Candida albicans VPS4 is required for secretion of aspartyl proteases and in vivo virulence.

Candida albicans secretes aspartyl proteases (Saps) during infection. Although Saps are secretory proteins, little is known about the intracellular trafficking and secretion of these proteins. We previously cloned and analyzed the C. albicans pre-vacuolar protein sorting gene VPS4, and demonstrated that extracellular Sap2p is absent in the culture supernatants of the vps4delta null mutant. We therefore investigated the role of the C. albicans pre-vacuolar secretion pathway in the trafficking of Sap4-6p and in vivo virulence. The C. albicans vps4delta mutant failed to produce extracellular Sap4-6p. Next, when tested in a mouse model of disseminated candidiasis, the vps4delta mutant was greatly attenuated in virulence. Histopathological analysis indicated that infection with the vps4delta mutant did not cause renal microabscess formation, in contrast to the wild-type strain. Our results imply that VPS4 is required for extracellular secretion of Sap4-6p, and that C. albicans requires an intact pre-vacuolar secretory pathway for wild-type virulence in vivo.

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

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 non-specific 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.

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4.

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.

Intracellular trafficking of fluorescently tagged proteins associated with pathogenesis in Candida albicans.

Proteins that enter the secretory pathway play important roles in virulence and pathogenesis in Candida albicans, but our understanding of the trafficking of these proteins is in its early stages. In Saccharomyces cerevisiae, dominant negative alleles of YPT1 and SEC4 interrupt secretory traffic at pre- and post-Golgi steps, respectively. We therefore used a dominant negative genetic approach to examine the intracellular trafficking of several proteins associated with virulence or azole resistance. When the dominant negative ypt1(N121I) allele of C. albicans was overexpressed, yellow-fluorescent protein (YFP) tagged forms of two plasma membrane transporters (Cdrlp and Ftrlp) and the vacuolar membrane ABC transporter Mltlp accumulated in intracellular structures that appeared related to the ER, but localization of Cdc10p and Int1p was unaffected. When the dominant negative sec4(S28N) allele of C. albicans was overexpressed, Cdrlp and Ftrlp accumulated intracellularly, and localization of Mltlp, Cdc10p and Int1p was unaffected. These results imply that (i) Cdrlp and Ftrlp are transported to the plasma membrane by the general secretory pathway, (ii) Mlt1p enters the secretory pathway but is diverted to the vacuole at an early post-Golgi step, and (iii) like Cdc10p, Int1p does not enter the general secretory pathway.