
38.52
70.34
27.57
28.79
14.64
50.72
28.45
32.88
29.16
20.55
19.25
60.33
31.49
23.9
24.41
14.34
44.9
47.05
38.88
27.25
33.97
23.92
46.22
1.6E-09
1.7E-05
0.047
0.024
0.044
0.033
0.003
1.5E-05
1.6E-09
0.00047
3.6E-07
0.007
0.0055
0.034
3.5E-05
0.0066
0.0026
0.0053
0.029
0.019
1.5E-06
0.0012
0.0043
0.0036
0.037
0.00017
6.8E-05
0.0008
0.012
0.0019
0.016
0.00005
991.9904
979.1095
628.3077
2
3
2
59.53
64.96
44.79
0.000004
1.2E-06
0.00007
Chapter 5
LysC
Q3E842
Ym122
AspN
Not assigned
P36135
Uth1
LysN
ASSTSTSASASSSIK
DGAVVIPAATTATSAAA
AAASQITDGQIQASK
STAAAVSQITDGQVQAAK
STAAAVSQITDGQVQAAK
STAAAVSQITDGQVQAAK
KSTAAAVSQITDGQVQAA
KSTAAAVSQITDGQVQAA
1
2
1
1
2
3
1
2
767.3595
905.9436
825.9122
954.481
1035.5064
1116.5331
2
2
2
2
2
2
115.38
50.22
50.07
54.26
37.21
31.48
4.2E-12
1.5E-05
1.6E-05
1.1E-05
0.00074
0.0026
767.359
954.4803
1035.505
2
2
2
80.85
101.22
99.72
1.2E-08
2.5E-10
4E-10
Chapter 5
126
127
Crh1
P53301
Gas1
Cis3
P47001
P22146
Ccw14
O13547
Egt2
Aga1
P32323
P42835
Protein
Acc.
ISPTSANTK
ISPTSANTK
ISPTSANTK
ISPTSANTK
ISPTSANTK
ISPTSANTK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
SYSATTSDVACPATGK
TLDDFNNYSSEINK
LTEATATDK
LTEATATDK
LTEATATDK
LTEATATDK
TTGCTPDTALATSFSEDFSSSSK
TTGCTPDTALATSFSEDFSSSSK
TTGCTPDTALATSFSEDFSSSSK
TTGCTPDTALATSFSEDFSSSSK
ISSSASKTSTNATSSSCATPSLK
ISSSASKTSTNATSSSCATPSLK
APSSEESSSTYVSSSK
APSSEESSSTYVSSSK
SWVSSMTTSDEDFNK
SWVSSMTTSDEDFNK
Sequence
1
1
2
2
3
3
1
1
2
2
3
3
1
1
1
2
2
1
1
2
2
4
4
1
1
1
1
# Man
2
2
2
2
2
2
2
2
2
702.827
706.835
896.4004
900.4071
977.4256
981.4329
911.4036
2
2
3
3
3
3
3
3
2
2
2
2
z
540.7746
544.7822
637.2988
641.3052
858.3748
861.0431
912.3906
915.0616
979.1105
984.4546
897.8909
901.8982
948.3945
952.4017
m/z
3.7E-05
0.01
No MS2
1.2E-07
0.00097
0.0042
0.00043
0.0023
No MS2
No MS2
0.031
No MS2
3.2E-05
2.2E-05
8.7E-07
0.0073
No MS2
1.6E-05
4.6E-05
42.39
20.96
20.97
32.75
32.75
31.27
31.27
23.16
23.17
20.69
20.69
52.02
52.02
51.59
51.59
29.61
29.53
27.21
27.21
ETD
RT
Expect
[min]
2.2E-06
46.04
0.00026
46.04
1E+08
1E+07
1E+07
3E+07
2E+07
2E+07
2E+07
7E+07
7E+07
8E+06
8E+06
5E+07
5E+07
2E+07
2E+07
5E+06
6E+06
1E+07
1E+07
1E+07
1E+07
AUC
1.1
1.1
1.1
1.1
1.0
1.1
1.0
0.8
1.1
0.9
Ratio
L/H
540.7743
544.7812
621.8009
625.8077
702.8266
706.8337
896.3999
900.4067
977.4266
981.4335
1058.4523
1062.4595
911.4016
556.2713
560.2785
637.2986
641.3047
1287.056
1291.0623
912.3924
915.062
979.11
984.4546
897.8909
901.8977
948.3967
952.4008
m/z
extracted AUCs. For each peptide, the light and heavy version is shown with the corresponding retention time (RT).
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
2
2
2
2
z
4E-06
8.8E-07
0.00002
2.9E-05
3.7E-05
0.00006
1.7E-10
2.1E-11
8.4E-08
2.2E-11
1.9E-09
2.8E-07
2.8E-09
1.8E-05
4.6E-05
0.00016
0.00009
1.3E-08
5.1E-07
No MS2
0.016
3.7E-07
No MS2
4.7E-12
1.3E-15
22.49
22.49
23.09
23.09
21.06
21.06
32.46
32.46
31.89
31.89
30.46
30.46
42.08
23.54
23.54
21.06
21.06
51.82
51.82
51.36
51.46
29.19
29.19
26.83
26.83
HCD
RT
Expect
[min]
No MS2
45.81
0.024
45.81
7E+07
7E+07
1E+08
1E+08
1E+07
1E+07
3E+07
3E+07
1E+07
1E+07
2E+07
2E+07
1E+08
5E+06
4E+06
7E+06
6E+06
2E+07
2E+07
1E+07
1E+07
7E+06
1E+07
2E+07
2E+07
1E+07
1E+07
AUC
1.1
1.3
1.0
1.0
1.1
1.1
1.2
1.2
0.9
1.0
0.7
1.1
1.0
Ratio
L/H
curve (AUC) was determined manually from a XIC chromatogram of the corresponding m/z. Light (L) to heavy (H) ratios were calculated manually from the
Table S2. O-mannosylated peptides quantified by SILAC ETD- and HCD-MS/MS. The peptides were produced by a LysC digest. The area under the
Chapter 5
Hpf1
Hsp150
Q05164
P32478
128
Q01589
Sed1
PIR sequences
Gas3
Q03655
5
5
6
6
1
1
3
3
4
4
2
2
5
5
6
6
4
4
2
2
AAAVSQIGDGQIQATTKTTSAK
AAAVSQIGDGQIQATTKTTSAK
AAAVSQIGDGQIQATTK
AAAVSQIGDGQIQATTK
GTTTKETGVTTK
GTTTKETGVTTK
GTTTKETGVTTK
GTTTKETGVTTK
SEAPESSVPVTESK
SEAPESSVPVTESK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESK
SEAPESSVPVTESK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
SEAPESSVPVTESKGTTTK
4
4
1
1
2
2
4
4
2
2
3
3
1
STAAAVSQIGDGQVQATTTTLAPK
STAAAVSQIGDGQVQATTTTLAPK
ASSAISTYSK
ASSAISTYSK
ATSLTTAISK
ATSLTTAISK
SYTTVTSEGSK
SYTTVTSEGSK
ANSLNELDVTATTVAK
ANSLNELDVTATTVAK
ANSLNELDVTATTVAK
ANSLNELDVTATTVAK
TLDDFNNYSSEINK
2
2
3
3
2
2
3
3
861.7262
867.0703
885.9041
889.912
915.7435
921.088
3
3
2
2
3
3
2
2
2
2
2
2
2
2
2
804.8785
808.8838
932.4502
937.7954
991.9907
995.9976
988.8087
991.4799
588.7847
592.7927
658.8364
662.8452
904.3823
908.3908
985.9844
989.9914
915.4102
0.0025
No MS2
0.037
No MS2
0.041
No MS2
No MS2
8.4E-06
0.0084
No MS2
0.0072
0.00077
0.033
0.0085
0.0024
No MS2
0.018
No MS2
0.037
No MS2
1.1E-05
1.7E-05
1.7E-05
28.87
28.88
30.35
30.35
28.39
28.47
31.1
31.1
33.47
33.47
35.43
35.51
38.57
38.57
26.63
26.63
31.19
31.27
24
24.09
41.81
41.89
42.39
1E+07
2E+07
1E+08
1E+08
1E+07
1E+07
1E+08
9E+07
6E+06
8E+06
2E+07
2E+07
2E+07
2E+07
6E+06
3E+06
2E+07
1E+07
3E+06
2E+06
2E+07
2E+07
1E+08
1.0
1.1
1.0
1.2
0.9
0.8
0.9
1.5
1.5
2.0
1.0
1.1
678.6416
683.9854
732.6603
738.0025
804.8767
808.8837
807.7086
813.0529
861.7263
867.069
885.9036
889.9105
915.7428
921.0871
969.7611
975.1057
932.4506
937.7953
991.9924
995.9978
588.7852
592.7927
658.8365
662.8437
985.9845
989.9938
1067.0111
1071.0183
915.4084
3
3
3
3
2
2
3
3
3
3
2
2
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
0.00048
No MS2
No MS2
0.0048
7.8E-08
1.3E-07
0.0027
No MS2
0.0023
0.1
4E-07
4.2E-07
0.0075
0.0092
0.027
No MS2
0.0011
No MS2
No MS2
0.00039
0.025
No MS2
0.00044
0.0013
8.4E-06
No MS2
5.6E-06
0.00002
3E-09
20.57
20.57
20.57
20.57
30.87
30.87
29.19
29.19
28.52
28.52
30.05
30.05
28.09
28.09
27.31
27.31
33.28
33.28
35.17
35.17
26.38
26.38
30.96
30.96
41.5
41.5
40.39
40.39
42.08
4E+06
3E+06
1E+07
1E+07
1E+08
1E+08
9E+06
9E+06
2E+07
2E+07
2E+08
2E+08
2E+07
2E+07
1E+07
8E+06
8E+06
8E+06
2E+07
2E+07
9E+06
5E+06
3E+07
2E+07
2E+07
2E+07
4E+07
3E+07
1E+08
1.2
1.1
1.1
1.0
1.0
1.1
1.0
1.2
1.0
1.0
1.4
2.1
1.1
1.1
1.1
Chapter 5
Tip1
Tir1
Ym122
P27654
P10863
Q3E842
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ASSTSTSASASSSIK
ITSAAPSSTGAK
ITSAAPSSTGAK
ITSAAPSSTGAK
ITSAAPSSTGAK
ITSAAPSSTGAK
ITSAAPSSTGAK
MLTMVPWYSSRLEPALK
MLTMVPWYSSRLEPALK
TSAISQITDGQIQATK
TSAISQITDGQIQATK
LPWYTTRLSSEIAAALASVSPASSE
AASSSEAASSSK
LPWYTTRLSSEIAAALASVSPASSE
AASSSEAASSSK
LPWYTTRLSSEIAAALASVSPASSE
AASSSEAASSSK
LPWYTTRLSSEIAAALASVSPASSE
AASSSEAASSSK
1
1
2
2
3
3
4
4
5
5
767.3604
771.3665
848.3851
852.3927
929.4121
933.4201
2
2
2
2
2
2
2
2
4
993.4905
997.4973
1002.9931
2
4
2
2
2
2
999.4829
2
3
3
707.8432
711.851
788.8693
792.8774
1282.9677
1
1
1
2
2
3
3
1
1
2
2
1278.2889
1
No MS2
2.2E-05
0.00015
2.1E-06
0.0031
0.0077
0.00073
0.0012
0.00094
No MS2
0.00039
No MS2
No MS2
1.7E-06
0.00036
2.6E-08
24.35
24.26
23.75
23.84
23.21
23.22
40.99
40.99
23.58
23.25
20.96
20.97
63.66
63.66
67.47
67.54
6E+06
5E+06
9E+06
7E+06
1E+07
1E+07
8E+06
8E+06
2E+07
2E+07
2E+06
2E+06
8E+07
1E+08
6E+06
6E+06
129
1.2
1.2
1.1
0.9
1.1
0.9
1.2
1.0
767.3595
771.3677
848.3869
852.3928
929.4123
933.419
1010.4383
1014.4448
1091.4646
1095.4733
728.7074
733.3861
993.4922
997.4976
626.8162
630.8235
707.8428
711.8497
1336.9881
1332.3079
1282.969
1278.2901
2
2
2
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
3
3
3
3
7.5E-08
No MS2
3E-08
1.1E-10
3.2E-09
3.5E-09
2.5E-07
1.9E-09
1.7E-07
No MS2
0.0068
No MS2
No MS2
2.3E-05
1.2E-06
2.2E-08
1.9E-06
4.4E-07
No MS2
0.00046
2.4E-05
6.6E-05
24.02
24.02
23.54
23.54
22.02
22.02
21.38
21.38
21.06
21.06
56.48
56.48
40.7
40.7
23.7
23.7
23.25
23.25
63.57
63.57
67.38
67.38
6E+06
6E+06
7E+06
6E+06
8E+06
7E+06
8E+06
8E+06
3E+06
3E+06
3E+07
4E+07
7E+06
9E+06
1E+07
2E+07
2E+07
2E+07
8E+07
8E+07
7E+06
8E+06
1.1
1.1
1.1
1.2
1.1
0.7
0.8
0.9
0.7
1.0
1.1
Chapter 5
130
Chapter 6
Discussion and
future perspectives
131
Chapter 6
The aim of this PhD thesis was to characterize defense strategies of fungi against other
microbes, in particular bacteria, at the peptide and protein level. We focused on cell wall and
secreted antibacterial proteins.
O-mannosylated cell wall proteins of S. cerevisiae
Besides a number of other biological functions, the cell wall of fungi acts as a protective line for
competing microbes. O-mannose (O-Man) glycans are one type of glycosylation found on cell
wall proteins (CWPs) crucial for its integrity. We identified 24 unique O-mannosylated CWPs,
combining a protein specific cell wall extraction workflow with electron transfer dissociation
(ETD) and higher-energy collisional dissociation (HCD) mass spectrometry (MS).
The analysis revealed an enormous heterogeneity of this type of glycosylation, which was so far
not shown for another protein modification. An in-depth description of the complex pattern and
stoichiometry of O-mannosylations is not possible with the currently available technologies.
When restricted to a certain level of complexity, a relative quantitative analysis by implementing
SILAC (stable isotope labeling by amino acids in cell culture) is feasible, as we demonstrated in
our studies. However, different steps of the cell wall extraction workflow should be optimized
further to achieve a higher reproducibility. For example, a pre-treatment of the cell wall with
hydrolytic enzymes (glucanases, chitinases) could release CWPs and preserves O-Man glycans.
ETD has still a very low efficiency in generation of fragment ions in contrast to HCD. Therefore, if
the localization of the mannoses is not of interest, an HCD based approach combined with a
neutral loss scan is as efficient as an ETD fragmentation.
The SILAC-MS workflow is a valuable tool for an analysis of, for example, pmt (protein
mannosyltransferase) mutant strains. A relative quantification of specific O-Man sites can
answer questions about the functionality and regulation of this modification upon intra- and extracellular stimuli. One of the most interesting open questions is the interaction of Omannosylations and other glycan modifications. Recently, it could be shown that the mucin
domain of α-dystroglycan is heavily occupied by O-GalNAc and O-Man moieties, both attached
to serine and threonine residues 1. ETD and HCD measurements revealed that specific sites
serve as acceptors for both types of O-glycoconjugates. These findings indicated a certain
competition in site occupancy of O-GalNAc and O-Man glycans in higher organisms. An
interaction between N-glycans and O-mannosylations was previously found on the yeast cell wall
protein 5 (Ccw5/Cis3) 2. In this case, Pmt4 transfers a mannose to a particular threonine side
chain, which prevents the attachment of the N-glycan in a NAT motif. In our studies, we could
confirm that this threonine position and adjacent regions are heavily modified by O-Man glycans.
On Ccw12, we identified O-mannosylations in close proximity to an N-glycan motif (N81), which
was shown to be modified by an N-glycan structure 3. In a targeted MS approach, it would be
132
Chapter 6
feasible to combine the analysis of N-GlcNAc and O-Man residues, as we cleave the N-glycans
in our workflow to a single GlcNAc moiety.
Bacterial-fungal interactions
An appropriate model system is crucial for studying the interactions of bacteria and fungi. We
tested several substrates, such as glass wool, sand, or vermiculite, to grow C. cinerea on and to
extract enough material for the analysis of secreted proteins. Finally, we selected inert
borosilicate glass beads to cultivate the fungus on a solid surface and to extract medium
repetitively. This setup allowed for a co-cultivation with bacteria that were added to the medium
underneath the fungus. We found a strong interaction between C. cinerea and different bacteria,
such as B. subtilis and P. aeruginosa. The bacterial species tested are not necessarily relevant
for the fungus on herbivorous dung. Nevertheless, they represent model systems to study
bacterial-fungal interactions at the molecular level. It would be interesting to extract the
secretome of C. cinerea grown on a substrate resembling better is natural environment. Its
physiology and certainly also its protein expression profile vary strongly when grown on horse
dung instead of artificial medium. For example, the dung could be placed on top of a glass
beads layer, separated by a membrane selectively permeable for low molecular mass
substances. The medium that connects the beads layer and the dung would allow for an easy
extraction of secreted molecules underneath the dung.
Antimicrobial peptides and proteins
The analysis of the secretome and a newly developed purification workflow revealed a set of
antibacterial peptides and proteins secreted by C. cinerea. Two AMPs (CC92 and copsin) and
the lysozyme CC49 were expressed in Pichia pastoris and the antibacterial activity was shown in
a standard disk diffusion assay on B. subtilis. For each of these three proteins, several
homologous proteins were identified in the genome of C. cinerea and one of them, CC82, was
heterologously expressed in P. pastoris.
Discovery strategies for new lead molecules are of great importance and are a key step in
development of medical drugs. Nowadays, high-throughput genome mining is one of the most
prominent ways for the identification of genes or gene clusters that code directly for an antibiotic
or for enzymes catalyzing the synthesis of a secondary metabolite. Our strategy was a
combination of a ‘classical approach’ of fractionation with a quantitative mass spectrometry
measurement, which led to the identification of copsin. It would be certainly possible to perform a
genome wide search for specific cysteine patterns to identify these AMPs. However, a
fractionation and identification at the protein level allowed us to select for criteria such as
temperature stability, solubility, protease sensitivity, and a maximum of activity in vitro.
133
Chapter 6
The wide range of antimicrobial proteins encoded by C. cinerea is not surprising. As fungi lack
an adaptive immune system, they depend strongly on a diversified and highly effective innate
immune system. Especially as a late fruiter on herbivorous dung, C. cinerea interacts and
competes with a heterogeneous group of fungi and bacteria for nutrients and space. C. cinerea
and most other fungi are referred to as saprotrophs. Per definition, saprophytic nutrition is a
process of extracellular digestion and uptake of dead organic matter. This lifestyle requires the
secretion of numerous digestive enzymes, such as proteases, lipidases, and glycosidases.
Indeed, the analysis of the C. cinerea secretome revealed a diversity of these hydrolytic
enzymes. For copsin and very likely also for CC82 and the lysozyme CC49, we found a lytic
effect on bacteria. In the context of its saprophytic lifestyle, an induced lysis of bacterial cells
could provide the fungus with essential metabolites from bacteria. To study the potentially
synergistic effects of digestive enzymes, secondary metabolites, and AMPs, an appropriate
model system is needed that allows for a quantitative measurement of secreted substances. The
beads system together with a quantitative MS-SILAC approach can be a powerful tool to perform
such an analysis. Furthermore, genetic approaches for targeted mutations of genes are
necessary to study the impact of relevant proteins and AMPs on this defense network. The effect
of changed expression levels of AMPs were studied for species of the cnidarian Hydra. It was
shown that AMPs are involved in selection and regulation of a suitable bacterial community 4.
Time and space are two parameters that were not addressed, so far. From an ecological and
energetic point of view, in makes little sense that the fungus secretes all of these antibacterial
substances constitutively over its entire mycelium. It can be expected that particular bacteria and
other microbes trigger the secretion of AMPs and enzymes locally. One example is the bubble
protein, a defensin from the ascomycete Penicillium brevicompactum 5. It is specifically secreted
into exudate bubbles on the fungal surface, where it reaches high local concentrations and acts
antifungal. Even though technically very challenging, microscopy is one strategy to monitor the
secretion of antibacterial proteins in space and time. It was possible to attach a polyhistidine-tag
at the C-terminal end of copsin, which did not dramatically affect the antibacterial activity.
Whether eventually also a fluorescent tag can be added has to be tested for copsin and the
other proteins identified.
A feature common to most defensins is the high density of charged amino acids. The
amphipathic structure of many AMPs is often related to cell membrane perturbation
mechanisms. Even though fungal defensins characterized contain a high number of charged
side chains, it could not be shown that they are essential for antibacterial action by membrane
pore formation. Lipid II was determined as the molecular target of copsin located at the outer
surface of the bacterial membrane. The third position in the pentapeptide side chain of lipid I was
shown to be crucial for binding, independently of whether it is a lysine, DAP (diaminopimelic
acid), or elongated by a pentaglycine interpeptide bridge. According to the acquired data, we
134
Chapter 6
assume that the pyrophosphate moiety and the lipid tail are also involved in binding. As for
plectasin, NMR based binding studies of lipid II and copsin could provide more insights in the
binding pattern. Mechanistically, copsin can block further steps in peptidoglycan synthesis, such
as transglycosylation and transpeptidation and most likely interferes with the membrane integrity.
Furthermore, an accumulation of a polypeptide, such as copsin or plectasin, close to the
bacterial membrane could also induce a fatal stress response, as it was demonstrated for
mammalian peptidoglycan recognition proteins (PGRPs) 6. PGRPs bind to peptidoglycan and
can activate the CssR-CssS two component system in Gram positive bacteria, which triggers
removal of misfolded or aggregated proteins at the extracellular side of the cytoplasmic
membrane. A constitutive induction of this system that also exists in Gram negative species
induces a membrane depolarization and production of toxic radicals, consequently leading to cell
death.
In contrast to target structures, often little is known about the path of reaching these molecules.
The outer membrane of Gram negative bacteria or teichoic acid in the cell wall of Gram positive
species are known to be involved in attracting or repelling AMPs. A more extensive description
of such off-target interactions would be important to explain the often very distinct antibacterial
profiles of defensins, also found for copsin.
Heterologous expression of AMPs and pharmaceutical applications
A profound understanding of the antibacterial mechanism is important for further optimization of
the activity of an AMP. However, other topics dominate in literature of antibiotic drug
development, such as a low solubility, toxicity, or a short half-life intravenously. The biggest issue
in the field of antibiotics is the low profit made on the market. Nowadays, an intravenous
treatment with a commonly applied daily dose of 2 g of vancomycin costs in the range of CHF 90
(http://kompendium.ch). The generally high amounts of antibiotics needed for systemic
treatments require an optimized and high yield manufacturing procedure. This task should be
addressed as early as possible in research and development procedures, to reduce the overall
cost of a project. In a first attempt, copsin was produced in the Pichia pastoris system. A small
scale bioreactor expression yielded 20 mg/l purified and active peptide, a rather low value in
comparison to other fungal proteins expressed in Pichia. Further optimization of the expression
system is necessary and can be achieved at different steps. A multicopy strain optimized for
secreted proteins would be desirable, but is often laborious to construct. In bioreactor cultivation,
different parameters can be adjusted, such as pH, feeding rate, or time of cultivation. For
example, a reduction of temperature and aeration showed unexpectedly a strong increase in
yield for different proteins. Unfortunately, we found hyperglycosylation of copsin, a problem of the
Pichia system that was already discussed for the lysozyme CC49. In one stock, we identified 1015% of copsin modified by O-hexoses mainly at the C-terminal end. Such glycan structures can
135
Chapter 6
lead to a strong antigenic reaction when introduced intravenously and are commonly rapidly
cleared from the blood stream in the liver 7. As we identified these, most likely, mannose
structures only in one sample, it is possible that the hyperglycosylation depends on the culturing
conditions. More straight forward improvements could be achieved in downstream processing.
We estimated that up to 90% of the active peptide is lost during purification. One of the biggest
obstacles is an efficient separation of the massive amount of cells from culture supernatant and
peptide, which was done by a centrifugation step. A cross flow filtration system is an option for a
rapid separation of cells. An integrated downstream workflow could include a sedimentation step
in the bioreactor, followed by cross flow separation of remaining cells and a direct capturing and
purification on a cation exchange column. A longer sedimentation time is acceptable, due to the
high stability of copsin and would allow for an additional wash of the cells. However, other
expression hosts should be tested, such as Aspergillus oryzae, which was successfully used for
the heterologous production of plectasin. Further possibilities are mammalian systems, in
particular, immortalized Chinese Hamster Ovary (CHO) cells. CHO cells are well established for
the expression of protein therapeutics with final product titers of 1-5 g/l, routinely obtained in
fedbatch cultures 8. A critical factor in these higher organisms is the correct processing of the
pro-peptide, since Kex protease recognition sites can vary strongly. All expression systems have
in common that many parameters have to be determined in a matter of trial and error, which is
often very time consuming.
Today, antibiotics in clinical pipeline originate predominantly from secondary metabolites and
synthetic molecules 9. Based on the available knowledge and experience, it is not easy for AMPs
to compete with these conventional antibiotics. Nevertheless, AMPs could provide a new
generation of antibacterial drugs with additional effects on the immune system.
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Chapter 6
References
1.
Vester-Christensen, M. B. et al. Mining the O-mannose glycoproteome reveals cadherins as
major O-mannosylated glycoproteins. Proc. Natl. Acad. Sci. U. S. A. 1–6 (2013).
doi:10.1073/pnas.1313446110
2.
Ecker, M. et al. O-mannosylation precedes and potentially controls the N-glycosylation of a
yeast cell wall glycoprotein. EMBO Rep. 4, 628–32 (2003).
3.
Ragni, E., Sipiczki, M. & Strahl, S. Characterization of Ccw12p , a major key player in cell wall
stability of Saccharomyces cerevisiae. Yeast 24, 309–319 (2007).
4.
Franzenburg, S. et al. Distinct antimicrobial peptide expression determines host speciesspecific bacterial associations. Proc. Natl. Acad. Sci. U. S. A. 110, E3730–8 (2013).
5.
Seibold, M., Wolschann, P., Bodevin, S. & Olsen, O. Properties of the bubble protein, a
defensin and an abundant component of a fungal exudate. Peptides 32, 1989–95 (2011).
6.
Kashyap, D. R. et al. Peptidoglycan recognition proteins kill bacteria by activating proteinsensing two-component systems. Nat. Med. 17, 676–83 (2011).
7.
Cregg, J. M., Cereghino, J. L., Shi, J. & Higgins, D. R. Recombinant Protein Expression in
Pichia pastoris. Mol. Biotechnol. 16, 23–25 (2000).
8.
Jayapal, K. P., Wlaschin, K. F. & Hu, W.-S. Recombinant Protein Therapeutics from CHO
Cells — 20 Years and Counting. Chem. Eng. Prog. 103, 40–47 (2007).
9.
Butler, M. S., Blaskovich, M. A. & Cooper, M. A. Antibiotics in the clinical pipeline in 2013. J.
Antibiot. (Tokyo). 66, 571–91 (2013).
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Curriculum vitae
Andreas Essig
Address: Ausserdorfstrasse 147, 5276 Mettauertal
Nationality: Swiss
Date of birth: 10.06.1982
Education
12/2009 - present
PhD Thesis, Group Markus Aebi
Institute of Microbiology, ETH Zürich
2007 - 2009
Master of Science in Biology, Major in Systems Biology
ETH Zürich
2004 - 2007
Bachelor of Science in Biology, Major in Chemistry
ETH Zürich
2002 - 2004
Matura, Major in Natural Sciences and Mathematics
AKAD College Zürich
1998 - 2002
Graduate Electronics Engineer
ABB Switzerland
Work Experience
08/2002–10/2003
ABB Switzerland
Electronics engineer, Research and Development Communication
Products
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