MINIREVIEW
Analysis of the phylogenetic relationships and evolution of the cell
walls from yeasts and fungi
José Ruiz-Herrera & Lucila Ortiz-Castellanos
Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato,
Gto., México
Correspondence: José Ruiz-Herrera,
Departamento de Ingenierı́a Genética,
Unidad Irapuato, Centro de Investigación y de
Estudios Avanzados del Instituto Politécnico
Nacional, Apartado Postal 629, 36821
Irapuato, Gto., México. Tel.: 152 462 623
9600; fax: 152 462 623 45849; e-mail:
[email protected]
Received 19 May 2009; revised 8 September
2009; accepted 1 October 2009.
Final version published online 5 November
2009.
DOI:10.1111/j.1567-1364.2009.00589.x
Editor: Richard Calderone
Keywords
cell wall; evolution; chitin; chitosan; glucans;
glycoproteins.
Abstract
The fungal cell wall is a coherent structure formed by microfibrillar polysaccharides and amorphous material made of other polysaccharides and proteins. We
performed a phylogenetic analysis of covalent proteins and enzymes that synthesize fungal wall polysaccharides to determine the possible evolution of the wall
structure. It is suggested that the components that made up the archaic walls were
structural ones, forming a primitive girdle that retained noncovalently bound
proteins in the periplasm and allowed cell growth in hypotonic media. The
following hypothetical series of events in fungal wall evolution is suggested: (1)
Construction of a primitive wall made of chitin and chitosan by division 2 chitin
synthases and chitin deacetylases, respectively. (2) Appearance of class II chitin
synthase genes (CHS) after separation of Microsporidia. (3) Capture of a gene
encoding b-1,3-glucan synthase from an organism related to Plantae or Chromista
by horizontal transfer after separation of Chytridiomycota. (4) Appearance or
horizontal capture from Chromista of genes involved in b-1,6-glucan synthesis
after separation of Zygomycota. (5). Appearance of class III CHS genes. (6) After
split of Dikarya phyla, appearance in Ascomycota of class I CHS genes and the
capacity to synthesize covalently bound wall proteins.
YEAST RESEARCH
Introduction
The cell wall is the rigid outermost layer that covers the cells
of a number of organisms belonging to different taxa, both
prokaryotic and eukaryotic. In either type of cells, the
general functions of the wall are similar, being responsible
for the protection of cell integrity by providing support to
the cell membrane to withstand the difference in osmotic
pressure between the cytoplasm and the external medium. If
the cell wall is removed by enzymatic treatment or inhibition
of its synthesis by selective drugs, the resulting wall-less cells
(protoplasts) can survive only in isotonic or hypertonic
media. Besides this crucial role, the wall also protects the cell
from the action of enzymes and different deleterious compounds, it is responsible for the shape and morphogenesis of
the cell, and contains molecules responsible for the recognition of predators or hosts, or involved in the formation of
biofilms (see Ruiz-Herrera, 1992; Sentandreu et al., 2004).
If the functions are similar, the structure and chemical
composition of the cell walls of prokaryotes and eukaryotes
FEMS Yeast Res 10 (2010) 225–243
are basically different. Thus, the structure of the cell walls from
eukaryotic organisms is based on the presence of microfibrils formed by the association through hydrogen bonding
of long chains of linear polysaccharides. Characteristically,
microfibrillar polysaccharides are polymers of a single type
of sugar bound through glycosidic b-linkages. On the other
hand, the wall of prokaryotes does not contain microfibrillar
components, probably because of their smaller dimensions;
instead, its structure is made up in most prokaryotes of a
tridimensional net with a characteristic composition, the
sacculus. This is made of short oligosaccharides covalently
bound to oligopeptides: the peptidoglycan of bacteria, and
the archean pseudo-peptidoglycan.
Repeatedly, the structure of the eukaryotic cell wall has
been compared with synthetic composites made of a structural component responsible for the resistance to tensions,
and an amorphous component that confers resistance
to pressure and protects the structural component from
fracture and from the action of deleterious components
of the environment. The main structural microfibrillar
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226
components, depending on the type of organism, are cellulose, chitin and b-1,3-glucans. The chemical components
that constitute the amorphous part of the cell wall are different nonmicrofibrillar polysaccharides, proteins and lipids,
plus salts and pigments in small amounts (for reviews, see
Ruiz-Herrera, 1992; Sentandreu et al., 2004).
Prevalent ideas sustain the concept that all eukaryotic
organisms descend from an ancestral uniciliate eukaryote
(Cavalier-Smith, 2002, 2004; Steenkamp et al., 2006). Accordingly, at the time of appearance of cell-walled eukaryotic
organisms, the prokaryotic cell wall had to be substituted by a
completely different structure. The antecessors of the two most
abundant in number wall-bearing eukaryotic groups, Plantae
and Fungi kingdoms, separated early during eukaryote evolution (Cavalier-Smith, 2002, 2004), and developed cell walls
based on structurally similar, but chemically different microfibrillar components: cellulose for plants and chitin for fungi.
Both of them are linear polysaccharides made of a large
number (normally 4 2000 U) of either glucose for cellulose
or N-acetylglucosamine [2-acetamido-2-deoxy-D-glucose
(GlcNAc)] for chitin, joined by b-1,4-linkages. Because of the
crystalline arrangement of both polysaccharides that eliminates water from their structure, they are extremely insoluble,
and with a high tensile strength, higher for chitin than for
man-made fibers (steel, or carbon or boron fibers) (reviewed
by Ruiz-Herrera, 1992; Ruiz-Herrera & Ruiz-Medrano, 2004).
The structure and chemical composition of the fungal cell
walls has been the subject of a large number of studies. The
majority of these studies have been focused on ascomycetes,
and mostly on different yeasts, especially Saccharomyces
cerevisiae, giving rise to an almost generally accepted model
of the fungal cell wall. It has been concluded that the most
important structural polysaccharides of the fungal cell wall
are chitin and b-1,3-glucans that associate with b-1,6-glucans,
and with covalently and noncovalently bound mannoproteins
(see reviews and representative schemes in Lipke & Ovalle,
1998; Chaffin, 2008). Other polysaccharides present in lower
amounts appear to be specific of groups or species: a-glucans,
chitosan, polyuronides, galactans, etc. As indicated above,
lipids, pigments and salts are present in minor amounts (see
Ruiz-Herrera, 1992; Sentandreu et al., 2004).
Recently, and due to their important roles in the cell
wall, considerable attention has been focused on fungal wall
proteins, recognizing in general two different classes according to the way in which they associate with other wall
components: noncovalently bound and covalently bound
proteins. Noncovalently bound proteins are retained in the
wall by hydrogen, hydrophobic and/or ionic bonds, being
released into the medium in different proportions. Three
types of covalently bound proteins are generally recognized:
(1) glycosyl phosphatidyl inositol (GPI) proteins, which
contain the residue of a GPI substituent, and are bound to
b-1,6-glucans by a glycosidic linkage; (2) Pir proteins
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J. Ruiz-Herrera & L. Ortiz-Castellanos
(proteins with internal repeats), bound to b-1,3-glucans
through an alkali-labile linkage; and (3) proteins retained in
the wall by binding to other proteins through disulfide
linkages (Sentandreu et al., 2004; Chaffin, 2008).
Although the ascomycete wall constitutes the paradigm of
the fungal cell wall, it may be indicated that our experimental and in silico analyses of the structure of the cell wall
of the basidiomycete Ustilago maydis revealed significant
differences from the established model of the cell wall of
ascomycetes (Ruiz-Herrera et al., 2008), raising doubts on
the existence of a universal model of the fungal cell wall.
Probably the first study that correlated the chemical
composition of the cell wall with the then accepted taxonomic division of fungi was published by Bartnicki-Garcı́a
(1968). This author reported that the different fungal groups
possessed cell walls with distinctive polysaccharides.
Although the taxonomy of fungi has changed dramatically
(see Lutzoni et al., 2004; James et al., 2006; Hibbett et al.,
2007), the general concepts of that study are still valid. A
different type of study was reported by Bernabé et al. (2002),
who analyzed the evolution of characteristic polysaccharides of
chemotaxonomic value present in the cell walls of ascomycetes.
More recently, Lipke and colleagues (Coronado et al., 2006,
2007a, b) devised computational procedures to correctly
analyze the homology among fungal cell wall glycoproteins,
and utilized the data to determine the existence of a number
of motifs in common among cell wall proteins, and their
conservation in different fungal groups, in an attempt to
understand the evolutionary origin of the fungal cell walls.
In the present study, we have followed a different approach
in order to analyze the phylogenetic relationships of the cell
walls from the several fungal groups. Accordingly, we have
analyzed the homology of the enzymes responsible for the
synthesis of different wall polysaccharides, and the covalently
bound proteins that make up the fungal cell wall in members
of the different fungal phyla. Noncovalently bound wall
proteins were excluded from this analysis because of the
difficulty in deciding whether they truly form part of the wall
structure, are fortuitously retained in the wall because of some
physicochemical characteristics (size, charge, etc.) or are
mainly secreted into the medium. The data reported here
reveal that the model of the cell wall structure of ascomycete
yeasts cannot be extended to the walls of the rest of the fungal
taxa, and constitute an initial attempt to understand the
evolution of the cell wall structure in the kingdom Fungi.
Chitin
Chitin is the basic structural polysaccharide of the cell wall
from most fungi. As indicated above, chitin is a large polymer
made of N-acetylglucosaminyl residues bound through b-1,4glycosidic likages. Chains of the polysaccharide associate by
hydrogen bonds to form microfibrils. Three different forms of
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Fungal wall evolution
chitin exist in nature, depending on the arrangements of the
chains in the microfibrils: b, with parallel chains [i.e. all in the
same nonreducing ! reducing ends orientation (polarity)],
a, with antiparallel chains (in which the polarity of the chains
alternates) and g, where two parallel chains are adjacent to
one antiparallel chain. The most abundant form in nature,
also present in the fungal cell walls, is a-chitin (reviewed in
Ruiz-Herrera, 1992; Ruiz-Herrera & Ruiz-Medrano, 2004).
Chitin synthesis is catalyzed by enzymes known as chitin
synthases (Chs), which utilize UDP-GlcNAc as a sugar donor.
Despite some unconfirmed early reports (Kang et al., 1984;
Montgomery et al., 1984), chitin synthases have not yet been
isolated, possibly because of their hydrophobic characteristics.
Accordingly, their catalytic properties are known from the use
of membrane fractions or chitosomes (see Ruiz-Herrera &
Ruiz-Medrano, 2004), and their structure from their encoding
genes (reviewed by Ruiz-Herrera et al., 2002). A characteristic
of fungi is that they contain more than one chitin synthaseencoding gene (CHS) in a number that varies from three in S.
cerevisiae to 4 20 in several mucoromycete species. [According to the revised fungal taxonomy, the former Zygomycota
phylum was found to be polyphyletic, and was therefore
divided into several phyla. The designation Mucoromycotina
for the former Mucorales has been suggested (James et al.,
2006; Hibbett et al., 2007). In this paper, we used either
designation for facility.] Sequence analysis of chitin synthases
revealed the existence of five classes (reviewed by Ruiz-Herrera
et al., 2002), which some authors have later on extended to
seven (in this study, we used the classification into five
classes). A thorough analysis of the structure of Chs that
identified conserved motifs in chitin synthases revealed the
existence of two groups of enzymes, denominated divisions: 1,
including classes I–III, and 2, which includes classes IV and V
(Ruiz-Herrera et al., 2002). Division 1 enzymes possess an
activity-related motif named ‘chitin synthase 1,’ different from
the equivalent motif of enzymes belonging to division 2,
named ‘chitin synthase 2’ (see http://pfam.sanger.ac.uk/).
Taking into consideration that chitin is probably the most
characteristic compound of the fungal cell wall, we proceeded to analyze the phylogenetic relationships of chitin
synthases from fungi belonging to the different phyla.
The neighbor-joining method was used for the analysis
(see details in the legend of Fig. 1). The first interesting
observation of this analysis concerned Chs from division 1
(Fig. 1). Thus, it was observed that its three classes are
present only in ascomycetes (although some species, such as
S. cerevisiae, contain members of only two classes). On the
other hand, basidiomycetes do not possess class I enzymes.
Zygomycetes (members of the new phylum Mucoromycotina) and the chytridiomycete Batrachochytrium dendrobatidis
contain only class II Chs, whereas the microsporidian
Encephalitozoon cuniculi does not contain any Chs belonging
to division 1. Regarding class II enzymes, two subclasses
FEMS Yeast Res 10 (2010) 225–243
(a and b) are distinguished in basidiomycetes and mucoromycetes, but not in ascomycetes. The latter appearance of
class III is apparent. With regard to the new phylum
Glomeromycota (previously included among zygomycetes),
no genomes have been sequenced or are accessible. Nevertheless, a single complete CHS gene, belonging to class IV of
division 2, has been isolated and sequenced in Glomus
versiforme (Lanfranco et al., 1999).
Genes belonging to both Chsp classes, IV and V from
division 2, are represented in members of all phyla, except
the microsporidian E. cuniculi, whose single CHS gene
belongs to class IV (Fig. 2). This result may agree with the
phylogenetic analysis suggesting that class IV is more
primitive than class V. The rest of fungal phyla contain two
IV subclasses, but Mucoromycotina species (zygomycetes)
possess the largest numbers of enzymes and two V subclasses
the same as basidiomycetes, as compared with members of
the other phyla. The existence of subclasses may be indicative of gene duplication and differentiation occurring at
early periods during evolution. The absence of two IV
subclasses in ascomycetes may be attributed to loss of the
ancestor gene in this fungal group.
It is interesting to note that a large number of class V
enzymes possess a motif related to a myosin head of an
unknown function. The fact that enzymes with or without a
myosin head coexist in the same subclasses shown in Fig. 2
indicates that they do not belong to separate groups.
A phylogenetic comparison of Chs from fungi with
those from other organisms that also contain chitin, at least
during specific stages of their life cycles, Animalia, Chromista and Protozoa kingdoms, was very illustrative. These
enzymes formed groups clearly separated from the fungal
enzymes, but maintaining a close association with those
from division 2 (Fig. 3). In silico structural analysis of
the sequence of chitin synthases present in members
of Protozoa and Animalia showed that all of them possessed
a chitin synthase motif with a homology closer to the ‘chitin
synthase 2’ motif from fungal enzymes, and none contained
the myosin head motif, suggesting that they are more related
to fungal class IV chitin synthases. These data support the
concept that the primitive Chs genes were related to modern
class IV. Interestingly, chitin synthase genes present in
Choroviruses that infect Chlorella (Kawasaki et al., 2002)
are also related to fungal class IV enzymes, possessing the
‘chitin synthase 2’ and lacking the myosin motifs.
Noticeable exceptions to these concepts are genes encoding chitin synthases from Oomycetes (Chromista). These are
related to fungal division 1 (see Ruiz-Herrera et al., 2002), and
it is important to notice that those from Aphanomyces euteiches
encode smaller fungal enzymes that synthesize a peculiar form
of water-soluble chitin of a very low Mr (Badreddine et al.,
2008). Whether this is a general characteristic of chitin
synthases and chitin of oomycetes remains to be determined.
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228
J. Ruiz-Herrera & L. Ortiz-Castellanos
Fig. 1. Evolutionary relationships of fungal chitin
synthases belonging to division 1. The MEGA4 program
(Tamura et al., 2007) was used to obtain the
dendrogram generated by the ‘neighbor-joining’
method (Saitou & Nei, 1987), with 1000 bootstraps
(Felsenstein, 1985). Data in the phylogenetic
dendrogram were drawn to scale with the branch
length in the same proportion as the evolution distance.
The evolution distance was computed using the
Poisson correction method (Zuckerkandl & Pauling,
1965). The abbreviations used are indicated in Table 1.
Asco, ascomycetes; basidio, basidiomycetes; Chytridio,
chytridiomycetes; and Mucoro, mucoromycetes
(zygomycetes).
Chitosan
Chitosan is a polysaccharide of a basic nature made up
mostly of glucosamine and a variable number of GlcNAc
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residues all bound through b-1,4-linkages. Chitosan is not
synthesized by a transglycosylation reaction from a glucosamine-activated donor, but through the deacetylation of
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Fungal wall evolution
Table 1. Species analyzed in this study
Fungi
Ascomycota
Ajellomyces capsulata, AJCN
Ashbya gossypii, ASHGO
Aspergillus clavatus, ASPCL
Aspergillus fumigatus, ASPFU, ASPFC
Aspergillus niger, ASPNG
Aspergillus oryzae, ASPOR
Aspergillus terreus, ASPTN
Candida albicans, CANAL
Candida glabrata, CANGA, CAG
Candida guilliermondii, PGUG
Candida lusitaniae, CLUG
Candida parapsilosis, CANPA
Candida tropicales, CTRG
Chaetomium globosum, CHAGB
Coccidioides immitis, COCIM
Colletotrichum lindemuthianum, COLLN
Cordyceps militaris, CORMI
Debaryomyces hansenii, DEBHA
Emericella nidulans, EMENI
Exophiala dermatitidis, EXODE
Flammulina velutipes, FLAVE
Fusarium solana, FUSSO
Issatchenkia orientalis, ISSOR
Kluyveromyces lactis, KLULA
Lodderomyces elongisporus, LODEL, LELG
Magnaporthe grises, MAGGR
Neosartorya fischeri, NEOFI
Neurospora crassa, NEUCR, NCU, NC
Paracoccidioides brasiliensis, PARBR
Phaeosphaeria nodorum, PHANO
Pichia guilliermondii, PICGU
Pichia stipitis, PICST
Pneumocystis carinii, PNECA
Saccharomyces cerevisiae, YEAST, Sc
Schizosaccharomyces pombe, SCHPO
Sclerotinia sclerotiorum, SCLS1
Vanderwaltozyma polyspora, VANPO
Yarrowia lipolitica, YARLI
Basidiomycota
Coprinus cinereus, Cc, COPC
Cryptococcus neoformans, Cn, CRYNE
Laccaria bicolor, LACBI
Malassezia globosa, MALGO
Melampsora laricis-populina, Mellp
Phanerochaete chrysosporium, Phc, Phchr
Pleurotus nebrodensis, 9AGAR
Postia placenta, Pospl
Puccinia graminis, Pgr
Sporobolomyces roseus, Sr, Sp, Sporo
Ustilago maydis, Um, USMTA
Mucoromycotina
Gongronella butleri, 9FUNG
Mucor circinelloides, Mucci, RHIRA
Mucor rouxii, MUCRO
Phycomyces blakeesneanus, PHYBL
Rizhopus oryzae, RO3G
FEMS Yeast Res 10 (2010) 225–243
Table 1. Continued.
Rhizopus stolonifer, RHIST
Chytridiomycota
Batrachochytrium dendrobatidis, BDEG
Microsporidia
Encephalitozoon cuniculi, ENCCU, ECU
Animalia
Aedes aegypti, AEDAE
Anopheles gambiae, ANOGA
Brugia malawi, BRUMA
Caenorhabditis elegans, CAEEL
Culex quinquefasciatus, CULQU
Dirofilaria immitis, DIRIM
Drosophila melanogaster, DROME, DMEL
Drosophila pseudoobscura, Dpse
Lottia gigantea, Lotgi
Lucilia cuprina, LUCCU
Manduca sexta, MANSE
Meloidogyne artiellia, MELAT
Nematostella vectensis, NEMVE
Papilio xuthus, 9NEOP
Spodoptera exigua, SPOEX
Tribolium castaneum, TRICA
Amoebozoa
Entamoeba histolytica, EHI
Chromista
Heterokontophyta
Phaeodactylum tricornutum, Phatr2
Phytopthora ramorum, Phyra
Phytophthora sojae, Physo
Thalassiosira pseudonana, Thaps
Haptophyta
EmiliInia huxleyi, EMIHU
Stramenopiles
Aureococcus anophagefferens, AURAN
Plantae
Arabidopsis thaliana, ARATH
Gossypium hirsutum, GOSHI
Hordeum vulgare var. distichum, HORVD,
Lolium multiflorum, LOLMU
Nicotiana alata, NICAL
Oryza sativa subsp. Japonica, ORYSJ
Physcomitrella patens, PHYPA
Vitis vinifera, VITVI
Chlorophyta
Chlamydomonas reinhardtii, CHLRE3
Chlorella NC64A, CHLNC64A
Ostreococcus tauri, OSTTA
Volvox carteri, VOLCA1
Protista
Percolozoa
Naegleria gruberi, NAEGR
Others
Virus
Chlorovirus 1, 9PHYC
Abbreviations after the name of each species were used to identify them
in the dendrograms.
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230
J. Ruiz-Herrera & L. Ortiz-Castellanos
Fig. 2. Evolutionary relationships of fungal chitin synthases
belonging to division 2. See details in the legend of Fig. 1.
nascent chitin by specific deacetylases (chitin deacetylases)
before this polymer reaches its microfibrillar crystalline
structure (Davis & Bartnicki-Garcia, 1984a, b; Calvo2009 Federation of European Microbiological Societies
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Mendez & Ruiz-Herrera, 1987). The existence of genes
encoding this enzyme may therefore be taken as evidence
suggesting the presence of chitosan in an organism, at
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Fungal wall evolution
least at some developmental stage. Chitin deacetylases are
not specific of fungi; genes encoding the enzymes are also
present in animals and protozoa, revealing their ancient
origin.
Regarding fungi, chitosan is the characteristic component
of the cell wall from Mucoromycotina (zygomycetes) species,
and was originally identified in a member of this taxon
(Phycomyces blakesleeanus; Kreger, 1954). Nevertheless, this
polysaccharide is not specific of this group of fungi, and it
was also found to be present in ascomycetes and basidiomycetes. In ascomycetes, it has been detected in the ascospores
of S. cerevisiae (Briza et al., 1988) and Schizosaccharomyces
pombe (Matsuo et al., 2005), but not in the vegetative walls
of S. cerevisiae or Candida albicans (Banks et al., 2005),
whereas in contrast, small amounts of chitosan were detected in the walls of the vegetative forms of S. pombe
(Sietsma & Wessels, 1990) and Aspergillus niger (Pochanavanich & Suntornsuk, 2002); besides, it is likely to exist in
other ascomycete species where genomic analysis revealed
the presence of a variable number of genes encoding chitin
deacetylases. In basidiomycetes, there are reports citing their
presence in Lentinus edodes and Pleurotus sajo-caju (Crestini
et al., 1996; Pochanavanich & Suntornsuk, 2002), and in the
cell walls of the vegetative form of Cryptococcus neoformans
that contained higher amounts of chitosan than chitin
(Banks et al., 2005; Baker et al., 2007). Genome analyses of
other basidiomycete species also showed the existence of
genes encoding chitin deacetylases, suggesting the wide
distribution of chitosan in this group of fungi. In silico
analysis of the U. maydis genome revealed the existence of
several genes encoding chitin deacetylases (Ruiz-Herrera
et al., 2008), although previous analyses of the walls from
its vegetative forms (yeast-like and mycelium) failed to
reveal the presence of chitosan (Ruiz-Herrera et al., 1996).
These data suggest the possibility that in this and other
basidiomycete species, the polysaccharide may be synthesized at specific stages of the life cycle, other than the
vegetative ones, just as occurs with S. cerevisiae.
Our in silico analyses revealed that the single chytridiomycete and microsporidian species analyzed also contained
genes encoding chitin deacetylases. These results are further
evidence of the ancient origin of chitin deacetylases and
chitosan, but as would be expected from the respective levels
of chitosan in the cell wall, Mucoromycotina (zygomycetes)
species possessed not only the greatest number of enzymes
but also the largest number of subclasses of chitin deacetylases (followed by basidiomycetes) (see Fig. 4). These
features are suggestive of repeated processes of gene duplication along evolution in these phyla.
Fig. 3. Evolutionary relationships of chitin synthases from fungi and
other taxonomic groups. See details in the legend of Fig. 1.
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232
J. Ruiz-Herrera & L. Ortiz-Castellanos
Fig. 4. Evolutionary relationships of
fungal chitin deacetylases. See details
in the legend of Fig. 1.
b-1,3-Glucans
b-1,3-Glucans are polysaccharides widely distributed in
fungi and with different cellular locations. There exist
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intracellular and cell wall-bound b-1,3-glucans with different degrees of polymerization and physicochemical characteristics, but in the majority of fungal species belonging to
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Fungal wall evolution
the clade Dikarya (see Lutzoni et al., 2004; James et al.,
2006; Hibbett et al., 2007), this polysaccharide constitutes
the most abundant component of the cell walls (see
Ruiz-Herrera, 1991, 1992 for a discussion), the structure of
the S. cerevisiae cell wall b-1,3-glucan being the most studied
(see Manners et al., 1973). Most of the cell wall b-1,3-glucan
is soluble in alkali, leaving an insoluble residue that was
originally found to be covalently bound to chitin in A. niger
(Stagg & Feather, 1973) and later on in other fungi (Surarit
et al., 1988). b-1,3-Glucan synthesized by protoplasts or in
vitro by cell-free extracts from S. cerevisiae appears in the
form of microfibrils (Kreger & Kopecká, 1975; Larriba et al.,
1981; Kopecká & Kreger, 1986), and it is also considered to
exist in this microfibrillar form within the cell wall. It is
important to notice that Mucoromycotina (zygomycetes) do
not contain b-1,3-glucans in their vegetative stages, but only
in the sporangiospore wall (see Bartnicki-Garcia & Reyes,
1964; Bartnicki-Garcı́a, 1968), and that in contrast to chitin
and chitosan, b-1,3-glucans are restricted to fungi in the
clade Opisthokont, being also present in members of the
Plantae and Chromista kingdoms.
Synthesis of b-1,3-glucan is catalyzed by specific synthases
that utilize UDP glucose as a glucosyl donor. In contrast to
chitin synthases, some fungal glucan synthases have been
purified to a reasonable degree (see Ruiz-Herrera et al., 2004
for a review). Interestingly, b-1,3-glucan synthases, independent of their origin, display important structural differences
from the rest of the b-glucosyl transferases, suggesting their
probable distinct phylogenetic origin (Campbell et al., 1997,
reviewed by Douglas, 2001). The number of b-1,3-glucan
synthase-encoding genes in fungal species is variable; there are
species that contain a single b-1,3-glucan synthase such as
U. maydis, Sporobolomyces roseus and Malazessia globosa,
whereas others contain as many as four, as occurs in S. pombe.
Noticeably, our in silico analyses revealed that the chytridiomycete and microsporidian species analyzed do not possess
homologues of genes encoding b-1,3-glucan synthases.
Phylogenetic analysis of fungal b-1,3-glucan synthases
revealed that in contrast to the results obtained with chitin
and chitosan, ascomycetes contain a larger number of enzyme
classes than the other fungal taxa, revealing several processes of
gene duplication and differentiation only in that phylum
(Fig. 5). A phylogenetic analysis of the relationships existing
between the fungal enzymes and those from the rest of the
taxonomic groups showed that the former appeared as a compact group clearly separated from the rest, their closest
relatives being b-1,3-glucan synthases from algae (not shown).
b-1,6-Glucans
As described above for b-1,3-glucans, b-1,6-glucans constitute
a large group of polysaccharides with different molecular
sizes, structures, chemical characteristics and cellular locations
FEMS Yeast Res 10 (2010) 225–243
(for a review, see Ruiz-Herrera, 1992). The most widely
studied b-1,6-glucan is the one present in the yeast wall, a
highly branched glucose polymer with an average of c. 350
monomers, covalently linked to b-1,3-glucan and corresponding to about 10% of the total glucans in the S. cerevisiae
cell wall (Shahinian & Bussey, 2000). The presence of b-1,6glucans is restricted to fungi and Chromista, being present in
the latter kingdom in species belonging to oomycetes, for
example Phytophthora (Bartnicki-Garcia & Lippman, 1967;
Zevenhuizen & Bartnicki-Garcia, 1969), Pythium (Blaschek
et al., 1992) and Achlya (Reiskind & Mullins, 1981) (reviewed
by Ruiz-Herrera, 1992). b-1,6-Glucans in fungi have been
studied mostly in ascomycetes, and only a few analyses have
reported their presence in basidiomycetes: higher basidiomycetes such as Sclerotium rolfsii and Schizophyllum commune
(see Rau, 2004), some in relation to their putative therapeutic
properties (e.g. Daba & Ezeronye, 2003) and in the cell wall of
U. maydis (Ruiz-Herrera et al., 2008). No report exists on the
presence or absence of b-1,6-glucans in chytridiomycetes or
microsporidians. Preliminary experiments showed their absence in the cell walls of sporangiospores (walls from their
vegetative stages have no glucose polymers) from P. blakesleeanus and Rhizopus oryzae (unpublished data).
A search for genes encoding enzymes involved in b-1,6glucan synthesis was performed in S. cerevisiae through the
isolation of mutants resistant to the killer toxin K1 (kre)
(Al-Aidroos & Bussey, 1978; Boone et al., 1990; Brown et al.,
1993; reviewed by Shahinian & Bussey, 2000), taking into
consideration that b-1,6-glucan is the receptor for this toxin
(Bussey et al., 1979; Hutchins & Bussey, 1983). The results
obtained demonstrated that the synthesis of b-1,6-glucan is
a complex process where at least 10 distinct proteins (some
with several homologues) are involved: Kre1p, Kre5p, Kre6p,
Kre9p, Kre11p, Cne1p, Cw41p/gls1p, Knh1p, Rot2p/Gls2p
and Skn1p. These proteins have different cellular locations,
and their specific functions are still controversial and
unclear. Mutation of several of them leads to reduction in
the levels of b-1,6-glucans, severe alterations in morphology
and growth and in some cases they are synthetically lethal
(see Table 2). Considerable confusion exists in the field
because an acceptable mechanism for b-1,6-glucan synthesis
is still missing, and due to the fact that to date the real b-1,6glucosyltransferase(s) involved and even the glucosyl donor
are unknown.
Homologues of all the above-described proteins have
been described in other ascomycetes (e.g. Mio et al., 1997)
and we identified them by an in silico search. On the other
hand, all the basidiomycete species analyzed lacked homologues for genes encoding Kre1p, Kre9p, Kre11p and Cne1p.
In the zygomycete and the chytridiomycete species analyzed,
in addition to these genes, homologues of KRE6 and SKN1
genes were also absent. Interestingly, E. cuniculi, the single
member of the Microsporidia phylum analyzed, did not
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234
J. Ruiz-Herrera & L. Ortiz-Castellanos
Fig. 5. Evolutionary relationships of fungal
b-1,3-glucan synthases. See details in the
legend of Fig. 1.
contain homologues of any of the enzymes putatively
involved in b-1,6-glucan synthesis (Table 3). It is interesting to recall that as mentioned above, b-1,6-glucan was
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identified in the wall of the basidiomycete U. maydis
(Ruiz-Herrera et al., 2008), suggesting that not all of the 10
proteins identified in S. cerevisiae and ascomycetes in
FEMS Yeast Res 10 (2010) 225–243
235
Fungal wall evolution
Table 2. Characteristics of the proteins described to be involved in b-1,6-glucan biosynthesis in Saccharomyces cerevisiae
Gene product
Putative function and/or characteristics
Putative location
Kre1
Kre5
Kre6
Kre9
Kre11
Cne1
Cwh41
Knh1
Rot2
Skn1
Surface protein. Involved in glucan assembly. K1 killer toxin receptor?
Similarity to UDPG: glycoprotein glucosyltransferase. Protein initial glycosylation?
Similarity to glucosyl transferases. Type II membrane protein. Homologue of Skn1
Mutations result in an aberrant morphology and growth and mating defects
Subunit of transport protein particle involved in ER to Golgi transport
Calnexin/calreticulin-like. ER membrane protein involved in protein folding control
ER glucosidase I. Involved in b-1,6 glucan assembly and N-glycolylation
Functional homologue of Kre9. Overproduction restores kre9 defects
ER glucosidase II. Involved in b-1,6 glucan assembly and N-glycosylation
Similarity to glucosyl transferases. Type II membrane protein. Homologue of Kre6
Plasma membrane, cell wall GPI protein
ER-soluble protein
Golgi membrane
Soluble extracellular
Cytoplasm
ER membrane
Endoplasmic reticulum
Soluble extracellular
Endoplasmic reticulum
Golgi membrane
Table 3. Conservation of enzymes involved in the synthesis of the mannoproteins outer chain
Number of species with homology
Phylum (species analyzed)
Och1
Mnn9
Mnn1
Mnn2
Mnn5
Ascomycota (14)
Basidiomycota (5)
Mucoromycotina (3)
Chitridiomycota (1)
Microsporidia (1)
14 (e50–e137)
2 (e16–e23)
None
e12
None
14 (e58–0)
None
3 (e36–e50)
None
None
14 (e40–e126)
None
3 (e8–e15)
e15
None
14 (e33–e75)
2 (e5–e11)
3 (e7–e12)
e15
None
14 (e30–e62)
1 (e6)
3 (e11–e15)
e12
None
In species with more than one homologue, only the one with the highest homology is shown.
general are strictly required for the synthesis of the polysaccharide. Phylogenetic analyses of the four proteins putatively involved in the synthesis of b-1,6-glucans present in all
fungal phyla revealed their separation in the fungal groups
analyzed (not shown).
Glycoproteins
All bona fide extracellular proteins are glycosylated, hence
their denomination as glycoproteins. In eukaryotic organisms, there exist two main types of binding of the carbohydrate moieties to the proteins: O-glycosylation, where a
short chain of sugars binds to serine or threonine residues,
and N-glycosylation, in which the sugar chain binds to
asparagine. The relative proportion of the carbohydrate
moiety may vary from o 10% to 4 95% of the whole
glycoprotein Mr. Glycoproteins are essential for all eukaryotes, as shown by the observation that mutations or
inhibitors that block the early steps in N- or O-glycosylation
are lethal. Protein O- or N-glycosylation initiates cotranslationally during protein synthesis in the ER, and continues
during the transit of the protein through the Golgi.
O-glycosylation involves the addition of only a limited
number of different sugars depending on the organism
(at most five mannosyl residues in fungi) (see the review by
Goto, 2007). Synthesis of the most internal moiety (the inner
core) of the N-bound oligosaccharide is universal, but further
modifications and extension of this core depend on the type
of organism. Characteristically, in fungi, the size of the
FEMS Yeast Res 10 (2010) 225–243
carbohydrate fraction of the glycoprotein may extend, mostly
by the action of mannosyl transferases, to give rise to high Mr
molecules, constituting the wall fraction known as mannan.
The main sugar present in fungal glycoproteins is mannose,
but diacetylchitobiose is universally present, forming the
bridge between the asparagine residue of the protein and
the carbohydrate fraction. Other sugars may also be present
in variable amounts, depending on the fungal species (see
reviews in Ruiz-Herrera et al., 2004; Sentandreu et al., 2004;
Jigami, 2008). The first step in protein O-glycosylation in
S. cerevisiae (the best-studied model) involves transfer of a
mannosyl residue using dolichyl-phosphate mannose as a
substrate, whereas the substrate used for transfer of the rest of
the mannosyl units is GDP mannnose. The classical studies
from Ballou (e.g. Frevert & Ballou, 1985; Ballou, 1990) with
S. cerevisiae as a model demonstrated that synthesis of the
outer chain of the mannan moiety of the glycoproteins was
catalyzed by specific mannosyl transferases, most of them
different from the ones responsible for the synthesis of the
inner core oligosaccharide. Further work has revealed that
elongation of the inner core is initiated by addition of an
a-1,6-linked mannosyl residue by the enzymatic action of
Och1p mannosyltransferase, and it is further extended by the
sequential action of different mannosyl transferases to form
an oligosaccharide of variable length made up of a-1,6-linked
mannose, with a-1,2- and a-1,3-linked mannose branches,
plus some variable modifications (for a review of the enzymes
involved, see Jigami, 2008). In S. cerevisiae, growth of the
outer chain is catalyzed by an a-1,6-mannosyl transferase
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236
encoded by the MNN9 gene, and its branching is catalyzed by
two a-1,2-mannosyl transferases: Mnn2, catalyzing the addition of the first mannosyl residue, and Mnn5, responsible for
the addition of further mannosyl residues, whereas Mnn1 is
the mannosyl transferase involved in the attachment of a-1,3bound mannosyl residues.
In this study, we proceeded to the comparison of the
genes encoding the enzymes involved in the synthesis of the
outer chain of glycoproteins (mannan) in the several fungal
groups included in our analyses using the sequences of the
whole S. cerevisiae enzymes for comparison. As expected,
homologues of all the genes encoding the above-mentioned
enzymes were found to be present in the analyzed ascomycete species with homology values between e33 and e144.
On the other hand, no homologue for any of the genes was
identified in the microsporidian E. cuniculi. Analysis of
homology in species of the rest of the phyla yielded variable
results (see Table 3).
GPI proteins
GPI proteins are glycoproteins that contain a GPI anchor,
which is added post-translationally to a motif located at their
C-terminus. The GPI anchor remains associated with the cell
membrane and its bound polypeptide protrudes into the
periplasmic space. GPI proteins are present in all eukaryotic
groups, mostly associated with the plasmalemma. De Nobel &
Lipke (1994) indicated their possible role within the fungal
cell wall, and later on a variable number of GPI proteins were
found to be covalently associated by a glycosidic linkage to b1,6-glucans in the cell wall of most of the ascomycete species
analyzed (e.g. Yin et al., 2005; Castillo et al., 2008). Regarding
the factors involved in their selective location at the plasma
membrane or the cell wall, Mao et al. (2008) obtained
evidence that a short stretch of amino acids at the C-terminus
of GPI proteins was involved in providing their specificity in
targeting to either location in C. albicans. Whether this is a
general characteristic in other fungi remains to be determined.
As would be expected, in silico analysis revealed the
presence of a number of GPI proteins in all species of the
ascomycetes, basidiomycetes, mucoromycetes and chytridiomycetes analyzed (see Ruiz-Herrera et al., 2008). In the
microsporidian E. cuniculi, at least one GPI protein extracted
from the spore wall with reducing agents has been described
(Xu et al., 2006). These data are evidence that GPI proteins are
distributed in all the fungal phyla. Nevertheless, repeated
proteomic analyses have failed to demonstrate the presence
of covalently bound GPI proteins in the cell wall of U. maydis
(J. Ruiz-Herrera & R. Sentandreu, unpublished data).
Synthesis of the GPI moiety occurs in the ER by a pathway
that is conserved in eukaryotic organisms, and independent
of the synthesis of the glycoprotein. In the final step
occurring at the plasmalemma, the characteristic C-terminal
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J. Ruiz-Herrera & L. Ortiz-Castellanos
signal of the protein is removed by a GPI transamidase and
replaced by the GPI moiety (for reviews, see Brown &
Waneck, 1992; Orlean & Menon, 2007; Pittet & Conzelmann, 2007). The transamidase complex is made up of five
components: Gaa1/GPAA, Gpi8/PigK, Gpi16/PigT, Gpi17/
PigS and Gab1/PigU (see Orlean & Menon, 2007). We
identified homologues for all the encoding genes in the
ascomycetes, basidiomycetes, zygomycetes and chytridiomycete species analyzed. The exception was the microsporidian
E. cunicili, which contained only a homologue of the Gpi8
gene (also involved in the synthesis of the Gpi moiety), a
result in agreement with its phylogenetic distance.
It has been suggested that transfer of the protein to the b1,6-glucan present in the wall occurs by a transglycosylation
reaction in which the GPI moiety is cleaved between the
mannosyl 1 and the glucosaminyl residues and transferred
to the terminal or an internal glucosyl residue of the glucan,
leaving the hydrophobic GPI residue attached to the plasmalemma. Nevertheless, the enzymes involved and the
mechanism of the reaction are still unknown (for a review,
see Orlean & Menon, 2007).
Pir proteins
Pir proteins are characterized by the presence of several
glutamine-rich repeats with the consensus sequence
Q[IV]XDGQ[IVP]Q, a signal peptide, a Kex2 cleavage site
and a C-terminal domain with four cysteine residues with
the structure-C-65/66-C-16-C-12-C. Pir proteins were initially identified in S. cerevisiae (Toh-e et al., 1993), and later
on reported to be present in a number of ascomycete species
(e.g. Martı́nez et al., 2004). It has been demonstrated that Pir
proteins play an important role in the structure and stability
of the cell wall of ascomycetes (Mrsa & Tanner, 1999;
Martı́nez et al., 2004). Pir proteins can be extracted from
the cell walls by treatment with dilute alkali at a low
temperature. The alkali-sensitive bond has been identified
as an ester linkage between the g-carboxyl group of glutamic
acid and the hydroxyl groups of glucose (Ecker et al., 2006).
Our BLAST analyses using the most conserved residue
(SQIGDGQVQATSAAT) failed to identify Pir proteins sensu
stricto in basidiomycetes or in other fungal species (RuizHerrera et al., 2008). In fact, these proteins appear to be
specific of ascomycetes in the entire living world. Nevertheless,
it must be stressed that other proteins, unrelated to Pir, and
containing different types of repeats, are present in the fungal
walls. Perhaps the best known of these are S. cerevisiae
flocculins (Flo1, Flo5, Flo9, Flo10 and Flo11), and Aga1,
Tir1, Tir4 and Dan4 (Verstrepen et al., 2005) and matingspecific yeast agglutinins (Lipke & Kurjan, 1992; Cappellaro
et al., 1994). Further examples of surface proteins containing
multiple repeats have been identified in U. maydis, among
them, repellent Rep1 and the atypical hydrophobin Hum3,
FEMS Yeast Res 10 (2010) 225–243
237
Fungal wall evolution
which confer hydrophobic properties to the cell wall of this
fungus (Teertstra et al., 2006), and Hum3 and Rsp1, proteins
involved in its pathogenic behavior (Müller et al., 2008). It is
therefore feasible that Pir-unrelated proteins containing
different repeats may be present in the walls of species
belonging to other fungal groups.
Evolutionary considerations
Modern analysis of the phylogeny and taxonomy of fungi
has revealed that the former Chytridiomycota and Zygomycota groups are not monophyletic (Lutzoni et al., 2004;
James et al., 2006; Hibbett et al., 2007). In the present
analysis, we mostly made use of data corresponding to those
fungi whose genomes have been sequenced and are available.
Because of this limitation, chytridiomycetes are represented
by a single species of the new phylum Chytridiomycota
(now separated from phyla Neocallimastigomycota and Blastocladiomycota), and zygomycetes by species belonging to
the new phylum Mucoromycotina. Also, a single species is
representative of microsporidians. On the contrary, the two
phyla belonging to the clade Dikarya, Basidiomycota and
Ascomycota, are over-represented.
Despite this possible source of criticism, we consider that
the data described here are valuable in the analysis of the two
main questions approached in this study: (1) is the cell wall
of S. cerevisiae a true paradigm of the structure of the fungal
cell wall? Or do we have to consider that the cell walls of
members of the different fungal groups have significant
differences and variations? and (2) can we obtain information on the evolution of the structure of the fungal cell wall
by means of this type of analysis?
Regarding the first question, we can conclude that the
data presented here are indicative that the use of the wall
from S. cerevisiae and related ascomycete yeasts as the
paradigm of the fungal cell wall is probably inadequate.
The only common feature of the cell walls from organisms
belonging to the several fungal phyla is their architecture,
which, as mentioned in the Introduction, depends on the
association of microfibrils of structural polysaccharides
with an amorphous material made up mainly of proteins
and other types of polysaccharides. For the second question,
it is clear that the analysis of the enzymes involved in the
synthesis of the most important polysaccharides and the
nature of the covalently linked proteins provided information that may suggest the steps occurring in the differentiation of the cell wall along the evolution of fungi.
The example of chitin synthases is illustrative of this
matter. Chitin is a polysaccharide specific of eukaryotes,
and its presence in eukaryote kingdoms (Fungi, Animalia,
Chromista and Protozoa) suggests that the appearance of
the enzymes responsible for its synthesis was an early event
in evolution, probably deriving from the ancestor of differFEMS Yeast Res 10 (2010) 225–243
ent b-glycosyl-transferases, as suggested by their common
structural features (see Ruiz-Herrera et al., 2002). Data of
the phylogenetic analysis of chitin synthases, and the relationship of division 2 Chs with chitin synthases from
members of other chitin-containing kingdoms, suggest that
this type of enzymes were the first ones to appear during
evolution. This suggestion is supported by the observation
that all fungal phyla contain enzymes belonging to this
division, whereas only ascomycetes contain all Chsp classes
from division 1, basidiomycetes lack one of them (class I)
and zygomycetes, and chytridiomycetes possess only one
(class II), whereas Microsporidia contain none. This observation is interesting, because it has been indicated that
microsporidians probably belong to the first diverging
fungal branch during fungal evolution (James et al., 2006).
Also, the absence of a class V chitin synthase in the microsporidian species analyzed may be taken as further evidence,
in addition to those discussed above, that class IV preceded
the appearance of class V chitin synthases. The possibility
that this deficiency in E. cunicili could be attributed to the
loss of a class V gene is possible, but it is not supported by
the phylogenetic analysis. Accordingly, it may be suggested
that the original class IV gene gave rise, through duplication
and modification, to a class V gene that most probably
acquired the myosin head motif, lost later on in some of the
enzymes belonging to different species.
Division 2 Chs enzymes have an average Mr larger than
division 1 Chs, and contain a large N-terminal tail without
conserved motifs (Ruiz-Herrera et al., 2002). It would not be
a wild speculation to consider that the first gene encoding a
division 1 enzyme (most probably belonging to class II CHS,
as supported by the phylogenetic analysis and the distribution of the different classes of Chs in the various fungal
phyla) also originated by gene duplication and modification,
a phenomenon amply represented in gene evolution. In the
present case, the modification would have involved deletion
of the region encoding the N-tail from a division 2 gene,
which left its catalytically important motifs untouched, and
its further variation during evolution. The later appearance
of genes encoding class III enzymes occurring only in the
common ancestor of Dikarya, and class I CHS genes only in
Ascomycota after its separation from Basidiomycota, would
have taken place by a similar duplication–modification
mechanism. As indicated above, in contrast to animals and
protists, members of the group of oomycetes (Chromista)
do not contain Chs related to division 2 fungal enzymes;
they only possess enzymes related to division 1. Two possible
explanations exist for this situation: their common ancestor
acquired the gene that gave rise to the two classes of Chs
present in the group by horizontal transfer from the
common ancestor of Chitridiomycota, Zygomycota and
Dikarya, or less likely, they lost the ancestor genes encoding
Chs related to division 2 and class III enzymes. Modern
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238
concepts on the evolution of eukaryotes strongly support
the first possibility (Cavalier-Smith, 2004). In this sense, it
is also important to recall the existence of the division
1-related CHS gene present in chloroviruses (Kawasaki
et al., 2002), which reminds us of CHS gene mobility from
fungal ancestors.
Chitin deacetylases exist in all fungal phyla, but only
zygomycetes appear to contain their product, chitosan, in all
their life-cycle forms, being, in some of them, the most
important wall component. In ascomycetes, chitosan has
been identified mostly in the sexual spores, interestingly
being synthesized from the product of enzymes belonging to
division 2 (Pammer et al., 1992), although isolated reports
have described its presence in the vegetative forms of
S. pombe and A. niger. Only a few reports have described
the presence of chitosan in the vegetative forms of some
basidiomycetes. The most thorough analyses correspond
to the pathogenic basidiomycete C. neoformans, which
contains higher amounts of chitosan than chitin in its cell
wall, interestingly also being synthesized from the product
of a division 2 enzyme (Chs3; Banks et al., 2005). It is
therefore not surprising that zygomycetes contain the highest number of genes encoding chitin deacetylases. The
presence of chitin deacetylases in all the fungal groups
analyzed, as well as in some insects and in amoeba, suggests
that their encoding genes appeared early in evolution, before
the separation of protozoa and opisthokonts, but, after their
split from the rest of the eukaryotic kingdoms, in which its
presence has not been reported. This suggestion is in line
with evolutionary data from Cavalier-Smith (2002).
Regarding the role of this apparently superfluous enzyme,
which might even be considered as deleterious because it
converts the stiff component responsible for the structure of
the cell wall into a less resistant (chemically and mechanically) component, it might be argued that the presence of a
chitosan layer in the cell wall of zygomycetes makes them
more resistant to chitinases, which are probably more
abundant in nature than chitosanases, and perhaps to some
other deleterious factors, because of its physicochemical
characteristics. Similarly, in C. neoformans, it was described
that chitosan is necessary for the integrity of the cell wall
(Baker et al., 2007) in analogy to yeast ascospores. In yeasts,
the polysaccharide is mostly restricted to the cell wall of
ascospores, and chitin deacetylases are required for its
assembly, and for providing its rigidity (Christodoulidou
et al., 1996, 1999). Mutation of the gene encoding the
deacetylase involved in chitosan formation in S. pombe
impairs ascospore formation (Matsuo et al., 2005). This
characteristic may be related to the resistance of a structure
that must remain dormant for long periods of time.
Accordingly, ascospores are more resistant than vegetative
cells to acid, alkali, ether, salt or heat stress as described by
Coluccio et al. (2008). These authors also reported that
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J. Ruiz-Herrera & L. Ortiz-Castellanos
ascospores from S. cerevisiae and S. pombe were resistant to
the passage through the digestive tract of Drosophila due to
their chitosan layer (and a polymer containing dityrosine in
S. cerevisiae), and suggested that this resistance is a mechanism for spore dispersal by means of insects that feed on
them. Accordingly, it may be hypothesized that the capacity
to turn on the gene(s) encoding chitin deacetylases during
sporulation, mostly in ascomycetes, appeared as a selective
factor during evolution.
In contrast to chitin and chitosan, b-1,3-glucans are more
abundant in the cell walls of ascomycetes and basidiomycetes than in zygomycetes, where the presence of the
polysaccharide is restricted to the sporangiospore wall. In
some chytridiomycetes, the existence of glucose-containing
polymers has been described (e.g., Sikkema & Lovett, 1984),
although their chemical structure has not been analyzed.
Because in silico analysis did not reveal the presence of a
homologue of a b-1,3-glucan synthase in B. dendrobatidis,
it may be suggested that the polysaccharide detected in other
chytridiomycete species is probably not b-1,3-glucan. This
result and the absence of b-1,3-glucan synthase also in
E. cuniculi suggest an interesting possibility of the presence
of the enzyme only in zygomycetes, ascomycetes and basidiomycetes.
Fungal b-1,3-glucan synthases, the same as those from
plants, algae and members of the kingdom Chromista, have
significant structural differences from the rest of the glycosyltransferases: they lack the characteristic conserved amino
acids (three Asp residues and the QXXRW motif), associated
with the catalytic activity of the rest of the b-glycosyl
transferase families including cellulose and chitin synthases
(Campbell et al., 1997; reviewed by Douglas, 2001), and the
UDP glucose-binding site (R/K) XGG from glycogen
synthases (Farkas et al., 1990). These characteristics and the
absence of b-1,3-glucan synthases in the rest of the opisthokont species make unlikely the possibility that acquisition of
these enzymes by fungi occurred by a parallel and independent process. It is more feasible that after separation of
microsporidians and chytridiomycetes from the fungal
evolutionary line, a gene encoding b-1,3-glucan synthase
from a precursor of the Plantae or Chromista kingdoms was
captured by the common ancestor of the rest of the fungal
phyla through a horizontal transfer process. As with chitosan in ascomycetes, the capacity to synthesize b-1,3glucans remained restricted to spores in zygomycetes as a
relic process of probable ecological advantage. On the other
hand, the polymer became an important component of the
cell wall in the Dikarya species. Knowledge of the factors
involved in the regulation of chitosan and glucan synthesis,
respectively, in ascomycetes or zygomycetes, would undoubtedly provide important information on the prevailing
ecological conditions under which the antecessors of these
fungi survived.
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Fungal wall evolution
As indicated previously, the presence of b-1,6-glucans in
opisthokonts is restricted to fungi, and its mechanism of
synthesis is complicated and has not been clarified, apparently involving at least 10 proteins in the process carried out
by ascomycetes (see the review by Shahinian & Bussey, 2000).
Because homologues of only six of these proteins (Kre5,
Kre6, Cne1, Cwh41/Gls1, Rot2/Gls2 and Skn1) are present in
basidiomycetes, it may be suggested either that the rest of the
proteins have other functions only indirectly related to
synthesis of the polysaccharide or that their roles are
redundant. The observation that mutation in ascomycetes
of some of the genes absent in basidiomycetes leads to an
aberrant morphology and growth defects (not observed of
course in normal basidiomycetes) suggests that the first
possibility is more likely. As indicated above, zygomycetes
and chytridiomycetes lack, in addition Kre6 and Skn1,
proteins with similarity to glycosyl transferases, whereas
Microsporidia lack homologues to all of them. These results
suggest that those two proteins might be indispensable for b1,6-glucan synthesis, and their absence would explain why
the polysaccharide is not present in these fungal phyla.
According to these data, it is likely that acquisition of the
capacity to synthesize this polysaccharide occurred in a
common ancestor of Dikarya after departure of zygomycetes
from the fungal evolution line. As already mentioned, b-1,6glucans are also present in chromista, and absent in plants
and the rest of the Opisthokont clade. Whether appearance
of the genes encoding the hypothetical catalytic peptide(s)
for b-1,6-glucan synthesis (homologues of KRE6 and SKN1?)
in Fungi and Chromista kingdoms was due to separate and
independent processes, or once present in one group they
were transferred to the precursor of the other one by a
horizontal transfer mechanism (in what direction?) as suggested above for b-1,3-glucans, remains to be determined.
Regarding the synthesis of mannans, the outer chain of
mannoproteins, it is apparent that drastic changes occurred
during evolution of the genes encoding the enzymes involved, as revealed by the variable degrees of homology
among the corresponding genes of the several fungal phyla.
These changes most probably gave rise to alterations in the
structure of the main types of outer chains from the
glycoproteins present in several fungal groups, and show
the differences existing with the ascomycete model, best
represented by S. cerevisiae.
The presence of covalently bound Pir and GPI proteins in
the walls of ascomycetes is an interesting feature. The
acquisition of the capacity to synthesize proteins that are
linked to the polysaccharide mesh of the cell wall through
b-1,6-glucans or b-1,3-glucans, respectively, undoubtedly
represented a landmark for a change in the organization of
the cell wall. The existence of Pir proteins in other fungi
besides ascomycetes has already been clearly discarded, but
as indicated, GPI proteins are present in all the fungal
FEMS Yeast Res 10 (2010) 225–243
groups, bound to the cell membrane through the GPI
moiety. Whether GPI proteins covalently bound to b-1,6glucans exist in fungi other than ascomycetes remains
unknown. It is unfortunate that, with the exception of U.
maydis, no analyses have been performed to detect the
presence of GPI proteins covalently bound to the cell wall
in basidiomycetes, zygomycetes, chytridiomycetes or microsporidians. Nevertheless, the absence of b-1,6-glucans in the
last three phyla makes the existence of the typical ascomycete cell wall-bound GPI proteins impossible. Identification
of the enzymes involved in the covalent attachment of GPI
proteins to b-1,6-glucans would be the ideal tool to determine by in silico techniques as to whether they exist in other
fungi besides ascomycetes.
Based on the data and ideas described above, tentatively,
we may schematically represent the evolution of the organization of the fungal cell wall as illustrated in Fig. 6.
According to this hypothetical scheme, the first step in the
development of the eukaryotic cell wall would have been the
acquisition of the capacity to synthesize microfibrillar polysaccharides, chitin in the case of the common ancestor of the
opisthokonts, by chitin synthases most probably related to
modern class IV enzymes. This feature would allow the
formation of an exoskeleton that permitted cell growth in
hypotonic media and retention of noncovalently bound
important proteins, some with enzymatic activity, in the
periplasmic space. This would have been followed by the
appearance of a more plastic wall component (chitosan) by
the activity of chitin deacetylases (it is important to recall
that these enzymes are also present in protozoa and animals). Once fungi separated from the other Opisthokont
phyla, duplication and modification may have occurred of
the gene encoding the class IV enzyme ancestor to give rise
to genes coding for class V chitin synthases. Next, when
Microsporidia departed from the fungal evolutionary line,
the appearance of class II CHS genes possibly, as discussed
above, through duplication and modification of a gene
encoding a division 2 enzyme, might have taken place. After
departure of chytridiomycetes from the main fungal evolutionary line, the common ancestor of the rest of the fungal
phyla would have captured a gene encoding b-1,3-glucan
synthase, and not just after separation of the phylum
Zygomycota that genes encoding class III Chs would have
evolved probably by the duplication and modification most
likely of a class II CHS gene. Also around this time genes
encoding catalytic enzymes involved in b-1,6-glucan synthesis would have appeared or would have been acquired by
horizontal transfer, as suggested above. Finally, after the split
of the Ascomycota and Basidiomycota phyla, the ancestor of
the former would have developed the capacity to covalently
bind exocellular proteins to b-glucans, and acquired class I
CHS genes possibly by the same mechanism as that suggested for the origin of class II and class III CHS genes.
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240
J. Ruiz-Herrera & L. Ortiz-Castellanos
Basidiomycota
Ascomycota
Multiple β1,3-GS
Covalent
Proteins
Multiple Cda
CHS I
?
β1,6-GS
CHS III
Zygomycota
Multiple Cda
Chytridiomycota
β1,3-GS
Other
Opisthokonts
CHS V
?
CHS II
Microsporidia
Fungi
Cda
CHS IV
Common Ancestor
In summary, our data point out that cell walls from the
different taxonomical groups of fungi have distinct features
that characterize them. No general model of the fungal cell
walls can be defined, because their structure in the fungal
phyla is a recapitulation of the evolutionary history of the
different compounds that make them up. This history
involves: (1) evolution of polysaccharide synthases, (2)
horizontal gene transfer of synthesizing enzymes, (3) development of systems for the selective regulation of specific
synthases during the life cycles of fungi, (4) important
changes leading to modification of the synthesized products
and (5) the capacity to associate different products through
covalent linkages, in order to give rise to a coherent
structure. Most importantly, although chemically and structurally the fungal cell wall may be different in the several
taxonomic groups, it faithfully fulfills its important functions in all of them.
Acknowledgements
This work was partially supported by CONACYT, Mexico
and by CONCYTEG from the State of Guanajuato, Mexico.
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