What are the origins of fungal cell walls?
The cell surface is at the front line of species adaptation. While the cell wall needs to maintain a basic functionality like other organelles, it is expected to evolve more quickly because it is directly exposed to biotic and abiotic selective forces. Cell surface structures are a defining feature of some of the deepest evolutionary lineages, including bacteria, archea, fungi, plants, and animals. Indeed, innovations in cell surface structures may be responsible for the success and persistence of major evolutionary lineages (5
). One way to test for a causal relation between the evolution of the cell wall and the cell as a whole is to study the evolutionary conservation of cell wall macromolecular components. For example, a key evolutionary innovation in a lineage should be conserved and shared by all descendants, without repeated gain and loss.
Cavalier-Smith has argued that fungal cell walls are derived from ancestral chitinous walls of desiccation-resistant cysts in many protist clades (7
). Certainly, chitinous cysts and spores are present in other eukaryote kingdoms, including Amoebozoa
(such as Entamoeba
]), and Excavata
]). In addition, S. cerevisiae
walls share extracellular disulfide cross-links and GPI-anchored cell adhesion molecules with Amoebozoa
, perhaps the earliest-diverged eukaryotes (Fig. ) (7
). Cavalier-Smith's hypothesis motivated our use of genomics to identify genes that are key to wall biogenesis and structure and to test which are conserved and which are subject to adaptive selection.
The deep roots for the ascomycete-basidiomycete split show large amino acid distances among all homologs in the fungi (25
) (Fig. ). This observation implies that cell wall genes could also have diverged broadly. Therefore, the identification of homologs in some cell wall proteins among distantly related organisms implies greater-than-average sequence conservation. In other words, anciently diverged fungal clades should retain sequence similarity only in the most conserved of the ancestral cell wall genes. The sequence comparisons summarized in this study demonstrate precisely that.
Prevalence of cell wall-related sequences in fungi.
The evolutionary distance between two fungal species is correlated with the divergence of their cell wall proteins (Fig. ). The Saccharomyces sensu stricto species have homologs for at least 166 of the 187 S. cerevisiae cell wall-related ORFs, with an amino acid identity of at least 86%. The number of conserved wall genes decreases in more distant fungal clades. Saccharomyces sensu lato yeasts have homologs for at least 70% of the S. cerevisiae cell wall-related proteins; filamentous ascomycetes had fewer. Among the homologs not found in filamentous ascomycetes were genes encoding extracellular proteins, including cell wall structural proteins, adhesins, and extracellular members of the GO classification “cell wall organization and biosynthesis.” Also not conserved between yeast and filamentous ascomycetes were many of the proteins involved in the cell wall stress response pathway.
The two basidiomycete genomes in our study had homologs for 68 of the S. cerevisiae
putative cell wall proteins (36%). The intracellular components were mostly conserved, except for the cell wall stress response pathway components. Among the cell wall-localized proteins, all but six of the glycosylases were conserved, as were lipases and proteases. Twelve GPI-anchored proteins from S. cerevisiae
had basidiomycete homologs. The large number of homologs to the Y. lipolytica
GPI proteins supports this concept of conservation of GPI-anchored proteins, as previously noted in genomic analyses of several fungi (12
). Thus, basidiomycetes share with S. cerevisiae
intracellular and extracellular activities that are critical for biosynthesis and assembly of fungal cell walls.
In the search of the nonredundant NCBI database, almost every major fungal phylum contained homologs of some S. cerevisiae cell wall proteins (Fig. ). (The single exception, Glomeromycota, is probably due to the small number of sequences in GenBank.) Thus, homologs of some of the S. cerevisiae cell wall-related proteins are likely to be present throughout the fungi.
Differences in divergence of functional classes of cell wall-related proteins.
A major finding of this analysis is that that the degree of conservation among cell wall proteins is strongly correlated with the cellular roles of those proteins (Fig. ). Many of the proteins that reside in S. cerevisiae
cell walls have diverged to the extent that homologs were not recognized in organisms outside of the Saccharomyces
sensu lato group. The least-conserved include the three Fit iron transport proteins and the largest class, homologs of unannotated S. cerevisiae
cell wall ORFs. Homologs of unannotated sequences were often found only in the sensu stricto group (Fig. ; see also Table S1 in the supplemental material). These poorly conserved proteins probably have important (45
) but noncatalytic roles in walls.
Among the adhesins, most have homologs in the Saccharomyces
sensu lato group but not in more-distant ascomycetes (Fig. ; see also Table S1 in the supplemental material). There is anecdotal evidence that adhesins are poorly conserved, because they are subject to sexual isolation and to diversifying selection for adaptation to different environments (16
). The structural cell wall proteins (in the CWP
, and DAN
families) are also poorly conserved. This observation may reflect the idea that this class is rich in proteins with very-low-complexity compositions. Within each sequence, only short segments are under selective pressure to retain sequence: the secretion and GPI signals and the relatively short glycosylation and transglycosylation sites (9
). The majority of each sequence has low complexity and would have evolved faster than other parts of the genome (49
Two protein classes show intermediate levels of conservation: the cell wall stress response pathways and cell wall biogenesis pathways. Their intermediate mean conservation scores result from their composition, a mixture of poorly conserved and highly conserved proteins. In general, those proteins resident in the walls are poorly conserved. Within these two classes, the plasma membrane and intracellular proteins are a mixture of conserved and nonconserved sequences (see Table S1 in the supplemental material).
Strongly conserved proteins.
In contrast, the strongly conserved functional classes are key biosynthetic and processing enzymes that must be useful in wall biosynthesis and assembly. These include the glycosylases, GPI synthetic enzymes, proteases, and lipases. These proteins include many orthologs that may function in homologous roles in wall biogenesis across the fungi. Among the most highly conserved are three sets of partially redundant glycosylases: the Chs chitin synthases, the Gas transglycosidases, and the Fks β-1,3-glucan synthases. Other highly conserved components were peptidases, phosphatases, lipases, and enzymes of the N-acetylglucosamine synthesis pathway (see Table S1 in the supplemental material).
It is not surprising that chaperone sequences and triose phosphate dehydrogenases (in the metabolic enzyme class) are strongly conserved, because these are genes whose sequences are highly conserved across the biome (3
). Their primary locations are intracellular. Although their role as cell wall proteins is less well known and somewhat controversial, it is well documented in two ascomycetous yeasts, S. cerevisiae
and C. albicans
). Given their controversial or auxiliary role in cell walls and their dual roles as wall resident and key metabolic activities, these classes could be excluded from our analysis (“chaperones” and “metabolism” in Fig. and ). That exclusion would in fact strengthen our key observation, that wall biosynthetic genes generally have conserved sequences and wall resident proteins do not.
Modes of sequence divergence in cell wall components.
In contrast to the homology of cell wall biosynthetic pathways, the actual composition of cell walls is lineage specific. This dichotomy is similar to the distinction in genomics between conserved “housekeeping” genes and a faster-evolving set of “accessory” genes (23
). Two evolutionary mechanisms could give rise to such diversity and lineage specificity in the “accessory-like” genes: either rapid sequence divergence or frequent loss and substitution of the proteins. In the first evolutionary scenario, a homologous core set of cell wall proteins exists, but they are conserved only in protein structure and function, not in sequence. For instance, the greatest mean amino acid distance between genomes of Saccharomyces
sensu stricto species is 16% (Fig. ). The S. cerevisiae
wall proteins generally have homologs within the sensu stricto group, and so the sequences conserved in this clade must have been conserved within the timescale corresponding to this difference. Rapid evolution would make homology unrecognizable in more distant groups. This result is consistent with the observation that many of these sequences are rich in low-complexity sequences, which are evolutionarily less constrained (49
). In such a case, proteins resident in fungal cell walls may have a single origin but have diverged among different fungal lineages, through either neutral or adaptive divergence.
In the second evolutionary scenario, fungal cell wall architecture is conserved as a whole, with frequent additions and deletions of specific macromolecular components during lineage diversification. In this case, fungal cell walls are more analogous to a cell organelle that has evolved independently among lineages with few underlying homologous components. Functional features of fungal cell walls have been maintained by natural selection, similar to the multiple emergences of fins among different vertebrate groups. These fins show functional homology and anatomic convergence despite their multiple origins. Possible fungal examples might be the relocalization of novel proteins to the wall following acquisition of secretion signals and GPI anchor sequences or the internal repeats that include Gln residues to be esterified to wall glucans (17
). Either of these additions could happen by recombination or by insertion of foreign DNA (53
Whether the cause is a rapid sequence divergence or frequent loss and substitution, it appears beyond dispute that fungal cell wall components evolve faster than do core metabolic protein sequences (Fig. ). Fast evolution in a large number of cell wall-related ORFs may be driven by adaptive divergence. Unlike other cell organelles, cell walls in fungi are in contact with the external environment, play a direct role in cell adaptation, and must have evolved as highly specialized and dynamic structures for colonization, host immune system evasion, signal transduction, transport, and structural maintenance. Fungi have continuously put these structures to the test through natural selection, and the selection continues. Cell wall proteins that are harmful or neutral to the cell can be mutated or removed from the genome. As a result, the organism loses a maladaptive factor and gains efficiency in its growth and replication rates. Given that natural selection favors improved fitness and that the organism can gain two benefits from a single such event, the likelihood of selection for such mutations and deletions increases.
Conservation of cell wall biogenesis.
Several classes of fungal cell wall-related proteins are common to eukaryotes: glycosylases, proteases, proteins needed for GPI synthesis, and the poorly characterized Pry proteins, as well as chaperones. The presence of these classes throughout the fungi and in several eukaryote kingdoms (Fig. ) implies that they are ancestral. These genes represent a conserved set of metabolic activities that were coordinated in the formation of cell walls. Many of the conserved glycosylases have plant homologs with annotations that show roles in plant cell wall biogenesis. These proteins include glucosidases Dse4, Spr1, and Exg1, mannosidases involved in glycoprotein biogenesis (Mns1 and Mnl1), and the Fks β-1,3-glucan synthase subunits. The roles in plant wall biogenesis include synthesis and processing of callose (β-1,3-glucan), cellulose, and glycoproteins.
The enzymes of chitin metabolism are extremely highly conserved. Two enzymes in the biosynthetic pathway for N
-acetylglucosamine (Gfa1 and YMR84w) are conserved in ascomycetes and basidiomycetes, as are chitin deacetylases (Cda1 and Cda2) (see Table S1 in the supplemental material). Fungal chitin synthases (Chs) are homologous to those in other fungi, metazoa, amoebozoa, and chromalveolata (Fig. ). This high degree of conservation is consistent with an ancestral and continuing role of chitin in cell walls of many eukaryotes (4
Given the conservation of wall biosynthetic sequences, the broad distribution of specific wall glycoconjugates, and the similarity in roles of plant and fungal glycosylases, it is reasonable to infer that a common ancestor to fungi and other eukaryotes was an organism with an extensive carbohydrate chemistry repertoire. The commonalities identified here suggest that each eukaryotic lineage has conserved the mechanism to generate diverse extracellular structures but has altered the products of the mechanism in their ancestor to form cell walls (in plants) or some other extracellular matrices (in metazoans). This conserved mechanism uses GPI synthesis and processing enzymes, carbohydrate-processing enzymes, and chaperones to enable the localization and proper folding of the proteins (3
In summary, genes that encode the proteins that make up fungal cell walls have evolved so fast that their homology is often not recognized, even within the Saccharomyces sensu lato group. This rapid divergence appears to be driven by the great diversity of strong selective pressures on cell interactions, including mating, colony and biofilm formation, pathogenesis, and immune escape. On the other hand, there is a conserved core of sequences that are involved in wall biogenesis throughout the fungi and in other eukaryote kingdoms. The conserved metabolic capabilities in Fig. are apparently ancestral. Their conservation implies that the ability to organize a wall may predate the divergence of the plants from the opisthokonts, including the fungi.