Biochem. J. (2012) 445, 205–212 (Printed in Great Britain)
205
doi:10.1042/BJ20112102
The saposin-like protein SPP-12 is an antimicrobial polypeptide in the
pharyngeal neurons of Caenorhabditis elegans and participates in defence
against a natural bacterial pathogen
Aylin HOECKENDORF, Mareike STANISAK and Matthias LEIPPE1
Zoological Institute, Zoophysiology, University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany
Caenopores are antimicrobial and pore-forming polypeptides in
Caenorhabditis elegans belonging to the saposin-like protein
superfamily and are considered important elements of the
nematode’s intestinal immune system. In the present study, we
demonstrate that, unlike the other members of the multifarious
gene family (spps) coding for caenopores, spp-12 is expressed
exclusively in two pharyngeal neurons. Recombinantly expressed
SPP-12 binds to phospholipid membranes and forms pores in a
pH-dependent manner characteristic of caenopores. Moreover,
SPP-12 kills viable Gram-positive bacteria, yeast cells and
amoebae by permeabilizing their membranes, suggesting a
wide-target cell spectrum. A spp-12 knockout mutant is more
susceptible to pathogenic Bacillus thuringiensis than wild-type
worms and is tolerant to non-pathogenic bacteria. By contrast,
SPP-1, a caenopore, whose gene is expressed only in the
intestine and reported to be regulated by the same pathway
as spp-12, is apparently non-protective against pathogenic
B. thuringiensis, although it also does display antimicrobial
activity. The transcription of spp-1 is down-regulated in wildtype worms in the presence of pathogenic B. thuringiensis and
a spp-1 knockout mutant is hyposusceptible to this bacterium.
This implies that SPP-12, but not SPP-1, contributes to resistance
against B. thuringiensis, a natural pathogen of the nematode.
INTRODUCTION
involving daf-2 (insulin-like receptor) and daf-16 (Forkhead box
protein O) that interfere with longevity, stress response and
innate immunity; and (ii) gene silencing of either or both in
a daf-2-mutant background reduced the lifespan of the worms
on Escherichia coli [22]. During the present study it became
evident that, whereas spp-1 is expressed in the intestinal tract,
spp-12 is localized exclusively to two pharyngeal neurons. This
allows the comparison of similar polypeptides with extremely
distinct tissue locations. Using recombinantly expressed proteins,
we characterized the activity of SPP-12 (8.6 kDa) and SPP1 (9.9 kDa) towards various targets in parallel. Moreover, we
analysed the effect of spp-12 and spp-1 knockouts on the survival
of C. elegans when exposed to potentially harmful bacteria using
the well-established comparative system of challenging worms
with pathogenic or non-pathogenic strains of the Gram-positive
soil bacterium Bacillus thuringiensis, which probably co-exists
with C. elegans in nature [2,23–25].
Invertebrate model organisms such as Drosophila and
Caenorhabditis elegans have become increasingly attractive to
analyse innate immune mechanisms and as surrogate hosts for
pathogens [1–5]. In its natural environment, rotting fruits and
fresh compost heaps [6], C. elegans is exposed to diverse bacteria,
fungi and amoebae, which serve as a food source, but may
also represent potential pathogens [7–9]. To defend itself against
microbes the nematode produces a set of antimicrobial peptides
and proteins (for recent reviews see [10–12]). The caenopore
family constitutes a large proportion of the worm’s arsenal of
antimicrobial effector proteins. The 28 coding genes (spps) in C.
elegans are either constitutively expressed or specifically induced
and potentially give rise to 33 caenopores [13]. Structurally,
the caenopores belong to the SAPLIP (saposin-like protein)
superfamily as exemplified by the structure of caenopore-5 (SPP5) [14]. SAPLIPs with antimicrobial activity can be found in
phylogenetically diverse organisms ranging from amoebae to
mammals [15–17] and are characterized by a compact and
stable three-dimensional structure consisting of five α-helices
connected by a conserved array of disulphide bonds [18–20]. The
antimicrobial activity of caenopores has been reported for only
a few members, i.e. SPP-1 [21] and SPP-5 [13]. Moreover, the
mode of action of SPP-5 was demonstrated to be pore formation
in bacterial cytoplasmic membranes [13].
Among the plethora of potential SAPLIPs of C. elegans, in the
present study we have chosen spp-12 and spp-1 for a more detailed
functional study, as it has been reported that: (i) expression of
both of these genes is regulated via the insulin signalling pathway
Key words: Bacillus thuringiensis, caenopore, host–microbe
interactions, innate immunity, invertebrate, pore-forming protein.
EXPERIMENTAL
Strains and culturing
The C. elegans strains used in the present study were
obtained from the Caenorhabditis Genetics Center (University
of Minnesota, Minneapolis, MN, U.S.A.; N2, spp-1 mutant) or
from the National Bioresource Project for the nematode (Tokyo’s
Women’s School of Medicine, Tokyo, Japan; spp-12 mutant).
Each mutant strain was backcrossed four times against the
wild-type N2: Spp-1(ok2703) with a 1000-bp deletion starting
in the promoter region 700 bp upstream and ends in the last
Abbreviations used: AL, asolectin; CL, cardiolipin; daf-2, insulin-like receptor ; GFP, green fluorescent protein; MBC, minimal bactericidal concentration;
MIC, minimal inhibitory concentration; NGM, nematode growth medium; NLP, neuropeptide-like protein; NSM/L, neurosecretory motorneuron; PC, L-αphosphatidylcholine; PE, L-α-phosphatidylethanolamine; PG, L-α-phosphatidylglycerol; PS, L-α-phosphatidylserine; qRT-PCR, quantitative real time
transcription PCR; SAPLIP, saposin-like protein; TFA, trifluoroacetic acid; TOF, time-of-flight.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
206
A. Hoeckendorf, M. Stanisak and M. Leippe
exon 300 bp downstream; spp-12(tm2963) with a 300-bp deletion
and a 20-bp insertion, only 43 bp of the coding sequence is
left; and the spp-12(tm2963);spp-1(tm2703) double mutant was
generated in the present study by crossing the single mutants.
C. elegans was grown on NGM (nematode growth medium)
plates seeded with E. coli OP50 cells according to standard
procedures [26]. The assays with B. thuringiensis spores were
performed on PFM (peptone-free medium) plates instead of
NGM. We used the pathogenic B. thuringiensis strain NRRL
B-18247 with nematicidal activity provided by the Agricultural
Research Patent Culture Collection (United States Department
of Agriculture, Peoria, IL, U.S.A.) and the non-pathogenic
B. thuringiensis strain DSM-350 obtained from the German
Collection of Micro-organisms and Cell Cultures (Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany). Bacillus megaterium (A.T.C.C. strain
14581), B. thuringiensis B-18247 and E. coli OP50 used for
antimicrobial activity assays were grown in LB (Luria–Bertani)
medium [27]. Dictyostelium discoideum Ax3-ORF cells were
cultured axenically in Ax medium [28], Saccharomyces cerevisiae
S150 in YPD medium [29], and human Jurkat T-cells in RPMI
1640 medium [30].
Tissue localization
The promoter and part of the spp-12 gene region including the
intron (encoding amino acid residues 1–97 out of 106) were
fused to gfp (green fluorescent protein) using fusion PCR [31]
and injected accompanied by the rol-6 (cuticle collagen rol-6)
plasmid pRF-4 (100 ng/μl DNA in total) into the gonad of young
adult hermaphrodites. Animals carrying extrachromosomal arrays
were mounted on to glass slides with 0.1 % NaN3 in M9 medium,
and immediately used for imaging. Images were acquired using
an AXIO Imager Z1 microscope with a AxioCam HRm camera
and Axiovision 4.7 software (Carl Zeiss).
Pharyngeal pumping, brood size, egg laying and population growth
At 1, 6 and 26 h after moving the worms to the assay plates, five
out of ten worms were randomly selected and their pharyngeal
pumping rate was measured. Two independent experiments were
performed at 20 ◦ C and the pumping rate was determined in triplicate for 20 s. The brood size of each of the five individual worms
was counted. Additionally, the eggs laid by the five worms in a
24 h period were counted for at least five replicates. Brood-size
and egg-laying experiments were performed at 15 ◦ C, 20 ◦ C and
25 ◦ C. For the population-growth assay, five worms were placed
on 120-mm-diameter NGM plates seeded with E. coli OP50 cells
and allowed to found a population at 20 ◦ C. After five days
all of the animals were counted. Two independent experiments
were performed in triplicate. Statistical analysis was performed
using the Mann–Whitney test, followed by Bonferroni correction
for the pharyngeal pumping and population growth assays.
Recombinant expression
Parts of the spp-12 and spp-1 sequences were ligated into the
pIVEX2.4a vector (Roche) using the KspI and PstI restriction
sites. This resulted in plasmids which encode fusion proteins
with the sequences shown below (underlined residues mark the
resulting amino acid sequence of the synthesized protein after
cleavage including a glycine residue preceding the N-terminal
residue of the SPP protein): SPP-12, MSGSHHHHHHSSGIEGRGSHGAFCHLCEDLIKDGKEAGDVALDVWLDEEIGSRCKDFGVLASECFKELKVAEHDIWEAIDQEIPEDKTCKEA
c The Authors Journal compilation c 2012 Biochemical Society
KLC; and SPP-1, MSGSHHHHHHSSGIEGRGNPANPLNLKKHHGVFCDVCKALVEGGEKVGDDDLDAWLDVNIGTLCWTMLLPLHHECEEELKKVKKELKKDIENKDSPDKACKDVDLC. These comprise an N-terminal His6 tag, a Factor Xa
cleavage site and the putative mature caenopore. Recombinant
expression was performed at 37 ◦ C overnight in E. coli BL21
(DE3) trxB (thioredoxin reductase) cells for SPP-12 and in E.
coli BL21 (DE3) pAPlacIQ cells for SPP-1 after induction with
1 mM IPTG (isopropyl β-D-thiogalactopyranoside). Bacteria
were resuspended in TBS (Tris-buffered saline; 50 mM Tris
and 150 mM NaCl, pH 7.2) and lysed by sonication at 60 %
duty cycle, for 1 min at 40 % power on ice (Sonoplus HD 2200
sonicator, MS-73 titanium microtip, Bandelin Electronic). After
centrifugation (40 000 g for 60 min at 4 ◦ C), the fusion protein
in the supernatant (soluble fraction) was purified by IMAC
[immobilized metal-affinity chromatography; Ni2 + -NTA (Ni2 + nitrilotriacetate) agarose; Qiagen] and subsequent reversed-phase
chromatography (500 mg; C18 SepPak, Waters). Purity was
checked by tricine SDS/PAGE (13 % gels) [32] and the protein
concentration was determined by recording the absorption at
280 nm in an UV spectrophotometer and use of a sequencespecific molar absorption coefficient. For the functional assays
determining liposome interaction and antibacterial activity the Nterminal His tag was removed by cleavage with Factor Xa (Roche).
The molecular masses of proteins before and after removal of His
tags were determined initially by MS in linear mode using a 4700
Proteomic Analyzer MALDI (matrix-assisted laser-desorption
ionization)–TOF (time-of-flight)-MS on a TOF/TOF mass
spectrometer (Applied Biosystems). MS analysis of the recombinant proteins was performed in detail to determine the
oxidative state of the cysteine residues by using an LTQ-Orbitrap
Velos mass spectrometer (Thermo Scientific). Calculation of
the theoretical molecular masses and isotopic distributions were
performed using the Isotope Distribution Calculator (http://
proteome.gs.washington.edu/software/IDCalc/). The average
masses of the cleaved recombinant products according to their
primary structures were calculated to be 8681.7673 Da and
9954.43 Da for SPP-12 and SPP-1 respectively.
Activity assays
Functional assays were performed with proteins dissolved
in 0.01 % TFA (trifluoroacetic acid) and this solvent was
monitored in all of the assays in parallel. Antimicrobial
activity was determined using a microsusceptibility assay
[27]. Permeabilization of the membranes of viable bacteria,
Dictyostelium amoebae and S. cerevisiae yeast cells using the
fluorescent dye SYTOX Green, cytotoxicity towards Jurkat
T-cells, and pore-forming activity using the liposome
depolarization assay was measured as described previously
[15,30,33]. The synthetic peptides alamethicin, melittin and
cecropin P1 served as the controls (all HPLC grade; Sigma–
Aldrich). Membrane association was shown by incubating
0.5 μg of proteins and 4 μg of liposomes of crude soya bean
phospholipids [AL (asolectin), Sigma–Aldrich] at 4 ◦ C for 1 h
in 20 μl of a solution of 50 mM Tris-maleate, 50 mM sodium
sulfate, 0.5 mM EDTA and 0.02 % NaN3 , pH 5.2, 7.4, or 9.0 [15].
After ultracentrifugation (100 000 rev./min for 30 min at 4 ◦ C;
A-100/18 rotor), the supernatants were collected, and the pellets
were washed with and finally resuspended in the aforementioned
buffers. Supernatant and pellet fractions were analysed by
tricine-SDS/PAGE. Permeabilization of liposomes with a defined
phospholipid composition was tested by the release of trapped
calcein from the lipid vesicles as described previously [30]. For the
preparation of the liposomes, pure PC (L-α-phosphatidylcholine)
Antimicrobial saposin-like protein in the pharyngeal neurons of C. elegans
Figure 1
207
Localization of spp-12 to the pharyngeal neurons
Animals that express spp-12p::spp-12(1-97)::gfp reveal GFP fluorescence exclusively in two pharyngeal neurons: the pharyngeal NSM/L and pharyngeal interneuron I6. (A) GFP fluorescence.
(B) DIC (differential interference contrast) microscopy of the pharyngeal metacorpus, isthmus and terminal bulb merged with the GFP fluorescence. Merged pictures of larger proportions of a worm
(C) and of an entire worm (D) demonstrate that spp-12 is exclusively expressed in the two pharyngeal nerves.
or 3:1 (w/w) PC/PE (L-α-phosphatidylethanolamine), PC/PG (Lα-phosphatidylglycerol), PS (L-α-phosphatidylserine), or PC/CL
(cardiolipin; diphosphatidylglycerol) were used (all of the lipids
were obtained from Avanti Polar Lipids, except CL which was
from Sigma–Aldrich). Liposomes made from AL were used as a
control. Untrapped calcein was removed by gel filtration using
a NAP-5 column (GE Healthcare Lifesciences). The increase
in fluorescence intensity due to liposome permeabilization after
the addition of polypeptides (250 nM) at pH 5.2 was monitored
fluorimetrically for 5 min. Fluorescence intensities corresponding
to 100 % of lysis were acquired from calcein leakage induced by
the addition of Triton X-100 to a final concentration of 0.1 %.
Survival and lifespan assays
Survival assays were conducted at 20 ◦ C with worms exposed to
the spores of pathogenic or non-pathogenic B. thuringiensis mixed
with E. coli OP50 cells (2×108 spores and 6×109 E. coli OP50
cells per ml) as an independent food source [34]. Spores were
prepared as described in [24]. Lifespan assays were performed
at 20 ◦ C and at 25 ◦ C on proliferative or non-proliferative (heatkilled) E. coli OP50 cells. For the heat killing of bacteria, fresh
overnight cultures were concentrated 2-fold and incubated at
37 ◦ C for 2 h with 50 μg/ml gentamycine followed by incubation
at 80 ◦ C for 3 h. After heat treatment, no viable cells remained.
Ten L4-staged hermaphrodites were transferred to each of the
five replicate plates per strain. Every other day the animals were
scored as alive or dead by gentle prodding with a hair. Worms
that died as a consequence of a bursting vulva or that crawled
off the agar were censored. The experiments were performed at
least three times and were statistically analysed using the software
GraphPad Prism. Survival data were compared using the log-rank
test. A P value of <0.05 was considered significantly different
from wild-type. Data are presented as means +
− S.E.M.
qRT-PCR (quantitative real-time PCR) analysis
Synchronized L4-staged N2 worms were placed on assay plates
for 24 h at 20 ◦ C or at 25 ◦ C. The plates were prepared as for
survival assays. Total RNA was extracted using RNAmagic,
and reverse transcribed using SuperScript III (Invitrogen). The
resulting cDNA was subjected to qRT-PCR analysis using
SYBR Green detection (SYBR Premix Ex Taq, TaKaRa) on
LightCycler (Roche Diagnostics). Oligonucleotides for qRT-PCR
were designed using OligoPerfect (Invitrogen). All CT values were
normalized against the control gene rpl-29, which did not vary
under the conditions being tested. All of the experiments were
repeated at least three times (biological replicates) and
were internally controlled (technical replicate). Expression
changes were obtained by calculating the relative expression
levels using the 2 − CT method.
RESULTS
Spp-12 tissue localization and first phenotypical characterization
of a spp-12 mutant
Although spp-1 is transcribed in the intestine [35], the spp12p::spp-12(1-97)::gfp construct displayed expression of spp12 exclusively in two pharyngeal neurons (Figure 1), which
are the pharyngeal NSM/L (neurosecretory motorneurons) and
pharyngeal interneuron I6 according to WormAtlas (http://www.
wormatlas.org). It is known that sensing of food bacteria by
the NSMs leads to an increased pumping rate of the pharynx,
decreased locomotion and an increase in egg laying [36]. To
test whether SPP-12 is involved in these processes, egg laying,
population growth and pharyngeal pumping were measured in
a strain carrying a loss-of-function allele of spp-12 (tm2963).
However, only marginal differences were observed in the mutant
when compared with wild-type worms: pharyngeal pumping of
the spp-12 mutant was slightly increased after 1 h (P = 0.0135),
but did not differ after 6 h and 24 h of incubation on assay
plates with E. coli OP50 cells. Egg laying of the mutant at 25 ◦ C
was decreased in the first 24 h of adulthood only (P = 0.0089).
Concerning the sum of egg laying of different generations
(population growth), we observed that the spp-12 mutant forms a
smaller population within 5 days if incubated with E. coli OP50
cells compared with the wild-type, but this difference turned out
to be not significant (P = 0.1000) (results not shown).
Functional analysis of SPP-12 in comparison with SPP-1
As the family name caenopores implies, it is assumed that
spps encode pore-forming peptides. To test whether SPP12, apparently localized in pharyngeal neurons, is indeed
c The Authors Journal compilation c 2012 Biochemical Society
208
Figure 2
A. Hoeckendorf, M. Stanisak and M. Leippe
Pore-forming activity of SPP-12 and SPP-1
(A) Time course of pore formation. Dissipation of valinomycin-induced diffusion potential in liposomes in 1 ml of cuvette buffer (50 mM Tris-maleate, 50 mM sodium sulfate, 0.5 mM EDTA and
0.02 % NaN3 , pH 5.2) after the addition (arrow) of 115 pmol of SPP-12 (grey), 100 pmol of SPP-1 (dark grey), 100 pmol of control peptide alamethicin (black) or peptide solvent 0.01 % TFA (light
grey) was recorded at pH 5.2. Pore-forming activity is reflected by increase of fluorescence as a function of time. (B) pH dependence of the pore-forming activity of SPP-12 (light grey) and SPP-1
(dark grey). Activities were measured in two independent experiments in cuvette buffer adjusted to various pH values. The values at pH 5.2 were set to 100 %. (C) Association of recombinant SPP-12
to liposomes under acidic, neutral and basic conditions. The polypeptide was incubated with liposomes in cuvette buffer adjusted to the respective pH at 4 ◦ C for 1 h. After ultracentrifugation,
polypeptide bound to membranes appeared in the pellet (P) or unbound in the supernatant (S) upon tricine-SDS/PAGE (silver-stained gel). (D) Lipid specificity of the membrane-permeabilizing
activity of SPP-12 (light grey) and SPP-1 (dark grey) measured by calcein release from preloaded liposomes of defined phospholipid composition. Results are the median +
− the range of three
measurements.
a pore-forming polypeptide as has been shown for SPP5 [13], we recombinantly expressed SPP-12 and analysed
its ability to associate with phospholipid vesicles and form
pores in artificial membranes. In parallel, we characterized the
intestinally located SPP-1 by monitoring its activity with the
recombinant form. MS analysis revealed that, for both of
the recombinantly expressed proteins, all of the six cysteine
residues are involved in disulphide bridges and that covalent
oligomerization did not occur (Supplementary Figures S1 and
S2 at http://www.BiochemJ.org/bj/445/bj4450205add.htm). In
the liposome depolarization assay, in which a valinomycininduced diffusion potential is disrupted when a pore-forming
protein is acting on the membranes, it became evident that
the activity of SPP-12 and SPP-1 is comparable with that of
alamethicin, the prototype of a pore-forming peptide (Figure 2A).
Accordingly, SPP-12 and SPP-1 are considered true caenopores.
The pore-forming activity of the proteins is dependent on a
mildly acidic pH, particularly that of SPP-12, which was not
detectable at pH 6 (Figure 2B). At neutral and basic pH,
binding of SPP-12 with phospholipid membranes was hardly
detectable by SDS/PAGE, but at mildly acidic pH, the majority
of the protein became membrane associated (Figure 2C). The
liposomes used was AL, a complex phospholipid mixture from
the soya bean. To analyse a potential specificity for particular
phospholipids, including those which are more representative for
bacterial membranes, we tested membrane permeabilization by
c The Authors Journal compilation c 2012 Biochemical Society
monitoring the release of calcein from preloaded vesicles of
defined phospholipid composition. Both of the proteins acted
preferentially on membranes that contained negatively charged
phospholipids that are frequently found in bacterial membranes
such as PG and CL (Figure 2D). In parallel, we determined the
antimicrobial activity of the two caenopores SPP-12 and SPP-1
against various micro-organisms. Several species, that the worm
feeds on in the laboratory or is potentially exposed to within its
natural environment and which may be ingested during nutrition,
were tested; representative Gram-positive and -negative bacteria,
yeast, and amoebae. Unlike SPP-5 [13], SPP-12 and SPP-1 are
not active against E. coli OP50 cells up to a concentration of
10 μM, but both are highly active against B. megaterium with
a MIC (minimal inhibitory concentration) and MBC (minimal
bactericidal concentration) of 275 nM. SPP-12 killed pathogenic
B. thuringiensis at higher concentrations (10 μM), whereas SPP1 did not (Table 1). Both of the proteins permeabilize the
cytoplasmic membranes of viable bacteria as evidenced by
the uptake of the membrane-impermeable fluorescent dye SYTOX
Green in live B. megaterium, which is able to enter the cell in the
presence of a membrane-permeabilizing agent. Comparable with
the well-known antimicrobial peptide cecropin P1, SPP-12 and
SPP-1 were active at submicromolar concentrations (Figure 3A).
Moreover, SPP-12 and SPP-1 permeabilized the membranes of
yeast cells as efficiently as the membrane-active and cytotoxic
peptide melittin (Figure 3B). Viable Dictyostelium amoebae were
Antimicrobial saposin-like protein in the pharyngeal neurons of C. elegans
Table 1
Antimicrobial activity of SPP-12 and SPP-1
Growth inhibiting and bactericidal activity of SPP-12 and SPP-1 was determined using a
microdilution susceptibility assay. >, no activity found at the concentration indicated.
Microbe
SPP-12
Gram-negative bacteria
E. coli OP50
Gram-positive bacteria
B. megaterium A.T.C.C. 14581
B. thuringiensis B-18247
SPP-1
MIC (μM)
MBC (μM)
MIC (μM)
MBC (μM)
>10
>10
>10
>10
0.275
10
0.275
10
0.275
>10
0.275
>10
affected in micromolar concentrations (Figure 3C). In regards to
their cytotoxicity, SPP-12 and SPP-1 were virtually inactive when
tested against human Jurkat T-cells (Figure 3D).
Survival of spp-12 and spp-1 mutants exposed to B. thuringiensis
The spores of the pathogenic B. thuringiensis strain B-18247
are associated with crystal toxins and after oral uptake together
with an independent food source they cause persistent infection
in C. elegans, which eventually kills the host [23]. The spp12 mutant was more susceptible to pathogenic B. thuringiensis
than the wild-type (P < 0.0001), indicating that SPP-12 is
instrumental in the defence response against this pathogen
Figure 3
209
(Figure 4A). Concomitant exposure to the food bacterium E.
coli OP50 precludes starvation and worms exposed to the
spores of non-pathogenic B. thuringiensis in a similar setting
did not differ significantly in their survival from the wild-type.
Conversely, the spp-1 mutant revealed an enhanced tolerance to
pathogenic B. thuringiensis (P < 0.0001; Figure 4B), suggesting
that SPP-1 is not beneficial for the worm upon challenge with
pathogenic B. thuringiensis. For the spp-12;spp-1 double mutant,
no difference in survival was observed when compared with the
wild-type (Figure 4C), which mirrors the equalization of opposite
phenotypes of the single mutants.
The surprisingly shorter lifespan of spp-1 mutant worms on
non-pathogenic B. thuringiensis compared with the wild-type,
however, may be explained by the simultaneous exposure to E.
coli OP50 cells. When grown on this bacterium alone, the spp-1
mutant, but not the spp-12 mutant and the spp-12;spp-1 double
mutant, died earlier than the wild-type (Supplementary Figures
S3–S5 at http://www.BiochemJ.org/bj/445/bj4450205add.htm).
This effect could be observed at 25 ◦ C and 20 ◦ C, but only when
worms fed on live E. coli (Supplementary Figure S4).
A transcriptional profiling of spp-12 and spp-1 with wildtype worms exposed to pathogenic and non-pathogenic B.
thuringiensis as in the survival assays revealed a massive downregulation of spp-1 upon challenge with the pathogen, indicating
again that spp-1 expression is not effective in defence against
this pathogen (Figure 5). However, we did not observe any
transcriptional regulation of the apparently protective spp-12
(Figure 5).
Permeabilization of membranes of various micro-organisms by SPP-12 and SPP-1 and evaluation of the cytotoxicity of the proteins
Membrane damage of viable bacteria, yeast cells and amoebae was measured fluorometrically using the dye SYTOX Green at pH 5.2. Binding of dye to DNA in membrane-compromised target
cells resulted in an increase of fluorescence. The antimicrobial activity of the polypeptides SPP-12 (䉱), SPP-1 (䊉) and the control peptides cecropin P1 (䊐) and melittin (䉫) are expressed as
the percentage of permeabilized cells. The cytotoxic activity of the polypeptides against Jurkat T-cells was determined fluorometrically using the dye AlamarBlue at pH 5.5, reflecting a decrease
of metabolic activity of the cells. (A) Membrane-permeabilizing activity after incubation of B. megaterium with each polypeptide at various concentrations for 60 min. (B) Permeabilizing effect on
S. cerevisiae after 210 min of incubation. (C) Permeabilization of D. discoideum amoebae after 60 min of incubation. (D) Cytotoxic activity against Jurkat T-cells after 180 min of incubation.
c The Authors Journal compilation c 2012 Biochemical Society
210
A. Hoeckendorf, M. Stanisak and M. Leippe
Figure 5
B. thuringiensis-induced expression profile of spp-12 and spp-1
Synchronized L4-staged N2 were exposed to pathogenic or non-pathogenic B. thuringiensis
spores (2×108 spores and 6×109 E. coli OP50 cells per ml) for 24 h at 20 ◦ C or 25 ◦ C.
Expression profiles were achieved by qRT-PCR after RNA isolation and cDNA synthesis. Relative
expression levels were calculated following the 2 − CT method. Mean and range of three
independent experiments are shown. Positive values indicate up-regulation and negative values
down-regulation after pathogen exposure when compared with non-pathogenic B. thuringiensis .
Spp-1 (dark grey) transcription was strongly down-regulated when the wild-type worms were
exposed to pathogenic B. thuringiensis at 20 ◦ C and 25 ◦ C, whereas a change in transcription
of spp-12 (light grey) was not detectable under these conditions.
Figure 4 Survival of the spp-12 , spp-1 and spp-12;spp-1 mutants exposed
to B. thuringiensis at 20 ◦ C
(A) Spp-12(tm2963) mutant nematodes ( and 䉱) were more susceptible to pathogenic B.
thuringiensis (䊏 and 䉱; P < 0.0001; n >250) when compared with the wild-type worms (䊏
and 䊐), but on non-pathogenic B. thuringiensis they were not affected (䊐 and ; P = 0.3179;
n >100). (B) Spp-1(ok2703) mutant worms (䊊 and 䊉) exhibited enhanced tolerance to
pathogenic B. thuringiensis (䊉 and 䊏; P < 0.0001; n >100), whereas on non-pathogenic
B. thuringiensis (䊊 and 䊐; P < 0.0001; n >120) they were more susceptible than the
wild-type (䊏 and 䊐). (C) Survival of spp-12(tm2963);spp-1(ok2703) mutant worms (䉫 and
䉬) on pathogenic B. thuringiensis (䉬 and 䊏; P = 0.1969; n >100) and non-pathogenic B.
thuringiensis (䉫 and 䊐; P = 0.5043; n > 110) did not differ from wild-type survival (䊏 and
䊐). The survival curves represent means +
− S.E.M. for three independent experiments performed
in quintuplicate.
DISCUSSION
The most intriguing finding of the present study was that we
localized the site of transcription of spp-12, the gene encoding
a potential caenopore, to the neurons. In contrast with other spp
genes, e.g. spp-1, spp-5 and spp-7, the gene products of which are
found primarily in the intestine [13,35,37], spp-12 was found to
c The Authors Journal compilation c 2012 Biochemical Society
be expressed exclusively in two pharyngeal neurons, namely the
NSM/L and I6. The observation that spp-12 is not transcribed in
the worm’s gut is plausible because spp-12 is one of the
four members of the caenopore gene family which misses an
ELT-2-binding site in the promoter region, indicative of an
intestinal location [13]. The NSMs are postulated to sense
bacteria with their small synapse-free processes ending next to
the cuticle of the pharyngeal lumen, and to transmit this signal
by serotonin secretions to the pseudocoelomic fluid ([36,38]
and http://www.wormatlas.org/). As serotonin secretion in the
presence of food leads to depressed locomotion, increased
pharyngeal pumping and enhanced egg laying, these parameters
were monitored to see whether SPP-12 is involved in the sensing
of bacteria. However, only slight differences in the spp-12 mutant
were observed in the present study. As the neurons that can
detect bacteria are redundant in C. elegans [40], it still cannot
be excluded that SPP-12 is involved in the sensing of potential
prey or of pathogens that are preferentially avoided using escape
mechanisms.
Concurrently, SPP-12 may serve as a sentinel protecting the
connection between the NSMs and the pharyngeal lumen against
invading bacteria. Also the I6, the other pharyngeal neuron
in which spp-12 is expressed, has a free subcuticular ending
between the pharyngeal muscles pm6 and pm7 [38], which
may need protection against bacterial assault. Although the
location of SPP-12 was somewhat unexpected, there are other
examples that demonstrate that antimicrobial proteins can be
found in the neurons of invertebrates. In the medicinal leech
Hirudo medicinalis, the antimicrobial peptides neuromacin and
Hm-lumbricin, which have biological activity against Grampositive bacteria, are produced in the central nervous system
by neurons and microglia cells and promote the regeneration of
injured neurons [41]. For C. elegans, it has been reported that
the genes lys-1 and lys-7, putatively encoding Entamoeba-type
lysozymes, are not only expressed in the intestine, but also in
head neurons as evidenced by gfp-reporter genes [35,42]. The
NSMs also produce several members of the NLPs (neuropeptidelike proteins), another very large family of potential antimicrobial
peptides [43,44].
One has to admit that in C. elegans only a very few of the
plethora of putative antimicrobial effector proteins and peptides
Antimicrobial saposin-like protein in the pharyngeal neurons of C. elegans
have been characterized at the protein level. The majority
of them have been associated with a defensive function by
indirect evidence such as transcriptional profiling after bacterial
challenge and phenotypic analysis of gene-silenced mutants.
Among those to which an antimicrobial activity have been
assigned experimentally by using recombinant proteins are NLP31 the gene of which is expressed in the hypodermis [45], ABF-2
[46], and the intestinal caenopores SPP-1 and SPP-5 [13,21].
Only the latter protein was characterized comprehensively with
regard to its mode of action [13] and tertiary structure [14]. In
the present study, for the first time we describe an antimicrobial
protein that is exclusively expressed in neurons. SPP-12 was
compared in the present study in parallel with SPP-1 and it was
found that both are indeed caenopores in that they exert poreforming activity and permeabilize the membranes of a broad
variety of viable target cells, including Gram-positive bacteria,
yeast cells and amoebae. In contrast with SPP-5, both are not
effective against E. coli. Cytotoxic activity towards human cells,
as shown for the structural relatives amoebapores and NKlysin [30] was negligible, indicating a broad, but nevertheless
selective, target cell spectrum. The pore-forming activity of
SPP-12, and less drastically of SPP-1, is dependent on mildly
acidic pH, and one may suggest that the internal histidine
residues are critically involved in mediating this pH preference
as was shown for amoebapores [20]. Although in amoebapores a
conserved single histidine residue at the C-terminus is critically
involved in activity, SPP-12 and SPP-1 contain three and four
histidine residues respectively, that may become protonated below
their pK a (pH 6.5) and may be responsible for a pH-dependent
electrostatic interaction with membranes. However, assuming the
same SAPLIP fold as shown for SPP-5 [14], in SPP-1 these
histidine residues are clustered, whereas in SPP-12 all point
in different directions. Collectively, SPP-12 and SPP-1 share
similar characteristics, they are situated next to each other in
a phylogenetic tree of the C. elegans SAPLIPs [13] and are
highly charged proteins (approximately 40 % of the residues are
charged at mildly acidic pH) with positively charged residues
interspersed along the entire sequence (Supplementary Figure
S6 http://www.BiochemJ.org/bj/445/bj4450205add.htm). Both
proteins preferentially permeabilize membranes with negatively
charged phospholipids, i.e. CL and PG, major components of
bacterial membranes, again suggesting an electrostatic interaction
in the initial phase of the SPPs’ attack on membranes. The pH
of the environment of the intestinal caenopores such as SPP-1
and SPP-5 is known to be acidic [13,47], whereas no information
exists about the conditions at which a secreted SPP-12 at the
interface between the pharynx and the pharyngeal neurons would
act.
SPP-12 is the only antimicrobial effector protein of
C. elegans, to date, for which the bactericidal activity towards
B. thuringiensis has been demonstrated in vitro, although much
higher concentrations are needed to kill the pathogen than for
B. megaterium. In the in vivo studies, it became evident that
spp-12 expression indeed has a protective effect upon challenge
with pathogenic B. thuringiensis, as the loss of the gene renders
the worms hypersusceptible. The transcriptional data could not
demonstrate any up-regulation of spp-12 upon challenge with B.
thuringiensis suggesting a constitutive expression of the gene.
However, the analysis revealed that spp-1 is down-regulated by
the worm in the presence of pathogenic B. thuringiensis. It is
suggestive to think that this is because the expression of this
gene is not of any value against this bacterium according to the
phenotype of the mutant worm and the lack of activity towards
this pathogen of the SPP-1 protein in vitro. A pathogen itself can
interfere with the immune system and it has been reported that
211
Pseudomonas aeruginosa down-regulates spp-1 transcription by
activating the DAF-2 signalling pathway to overcome the defence
system of C. elegans [48].
Unexpectedly, the spp-1 mutant survived longer on pathogens
than the wild-type worms. One may ask how the loss of an
antimicrobial polypeptide gene can be an advantage, particularly
as the costs for synthesis of such a small protein do not appear
high. The loss of a single antimicrobial factor such as SPP-1 may
increase the resistance of C. elegans in that the levels of other
defensive components are raised that are more relevant for coping
with a particular pathogen via the interconnected signalling
pathways. Similarly, the surprising finding has been reported that
stress resistance of C. elegans against particular oxidative-stresscausing agents is elevated when a peroxiredoxin gene instrumental
in protecting against environmental oxidative stress is lost [49].
In Anopheles, the loss of a lysozyme gene resulted in a better
protection against infestation by the insect stages of the malarial
parasite Plasmodium, presumably by enhancing basal immune
mechanisms [50]. Likewise, the lifespan extension of the spp-12
mutant fed on relatively non-pathogenic E. coli OP50 cells is most
likely not due to a trade-off (saving of costs) and other immune
effectors may overact when spp-12 is lost.
Nonetheless, one may think about an alternative explanation
for why the loss of spp-1 appear to have a positive effect on the
lifespan of worms challenged with pathogenic B. thuringiensis,
whereas spp-12 is required to cope with the pathogen. Possibly,
the synthesis of virulence factors by B. thuringiensis is triggered
by SPP-1 in the intestine and that is why the wild-type worms are
more susceptible than spp-1 mutants to the pathogenic bacterium.
In general, one has to consider that the multiplicity of potential
antimicrobial effector proteins in nematodes, and in particular the
diversity of caenopores and lysozymes in C. elegans, is enormous
[12,13,25,44]. The vast majority of them may act complementarily
and synergistically in the intestine of the worm to support nutrition
and to fight infection. Consequently, one may not expect that the
silencing or knock-down of one individual antimicrobial protein
gene will have a dramatic effect on survival. The extraordinary
location of SPP-12 may be the reason why an in-vivo effect upon
challenge with a pathogen was detectable in the present study.
Conclusively, the results of the present study emphasize that in
C. elegans antimicrobial proteins are not only instrumental in the
intestine against microbes engulfed via the food intake and
in the hypodermis against external invaders [11], but also in
pharyngeal neurons and indicate that SPP-12 is involved in the
defence against B. thuringiensis, a pathogen that the worm
encounters in its natural environment.
AUTHOR CONTRIBUTION
Aylin Hoeckendorf performed most of the experiments and wrote the first draft of the paper.
Mareike Stanisak recombinantly expressed the proteins and performed the majority of the
functional in vitro assays with the proteins. Matthias Leippe conceived the study, designed
the experiments and wrote the final version of the paper.
ACKNOWLEDGEMENTS
We thank Dr Thomas Roeder for valuable comments, Dr Hinrich Schulenburg for providing
B. thuringiensis strains and advice concerning the host–pathogen interaction system, Dr
Kerstin Isermann for her help concerning microinjection, Dr Marko Rohlfs for initial help
with statistics and Dr Christoph Gelhaus for initial mass spectrometric analysis. We are
particularly indebted to Christian Treitz and Dr Andreas Tholey for comprehensive mass
spectrometric analysis of the recombinant proteins finally used in the assays. We thank
Dr Joachim Grötzinger for structural comparison of SPP-12 and SPP-1 with the solution
structure of SPP-5. We thank the NIH (National Insitiutes for Health) National Center for
Research Resources-funded Caenorhabditis Genetics Center and the National Bioresource
Project for the Experimental Animal in Japan for the strains used.
c The Authors Journal compilation c 2012 Biochemical Society
212
A. Hoeckendorf, M. Stanisak and M. Leippe
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doi:10.1042/BJ20112102
SUPPLEMENTARY ONLINE DATA
The saposin-like protein SPP-12 is an antimicrobial polypeptide in the
pharyngeal neurons of Caenorhabditis elegans and participates in defence
against a natural bacterial pathogen
Aylin HOECKENDORF, Mareike STANISAK and Matthias LEIPPE1
Zoological Institute, Zoophysiology, University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany
EXPERIMENTAL
MS analysis of the recombinant proteins to determine the oxidative
state of the cysteine residues
The samples were loaded on to a PicoTip emitter and injected
offline with a nanospray ion source at spray voltage of 1.3 kV
and a capillary temperature of 197 ◦ C into an LTQ-Orbitrap
Velos mass spectrometer (Thermo Scientific). The automatic gain
control target for the Orbitrap mass analyser was set to 1×106 ,
the microscan number and maximum ion injection time were set
to 2 and 500 respectively. Full FTMS (Fourier transform mass
spectrometry) scans of the mass range from 400 to 2000 for
SPP-12 and from 1200 to 1600 for SPP-1 were acquired with
a resolution of 30000. As a control, the SPP-12 sample was
additionally measured after reduction with 10 mM TCEP [tris(2-carboxyethyl)phosphine] at 60 ◦ C for 30 min.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
A. Hoeckendorf, M. Stanisak and M. Leippe
Figure S1
MS analysis of SPP-12
(A) Spectra in the mass range 800–1800 m /z showing signals corresponding to the mass of SPP-12 with six cysteine residues oxidized to three disulfides from charge + 5 to + 10. The signals of
charge state + 7 and + 8 are zoomed in (B) and (D) respectively. The spectra of + 7- and + 8-fold charged SPP-12 after reduction with TCEP [tris-(2-carboxyethyl)phosphine] are shown in (C)
and (E) respectively.
c The Authors Journal compilation c 2012 Biochemical Society
Antimicrobial saposin-like protein in the pharyngeal neurons of C. elegans
Figure S2
MS analysis of SPP-1
(A) Mass spectrum showing the + 7- and + 8-fold charged signals of the completely oxidized form of SPP-1. (B) The + 7-fold charged signal is zoomed in showing m /z signals corresponding
to methionine sulfoxide and methionine sulfone modifications.
c The Authors Journal compilation c 2012 Biochemical Society
A. Hoeckendorf, M. Stanisak and M. Leippe
Figure S4
Figure S3
Lifespan of the spp-12 mutant on E. coli OP50 cells
(A) At 25 ◦ C, the spp-12 mutant () exhibited an extended lifespan (P < 0.0001; n >100)
when compared with the wild-type worms (䊐). (B) At 20 ◦ C, the spp-12 mutant () again lived
longer than the wild-type (䊐) (P = 0.0001; n >150). (C) On heat-inactivated bacteria at 20 ◦ C,
the mutant () has a shorter lifespan than wild-type (䊐) (P = 0.0223; n >140). Results are
means +
− S.E.M. of three independent experiments performed in quintuplicate.
c The Authors Journal compilation c 2012 Biochemical Society
25 ◦ C,
Lifespan of the spp-1 mutant on E. coli OP50 cells
(A) At
the lifespan of the spp-1 mutant (䊊) is shorter (P = 0.0086; n >100) than that
of the wild-type (䊐). (B) At 20 ◦ C, again the spp-1 mutant (䊊) had a shorter lifespan than the
wild-type (䊐) (P < 0.0001; n >150). (C) On heat-inactivated bacteria at 20 ◦ C, the lifespan
of the mutant (䊊) did not differ from that of wild-type (䊐) (P = 0.6872; n >100). Results are
means +
− S.E.M. of three independent experiments performed in quintuplicate.
Antimicrobial saposin-like protein in the pharyngeal neurons of C. elegans
Figure S5
Lifespan of the spp-12;spp-1 mutant on E. coli OP50 cells
(A) At 25 ◦ C, the double mutant (䉫) exhibited a lifespan extension (P < 0.0001; n >100)
compared with the wild-type (䊐). (B) At 20 ◦ C, spp-12;spp-1 mutant (䉫) did not differ
from the wild-type (䊐) lifespan (P = 0.9159; n > 120). (C) On heat-inactivated bacteria at
20 ◦ C, the spp-12;spp-1 mutant (䉫) had a shorter lifespan (P < 0.0001; n > 140) than the
wild-type (䊐). Results are means +
− S.E.M. of three independent experiments performed in
quintuplicate.
c The Authors Journal compilation c 2012 Biochemical Society
A. Hoeckendorf, M. Stanisak and M. Leippe
Figure S6
Multiple sequence alignment
The primary structures of SPP-12 (WormBase ID WP:CE13994) and SPP-1 (WormBase ID WP:CE35363) were compared with those of amoebapore A and SPP-5. Amino acid residues in one-letter
code are marked according to their properties in blue (cationic), red (anionic) or green (hydrophobic). Histidine residues are highlighted in white font on a dark blue background. The position of
the five helices in the solution structures of amoebapore A (PDB accession code 1OF9) [1] and SPP-5 (PDB codes 2JS9 and 2JSA) [2] are presented symbolically next to the respective sequence.
Disulphide bridges as located in both tertiary structures are depicted as connecting horizontal lines between the six conserved cysteine residues (yellow).
REFERENCES
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Received 30 November 2011/5 April 2012; accepted 23 April 2012
Published as BJ Immediate Publication 23 April 2012, doi:10.1042/BJ20112102
c The Authors Journal compilation c 2012 Biochemical Society