Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins

Abstract

The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption. CaMNN9 has significant homology with S. cerevisiae MNN9, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in Candida. CaMNN9 resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of CaMNN9 leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with beta-1,3-glucanase. Like all members of the S. cerevisiae MNN9 gene family the Camnn9Delta strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the Camnn9Delta strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of MNN9 gene family members CaVAN1 and CaANP1 in the Camnn9Delta strain showed that transcription of those genes is not affected in the absence of CaMNN9 transcription. Our results suggest that, while the role MNN9 plays in glycosylation in both Candida and Saccharomyces is conserved, loss of MNN9 function in C. albicans leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target.

FIG. 1

Amino acid alignments of the CaMNN9 product with protein sequences encoded by members of the S. cerevisiae MNN9 gene family. Boxes indicate amino acid identity. The putative membrane-spanning domains present in the CaMNN9 and ScMNN9 proteins are overlined. Arrows indicate sequences used to design primers for amplification of C. albicans genomic DNA.

FIG. 2

Chromosomal analysis of CaMNN9. Chromosomes isolated from C. albicans CAI4 and ATCC 10261 (A, lanes 1 and 2, respectively) were probed with the internal 600-bp ClaI/EcoRV fragment from p8A-KpnIΔ (B). Chromosomal designations are on the left. CaMNN9 resides on chromosome 3 in both strains.

FIG. 3

Analysis of expression of CaMNN9. Shown is a Northern analysis of total RNA (10 μg) isolated from C. albicans ATCC 10261 growing in either yeast (lane Y) or hyphal (lane H) growth phases. The blot was probed with the internal ClaI/EcoRV fragment of CaMNN9 (A), stripped, and reprobed with the S. cerevisiae actin gene (B).

FIG. 4

Strategy used for disruption of both alleles of the C. albicans MNN9 gene. (A) Restriction map of a genomic fragment containing CaMNN9. The 600-bp ClaI/EcoRV fragment was replaced with the 4.0-kb BglII/BamHI fragment carrying the hisG URA3 hisG cassette (see Materials and Methods). (B) Southern analysis of genomic DNA from strains obtained during the deletion process. DNA was digested with HindIII and separated by agarose gel electrophoresis. After transfer to a nylon membrane, the blot was probed with the 2.1-kb HindIII/KpnI fragment containing CaMNN9. Lane 1, SC5314 (wild type); lane 2, CAI4 (wild type, Ura⁻); lane 3, SS2+ (MNN9/mnn9, Ura⁺); lane 4, SS2− (MNN9/mnn9, Ura⁻); lane 5, SSCA-2 (mnn9/mnn9, Ura⁺); lane 6, SS19-4 (mnn9/mnn9, Ura⁻). The 3.5-kb band represents the wild-type MNN9 locus. The 6.9- and 4.0-kb bands correspond to the Camnn9::hisG-URA3-hisG and Camnn9::hisG loci, respectively.

FIG. 5

Morphology of yeast and hyphal growth forms of C. albicans wild-type and mnn9Δ strains. Yeast cells (Y) were grown in YPD to an OD₆₀₀ of 1.0. The dimorphic transition to hyphal growth was induced by the addition of serum to 10% and shifting the culture to 37°C. Hyphal samples (H) were observed after 4 h of incubation. Cells were viewed under a microscope with Nomarski optics.

FIG. 6

SDS-PAGE analysis of β-1,6-glucanase-soluble (A), β-1,3-glucanase-soluble (B), and SDS-soluble (C) proteins extracted from C. albicans SSCA-2 (Δmnn9) and SC5314 (wild type) cell walls. Controls are represented as untreated wild-type (lanes 1 and 5) and Δmnn9 (lanes 2 and 6) strain cell walls. Proteins isolated from wild-type (odd-numbered lanes) and mutant (even-numbered lanes) strain cell walls after treatment with either β-1,6-glucanase (lanes 3 and 4), β-1,3-glucanase (lanes 7 and 8), or SDS (lanes 9 and 10) were separated on 4- to 20% gradient gels and visualized by silver staining. Molecular mass standards (in kilodaltons) are shown on the left.

FIG. 7

RT-PCR (A) and Northern (B) analysis of expression of MNN9 family genes in C. albicans wild-type and Δmnn9 strains. RT-PCR was performed with RNA extracted from wild-type strain SC5314 (lanes 1 to 4) and Δmnn9 strain SSCA-2 (lanes 5 to 8). cDNA synthesis and subsequent amplification were done with primers specific for the C. albicans MNN9 (lanes 2 and 6), VAN1 (lanes 3 and 7), and ANP1 (lanes 4 and 8) genes. Control reactions (lanes 1 and 5) utilized ANP1-specific primers, and reaction mixtures contained no reverse transcriptase. Northern blot analysis was performed with total RNA (10 μg) isolated from strains SC5314 (lane 1) and SSCA-2 (lane 2), and the purified wild-type MNN9, VAN1, and ANP1 RT-PCR products were used as probes. The blot was stripped sequentially between hybridizations and was probed with the S. cerevisiae actin gene as a quantitative control for gel loading.