Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase

Sudoh, Masayuki; Watanabe, Miyuki; Mio, Toshiyuki; Nagahashi, Shigehisa; Yamada-Okabe, Hisafumi; Takagi, Masamichi; Arisawa, Mikio

doi:10.1099/13500872-141-10-2673

Cited by 13 publications

(6 citation statements)

References 19 publications

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“…Therefore, CaCHS1 is the first example of an essential chitin synthase gene. Although the variant gene CaCHS1A was able to complement the abnormal morphology of the S. cerevisiae Δ chs2 mutant (Sudoh et al ., 1995), the phenotype of the C. albicans conditional chs1 mutant was distinctly more severe than that of the S. cerevisiae chs2 mutation. This may reflect our observations that, in C. albicans , this enzyme is required for both septum formation and integrity of the lateral cell wall.…”

Section: Discussionmentioning

confidence: 99%

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

Munro

Winter²,

Buchan

et al. 2001

Molecular Microbiology

141

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CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura‐blaster protocol, then placing the remaining allele under the control of the maltose‐inducible, glucose‐repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Δchs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon‐like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.

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Section: Discussionmentioning

confidence: 99%

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

Munro

Winter²,

Buchan

et al. 2001

Molecular Microbiology

141

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“…Preliminary data from cloning and sequencing of an FKS homolog from C. neoformans suggests that this gene product is also remarkably conserved (23). Unlike genes of the FKS family, genes encoding proteins involved in chitin biosynthesis (CHS genes) are significantly divergent across different fungal genera (7), and three isozymes from C. albicans (CaChs1p, CaChs2p, and CaChs3p) are significantly different from one another (7,19,38,44,45).…”

Section: Discussionmentioning

confidence: 99%

“…The CHS2 (19) and CHS3 (44) genes play a role in chitin synthesis; there is evidence that CHS3 plays an important role in virulence in animal models of infection (8). A third gene (CHS1) has been identified, but its role remains unclear (38,45). Mutants in the C. albicans PHR1 gene are unable to conduct apical growth of either the yeast or hyphal growth form at alkaline pH.…”

Section: Discussionmentioning

confidence: 99%

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors

Douglas

D'Ippolito

Shei

et al. 1997

Antimicrob Agents Chemother

305

167

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Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

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“…Complementation analysis of the YpGALchsB plasmid and chs2 ‐deficient S. cerevisiae 4992H combination was tested. In contrast to Candida CHS1A [21] encoding a class II chitin synthase, A. nidulans ChsB could not rescue the abnormal morphology (unpublished data). Therefore, the evolutionary diversity of chitin synthase genes might be reflected in the failure of functional complementation.…”

Section: Resultsmentioning

confidence: 98%

Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth1

Tatsuno¹,

Yamada-Okabe²,

Takagi

et al. 2006

FEMS Microbiology Letters

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A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2+ ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 microM, and the K(m) for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2+ is used. These characteristics are the same as those of other chitin synthases.

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Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase

Cited by 13 publications

References 19 publications

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors

Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth1

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