WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37°C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Δ mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell–daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2Δ strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25°C and was not viable at 37°C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.     

Cytolocalization of the class V chitin synthase in the yeast, hyphal and sclerotic morphotypes of Wangiella (Exophiala) dermatitidis

Wangiella (Exophiala) dermatitidis is a polymorphic fungus that produces polarized yeast and hyphae, as well as a number of non-polarized sclerotic morphotypes. The phenotypic malleability of this agent of human phaeohyphomycosis allows detailed study of its biology, virulence and the regulatory mechanisms responsible for the transitions among the morphotypes. Our prior studies have demonstrated the existence of seven chitin synthase structural genes in W. dermatitidis, each of which encodes an isoenzyme of a different class. Among them, the class V chitin synthase (WdChs5p) is most unique in terms of protein structure, because it has an N-terminal myosin motor-like domain with a P-loop (MMD) fused to its C-terminal chitin synthase catalytic domain (CSCD). However, the exact role played by WdChs5p in the different morphotypes remains undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at 37 degrees C and consequently virulence. This report describes the expression in Escherichia coli of a 12kDa polypeptide (WdMyo12p) of WdChs5p, which was used to raise in rabbits a polyclonal antibody that recognized exclusively its MMD region. Results from the use of the antibody in immunocytolocalization studies supported our previous findings that WdChs5p is critically important at infection temperatures for maintaining the cell wall integrity of developing yeast buds, elongating tips of hyphae, and random sites of expansion in sclerotic forms. The results also suggested that WdChs5p localizes to the regions of cell wall growth in an actin-dependent fashion.     

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.     

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.     

A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30 degrees C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37 degrees C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Delta and the csr2Delta mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.     

Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature.

Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S(1)37C1 was isolated at 37.5 degrees C, S(1)38C5 was isolated at 38.5 degrees C, and S(1)39C6 and S(1)39C10 were isolated at 39.5 degrees C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S(1)37C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S(1)37C1 mutant to the S(1)39 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S(1)37C1 mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S(1)38C5 had these mutations plus another mutation in the 3D polymerase gene. The S(1)39 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S(1)38C5 and S(1)39 mutants from S(1)37C1, is probably responsible for the high neurovirulence of S(1)38C5 and S(1)39 mutants. The capsid region mutations may contribute to the neurovirulence of the S(1)39 mutants, which was the highest among the mutants.     

UGD1, encoding the Cryptococcus neoformans UDP-glucose dehydrogenase, is essential for growth at 37 degrees C and for capsule biosynthesis

We report the identification and disruption of the Cryptococcus neoformans var. grubii UGD1 gene encoding the UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Deletion of UGD1 led to modifications in the cell wall, as revealed by changes in the sensitivity of ugd1Delta cells to sodium dodecyl sulfate, NaCl, and sorbitol. Moreover, two of the yeast's major virulence factors-capsule biosynthesis and the ability to grow at 37 degrees C-were impaired in ugd1Delta strains. These results suggest that the UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C. neoformans. Consequently, deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDP-xylose. To differentiate the phenotype(s) associated with the UDP-glucuronic acid defect alone from those linked to the UDP-xylose defect, ugd1Delta mutants were phenotypically compared to strains from which the gene encoding UDP-xylose synthase (i.e., that required for synthesis of UDP-xylose) had been deleted. Finally, studies of strains from which one of the four CAP genes (CAP10, CAP59, CAP60, or CAP64) had been deleted revealed common cell wall phenotypes associated with the acapsular state.     

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.     

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.     

THTA, a thermotolerance gene of Aspergillus fumigatus

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

Temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.     

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.