Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition

Sudoh, Masayuki; Nagahashi, Shigehisa; Doi, Mikiharu; Ohta, Akio; Takagi, Masamichi; Arisawa, Mikio

doi:10.1007/bf00284688

Cited by 69 publications

(53 citation statements)

References 29 publications

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“…The differential expression of PbCHS genes in liquid cultures even within a class indicates that distinct regulatory mechanisms might operate on individual genes. Transcripts of 3.2-kb in class II and 4.7-kb in class IV observed in our Northern blots could be consistent with a single polypeptide containing the CHS function, as judged by comparison to full-length sequence data available in literature (Motoyama et al, 1994;Specht et al, 1996;Sudoh et al, 1993). However some explanation may be needed for the 8.2-kb mRNA in class IV.…”

Section: Discussionsupporting

confidence: 83%

Multiple genes for chitin synthase in the zygomycete fungus Phycomyces blakesleeanus.

Miyazaki

Ootaki

1997

J. Gen. Appl. Microbiol.

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Section: Discussionsupporting

confidence: 83%

Multiple genes for chitin synthase in the zygomycete fungus Phycomyces blakesleeanus.

Miyazaki

Ootaki

1997

J. Gen. Appl. Microbiol.

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“…Although the degree of similarity is relatively low, other experimental evidence obtained from studies of S. cerevisiae suggests that CHS3 encodes the catalytic subunit of CSIII (review in [1Ϫ3]). Recently, ScCHS3 homologs in other fungal species have been described such as YlCHS3 (Roncero, C., unpublished), AnCHSD [27], CaCHS3 [28], NcCHS4 [29], AfCHSF [30], UmCHS5 [31], CnCHS1 (Specht, EMBL accession no. AF021318) and MgCHS3 (Specht, EMBL accession no.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of Chs3p participation in chitin synthase III activity

Cos

Ford

Trilla

et al. 1998

European Journal of Biochemistry

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Chitin is a minor but essential component of the cell wall of Saccharomyces cerevisiae, with functions in septum formation in the vegetative life cycle and also in conjugation and spore cell-wall synthesis in the sexual cycle. Of the three chitin synthases present in yeast, chitin synthase III (CSIII) is responsible for the synthesis of most of the chitin found in the cell, including a chitin ring at early budding, chitin interspersed in the cell wall, and chitin laid down during the sexual cycle. We have tagged Chs3p, the putative catalytic subunit of CSIII, with the immunoreactive epitope of influenza virus hemagglutinin to follow expression of the protein. Little correlation was found between the levels of transcription and translation of Chs3p and in vivo function, supporting our previous conclusion that regulation of CSIII occurs at the posttranslational level. To identify possible regions of the protein involved in catalysis or regulation, mutations were generated in the QRRRW 'signature sequence' of chitin synthases. Arginine residue mutations in Chs3p, and in Chs1p and Chs2p, resulted in a loss of both function in vivo and enzymatic activity. Mutations in a serine residue adjacent to glutamine in Chs3p caused loss of function in vivo with a moderate decrease in CSIII activity, suggesting a regulatory role for the serine residue in chitin biosynthesis. Several truncations in the unique hydrophilic carboxy-terminal region of Chs3p identified a sequence of about 25 amino acids that is required for both function and in vitro activity. Since this region is not present in Chs1 or Chs2, it may be involved in the specific regulation of CSIII.Keywords : chitin synthase III; calcofluor resistance; morphogenesis; Saccharomyces cerevisiae.Chitin is a component of the yeast cell wall that, although only present in small amounts, is required for cell viability and has a major role in septum formation and cell division [1Ϫ3]. Three chitin synthases have been described in Saccharomyces cerevisiae, CSI, CSII and CSIII (see [1Ϫ3] for reviews), each with different functions. CSIII is responsible for synthesis of the chitin ring laid down at the base of an emerging bud; CSII is subsequently involved in the formation of the primary septum disk ; finally, CSI repairs damaged chitin during cell separation. Since CSIII also catalyzes the synthesis of chitin interspersed over all the cell wall and of that laid down during mating and sporulation, this enzyme is responsible for production of most of the chitin found in the cell [1].The different roles of chitin synthases require that each be regulated independently, so that it will execute its function at the required time and location in the cell. This regulation may take place during expression of the protein and/or posttranslationally. Some evidence of transcriptional regulation of the genes coding for the putative catalytic subunits of the three synthases (CHS1, CHS2 and CHS3) has been found [4]. However, further work showed that only CSII activity oscillates in the cell cycle...

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“…The hyphal cell wall has three to five times the chitin content of the yeast cell wall (4,5) and the cells have up to 10 times the in vivo chitin synthase activity (6). Three genes encoding chitin synthases have been cloned and sequenced in C. albicans (7,8,37). Two encode chitin synthase zymogens (CHSI and CHS2), homologous to the CHS genes of Saccharomyces cerevisiae.…”

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confidence: 99%

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

Gow

Robbins

Lester

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

139

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In the dimorphic fungus Cad Chitin is a fibrous polymer off-1,4-N-acetylglucosamine that constitutes a major structural component of the cell wall of many species of fungi, including those species that are pathogenic in humans. Because chitin is not found in mammals and its biosynthesis is normally essential for the shape and viability of the fungal cell (1, 2), chitin synthesis is an attractive target for the design of antifungal drugs. The most common medical mycosis is caused by Candida albicans, which is a diploid organism with no sexual cycle. This fungus is capable ofdimorphic growth where growth can occur by unicellular budding or filamentous hyphal extension and branch formation (3). The hyphal cell wall has three to five times the chitin content of the yeast cell wall (4, 5) and the cells have up to 10 times the in vivo chitin synthase activity (6). Three genes encoding chitin synthases have been cloned and sequenced in C. albicans (7,8,37). Two encode chitin synthase zymogens (CHSI and CHS2), homologous to the CHS genes of Saccharomyces cerevisiae. The third chitin synthase gene, CHS3, is homologous to the S. cerevisiae CSD2 (9) gene (also called CALI) and is apparently the structural gene for an enzyme, which in S. cerevisiae, does not require proteolysis for its activity. Northern blot analysis of the C. albicans CHS genes showed that CHS2 mRNA levels were elevated in cells undergoing hyphal development, whereas the CHS1 mRNA was expressed only at low levels early in germ-tube formation (8).The role of each of the chitin synthase activities described in S. cerevisiae has been investigated by disrupting each of the relevant genes (for a review, see ref. 10). Gene disruptions are more difficult in C. albicans because the organism is constitutively diploid and because stable multiply marked strains are required for sequential gene disruptions. Sequential gene disruption of HEM3 of C. albicans has been achieved with two genetic markers (11), but host strains with sufficient markers for the disruption of more than one structural gene are not yet available. In this study, we employed the "ura-blaster" protocol originally described by Alani et al.(12) for use with S. cerevisiae, to disrupt all alleles of the hyphal-specific C. albicans CHS2 gene. This method allows the sequential disruption of target alleles. by using Ura3 auxotrophy as a single selectable marker. Because the selectable marker can be regenerated after the disruption of each allele, this method overcomes the need for multiply marked host strains and is, therefore, ideally suited for the analysis of families of genes such as the CHS genes in C. albicans. We show that the chs2 null mutant was still able to form germ tubes, albeit with a reduced chitin content compared with the isogenic wild-type parent. A prototrophic chs2-null mutant was still able to cause disease in normal and immunosuppressed mice and had a similar virulence to the parental CHS2 strain. A preliminary report of the construction of the Achs2::hisG null mutant has a...

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Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition

Cited by 69 publications

References 29 publications

Multiple genes for chitin synthase in the zygomycete fungus Phycomyces blakesleeanus.

Multiple genes for chitin synthase in the zygomycete fungus Phycomyces blakesleeanus.

Molecular analysis of Chs3p participation in chitin synthase III activity

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

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