Defective Hyphal induction of a Candida albicans phosphatidylinositol 3-phosphate 5-kinase null mutant on solid media does not lead to decreased virulence

Abstract

A phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase gene (CaFAB1) of the most important human pathogenic yeast, Candida albicans, was cloned and sequenced. An open reading frame was detected which encodes a 2,369-amino-acid protein with a calculated molecular mass of 268 kDa and a relative isoelectric point of 6.76. This protein exhibits 38% overall amino acid sequence identity with Saccharomyces cerevisiae Fab1p. We localized the CaFAB1 gene on chromosome R. To determine the influence of the PI(3)P 5-kinase CaFab1p on processes involved in C. albicans morphogenesis and pathogenicity, we sequentially disrupted both copies of the gene. Homozygous deletion of C. albicans CaFAB1 resulted in a mutant strain which exhibited defects in morphogenesis. A Cafab1 null mutant had enlarged vacuoles, an acidification defect, and increased generation times and was unable to form hyphae on different solid media. The sensitivities to hyperosmotic and high-temperature stresses, adherence, and virulence compared to those of wild-type strain SC5314 were not affected.

FIG. 1.

Disruption of the FAB1 gene in C. albicans: restriction maps of plasmids pFX1, pDF2, and pFXU1, illustrating the strategy for disruption and reintegration of CaFAB1. For pFX1 the CaFAB1 gene (thick solid arrow, coding region; open boxes, noncoding regions) was cloned into pUC18, yielding plasmid pFX1. For pFD2 the 5′ and 3′ regions of CaFAB1 obtained by PCR were cloned in front of or behind the hisG-URA3-hisG cassette (4.1 kb) in the disruption vector pMB-7. For PFXU1 the URA3 gene (1.4-kb RsaI fragment from pMB-7) was cloned into pFX1 digested with XhoI approximately 2.3 kb downstream of the stop codon of CaFAB1. The labeled arrows indicate the locations of restriction sites, as follows: E, EcoRI; K, KpnI; P, PstI; Sa, SalI; Sc, SacI; Xb, XbaI; Xh, XhoI.

FIG. 2.

Southern analysis of XbaI-digested chromosomal DNA from the following C. albicans strains: parental strain CAI-4 (lane 1), CAF1 FAB1/fab1::hisG-URA3-hisG (lane 2), CAF2 FAB1/fab1::hisG (lane 3 ), CAF3 fab1::hisG-URA3-hisG/fab1::hisG (lane 4), CAF4 fab1::hisG/fab1::hisG (lane 5), and CAF5 fab1::hisG/FAB1-URA3 (lane 6). The blot was hybridized with the [α-³²P]dCTP-labeled XbaI insert of plasmid pFX1.

FIG. 3.

Multiple alignment of the FYVE finger domain from Fab1p of C. albicans (CaFab1), S. cerevisiae (ScFab1), S. pombe (SpFab1), A. thaliana (AtFab1), and mouse (MmFab1), as well as the Vps27 protein from S. cerevisiae (ScVps27) and the Vac1 homologue EAA1 from humans (HuEEA1). Zn²⁺-binding cysteines are indicated by asterisks. Amino acids that are involved in PI binding are indicated by plus signs.

FIG. 4.

Morphology of C. albicans fab1 null mutant CAF3, wild-type strain SC5314, and revertant strain CAF5. The use of Nomarski optics clearly revealed enlarged vacuoles and cells for CAF3 during yeast-phase growth compared to SC5314 and CAF5. The hyphal growing form of CAF3 also contains enlarged vacuoles in the mother cell and in the hyphae. Quinacrine staining revealed clear vacuoles in the yeast and hyphal growing forms of CAF3, indicating an acidification defect. In the apical regions of hyphae acidification was restored. In SC5314 and CAF5 we observed normally acidified vacuoles. Bar = 13 μm (for all panels).

FIG. 5.

Phenotypes of Cafab1 mutant strains on solid hypha-inducing media. Spider medium contained mannitol as a carbon source. YPD medium contained 10% FCS. The strains used were C. albicans SC5314 (wild type), CAF1 (Cafab1 heterozygous mutant), CAF3 (Cafab1 homozygous mutant), and CAF5 (CaFAB1 revertant). The strains were grown for 7 days at 37°C. Bars = 3.0 mm.

FIG. 6.

Pathogenicity of the Cafab1 mutants. C. albicans SC5314, CAF1 (FAB1/fab1::hisG-URA3-hisG), CAF3 (fab1::hisG-URA3-hisG/fab1::hisG), and CAF5 (fab1::hisG/FAB1-URA3) were tested in a mouse model of systemic candidosis. Survival of mice infected with 5 × 10⁴ (▪), 5 × 10⁵ (▴), and 5 × 10⁶ (•) cells was monitored for 21 days (n = 10).