Role of three chitin synthase genes in the growth of Candida albicans.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

Double chitin synthetase mutants from the corn smut fungus Ustilago maydis

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize ( Zea mays ) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3 / chs4 , and chs4 / chs5 . Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO²⁺, and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.     

Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.     

Genetic analysis of chs1+ and chs2+ encoding chitin synthases from Schizosaccharomyces pombe

To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database. The synthetic lethal phenotype of the S. cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S. pombe chs1+ or chs1+, indicating that both chs1+ and chs2+ in fact encode chitin synthase. The homothallic Deltachs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Deltachs2 strain had no noticeable phenotype. The chs1 chs2 double disruptant looked similar phenotypically to the Deltachs1 strain. The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth. The level of chs2+ mRNA increased just before the septum was formed. Levels of Chs2-13Myc synthesis also changed during the cell cycle. Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation.     

CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.     

Proper ascospore maturation requires the chs1+ chitin synthase gene in Schizosaccharomyces pombe

We have cloned chs1+, a Schizosaccharomyces pombe gene with similarity to class II chitin synthases, and have shown that it is responsible for chitin synthase activity present in cell extracts from this organism. Analysis of this activity reveals that it behaves like chitin synthases from other fungi, although with specific biochemical characteristics. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth. In contrast, chs1+ expression increases significantly during sporulation, and this is accompanied by an increase in chitin synthase activity. In addition, spore formation is severely affected when both parental strains carry a chs1 deletion, as a result of a defect in the synthesis of the ascospore cell wall. Finally, we show that wild-type, but not chs1-/chs1-, ascospore cell walls bind wheatgerm agglutinin. Our results clearly suggest the existence of a relationship between chs1+, chitin synthesis and ascospore maturation in S. pombe.     

Differential cell wall remodeling of two chitin synthase deletants Δchs3A and Δchs3B in the pathogenic yeast Candida glabrata

It is known that cell wall remodeling and the salvaging pathway act to compensate for an impaired or a damaged cell wall. Lately, it has been indicated that this mechanism is partly required for resistance to the glucan synthesis inhibitor echinocandin. While cell wall remodeling has been described in mutants of glucan or mannan synthesis, it has not yet been reported in a chitin synthesis mutant. Here, we describe a novel cell wall remodeling and salvaging pathway in chitin synthesis mutants, Δchs3A and Δchs3B, of the pathogenic yeast Candida glabrata. Electron microscopic analysis revealed a thickened mannoprotein layer in Δchs3A cells and a thickened chitin-glucan layer of Δchs3B cells, and it indicated the hypothesis that mannan synthase and chitin-glucan synthase indemnify Δchs3A and Δchs3B cells, respectively. The double-mutant CHS3A and MNN10, encoding α-1,6-mannosyltransferase, showed synergistic stress sensitization, and the Δchs3B strain showed supersensitivity to echinocandins. Hence, these findings support the above hypothesis of remodeling. Furthermore, unlike Δchs3A cells, Δchs3B cells showed supersensitivity to calcineurin inhibitor FK506 and Tor1p kinase inhibitor rapamycin, indicating that the Δchs3B strain uses the calcineurin pathway and a Tor1p kinase for cell wall remodeling.     

Chitin synthase III activity,but not the chitin ring,is required for remedial septa formation in budding yeast

Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall. In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential. However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa. We examined which of two CSIII functions, the formation of a chitin ring at bud emergence or of chitin in the remedial septa, was required for viability. By using cell cycle synchronization in combination with nikkomycin Z, a specific inhibitor of CSIII, we inhibited chitin synthesis in a chs2 mutant, during formation of either the ring or the remedial septa. The results show that only synthesis of the chitin during aberrant septa formation is essential for viability. Thus, the unique function of the chitin ring seems to be maintenance of the integrity of the mother-bud neck, as we recently found, and the importance of chitin in septum closure, both in normal and abnormal situations, is underlined.     

Selection in favor of nucleotides G and C diversifies evolution rates and levels of polymorphism at mammalian synonymous sites

The impact of synonymous nucleotide substitutions on fitness in mammals remains controversial. Despite some indications of selective constraint, synonymous sites are often assumed to be neutral, and the rate of their evolution is used as a proxy for mutation rate. We subdivide all sites into four classes in terms of the mutable CpG context, nonCpG, postC, preG, and postCpreG, and compare four-fold synonymous sites and intron sites residing outside transposable elements. The distribution of the rate of evolution across all synonymous sites is trimodal. Rate of evolution at nonCpG synonymous sites, not preceded by C and not followed by G, is approximately 10% below that at such intron sites. In contrast, rate of evolution at postCpreG synonymous sites is approximately 30% above that at such intron sites. Finally, synonymous and intron postC and preG sites evolve at similar rates. The relationship between the levels of polymorphism at the corresponding synonymous and intron sites is very similar to that between their rates of evolution. Within every class, synonymous sites are occupied by G or C much more often than intron sites, whose nucleotide composition is consistent with neutral mutation-drift equilibrium. These patterns suggest that synonymous sites are under weak selection in favor of G and C, with the average coefficient s approximately 0.25/Ne approximately 10(-5), where Ne is the effective population size. Such selection decelerates evolution and reduces variability at sites with symmetric mutation, but has the opposite effects at sites where the favored nucleotides are more mutable. The amino-acid composition of proteins dictates that many synonymous sites are CpGprone, which causes them, on average, to evolve faster and to be more polymorphic than intron sites. An average genotype carries approximately 10(7) suboptimal nucleotides at synonymous sites, implying synergistic epistasis in selection against them.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.     

Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae

Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P. W. (1986) Cell 46, 213-225). We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells. Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains. Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis. They differ sharply, however, in divalent cation specificity and in pH optimum. Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts. As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes. This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place. Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Mutation-selection models of codon substitution and their use to estimate selective strengths on codon usage

Current models of codon substitution are formulated at the levels of nucleotide substitution and do not explicitly consider the separate effects of mutation and selection. They are thus incapable of inferring whether mutation or selection is responsible for evolution at silent sites. Here we implement a few population genetics models of codon substitution that explicitly consider mutation bias and natural selection at the DNA level. Selection on codon usage is modeled by introducing codon-fitness parameters, which together with mutation-bias parameters, predict optimal codon frequencies for the gene. The selective pressure may be for translational efficiency and accuracy or for fine-tuning translational kinetics to produce correct protein folding. We apply the models to compare mitochondrial and nuclear genes from several mammalian species. Model assumptions concerning codon usage are found to affect the estimation of sequence distances (such as the synonymous rate d(S), the nonsynonymous rate d(N), and the rate at the 4-fold degenerate sites d(4)), as found in previous studies, but the new models produced very similar estimates to some old ones. We also develop a likelihood ratio test to examine the null hypothesis that codon usage is due to mutation bias alone, not influenced by natural selection. Application of the test to the mammalian data led to rejection of the null hypothesis in most genes, suggesting that natural selection may be a driving force in the evolution of synonymous codon usage in mammals. Estimates of selection coefficients nevertheless suggest that selection on codon usage is weak and most mutations are nearly neutral. The sensitivity of the analysis on the assumed mutation model is discussed.     

Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth

Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.     

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.     

Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast

How cell cycle machinery regulates extracellular matrix (ECM) remodeling during cytokinesis remains poorly understood. In the budding yeast Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. Here, we report that Dbf2, a conserved mitotic exit kinase, localizes to the division site after Chs2 and directly phosphorylates Chs2 on several residues, including Ser-217. Both phosphodeficient (chs2-S217A) and phosphomimic (chs2-S217D) mutations cause defects in cytokinesis, suggesting that dynamic phosphorylation-dephosphorylation of Ser-217 is critical for Chs2 function. It is striking that Chs2-S217A constricts asymmetrically with the actomyosin ring (AMR), whereas Chs2-S217D displays little or no constriction and remains highly mobile at the division site. These data suggest that Chs2 phosphorylation by Dbf2 triggers its dissociation from the AMR during the late stage of cytokinesis. Of interest, both chs2-S217A and chs2-S217D mutants are robustly suppressed by increased dosage of Cyk3, a cytokinesis protein that displays Dbf2-dependent localization and also stimulates Chs2-mediated chitin synthesis. Thus Dbf2 regulates PS formation through at least two independent pathways: direct phosphorylation and Cyk3-mediated activation of Chs2. Our study establishes a mechanism for direct cell cycle control of ECM remodeling during cytokinesis.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.     

Evidence of selection on silent site base composition in mammals: potential implications for the evolution of isochores and junk DNA.

It has been suggested that mutation bias is the major determinant of base composition bias at synonymous, intron, and flanking DNA sites in mammals. Here I test this hypothesis using population genetic data from the major histocompatibility genes of several mammalian species. The results of two tests are inconsistent with the mutation hypothesis in coding, noncoding, CpG-island, and non-CpG-island DNA, but are consistent with selection or biased gene conversion. It is argued that biased gene conversion is unlikely to affect silent site base composition in mammals. The results therefore suggest that selection is acting upon silent site G + C content. This may have broad implications, since silent site base composition reflects large-scale variation in G + C content along mammalian chromosomes. The results therefore suggest that selection may be acting upon the base composition of isochores and large sections of junk DNA.