Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation

Abstract

To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or nonspecific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation, and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.

Mutational Analysis of Candida albicans SNF7 Reveals Genetically Separable Rim101 and ESCRT Functions and Demonstrates Divergence in bro1-Domain Protein Interactions

“…Defects in both vesicle trafficking and pH sensing have been shown to affect hyphal formation (Davis et al 2000b;Gunther et al 2005;Palmer et al 2005;Bernardo et al 2008). Few of our alaninescanning snf7 alleles conferred strong defects in filamentation, which reinforces that few snf7 alleles completely abolished Snf7 function.…”

“…Filamentation-related snf7 phenotypes: Vesicle trafficking and Rim101 activation are both required for filamentation, a critical virulence trait of C. albicans (Bruckmann et al 2000;Kullas et al 2004;Palmer et al 2005;Bernardo et al 2008). We wished to further investigate the role of these processes and their relative contributions to filamentation.…”

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

“…We additionally analyzed vacuolar morphology in tetR-VMA2 cells with or without DOX using thin-section electron microscopy. Vacuolar morphology in the THE1-CIp10 strain, both with and without DOX, has been analyzed previously (20). The tetR-VMA2 cells without DOX addition displayed wild-type vacuolar morphology, as indicated by the presence of a single, large vacuole (Fig.…”

“…Therefore, we constructed the strain tetR-VMA2, a tetracycline-regulatable VMA2 mutant in which the VMA2 gene is repressed upon the addition of DOX (18). A strain from the THE1 background with URA3 reintegrated into the genome, THE1-CIp10, was used as an additional wild-type control (20). In order to confirm repression of VMA2 with DOX, we utilized RT-PCR to analyze VMA2 expression in the tetR-VMA2 strain (see Fig.…”

“…The ability of tetR-VMA2 to respond to various pHs was tested on complete synthetic media (CSM; 0.67% yeast nitrogen base without amino acids [YNB], 0.079% complete synthetic mixture, 2% glucose) agar plates buffered to pH 4.0 to 8.5 with or without DOX. The THE1-CIp10 control strain, a strain from the THE1 background with URA3 reintegrated into the genome (20), was used as an additional control in this and all subsequent experiments. THE1-CIp10 also was studied with and without DOX to control for the effect of DOX on this and all subsequent experiments.…”