Acute and recurrent vulvovaginal candidiasis (VVC) remains a significant problem in women of childbearing age. While clinical studies of women with recurrent VVC (RVVC) and animal models have provided important data about a limited protective role of adaptive immunity, there remains a paucity of information on the protective mechanisms or factors associated with susceptibility to infection. In the present study, an intravaginal live Candida challenge in healthy adult women showed a differential susceptibility to symptomatic VVC, where 3 (15%) of 19 women with no history of VVC acquired a symptomatic infection compared to 6 (55%) of 11 women with an infrequent history of VVC. Furthermore, these studies revealed that protection against infection is noninflammatory while symptomatic infection correlates with a vaginal infiltration of polymorphonuclear neutrophils (PMNs) and a high vaginal fungal burden. Thus, the presence of symptomatic infection appears more dependent on host factors than on properties of the organism. Finally, vaginal lavage fluid from women with a symptomatic infection, but not those asymptomatically colonized, promoted the chemotaxis of PMNs. These results suggest that rather than RVVC/VVC being caused by an aberrant adaptive immune response, symptoms that define infection appear to be due to an aggressive innate response by PMNs.Vulvovaginal candidiasis (VVC), caused primarily by Candida albicans, remains a significant problem in women of childbearing age (28). C. albicans is a commensal organism of the gastrointestinal and reproductive tracts (8). Several exogenous factors predispose menarchal women to acute VVC, including hormonal modulations associated with pregnancy, the luteal phase of the menstrual cycle, high-dose oral estrogen contraception, and hormone replacement therapy, and nonhormonal factors such as antibiotic usage and uncontrolled diabetes mellitus (29). There is also a population of women (5 to 10%) who suffer from recurrent VVC (RVVC), which is defined as three or more episodes per annum (28). While most women with RVVC have no known predisposing factors, hormonal or otherwise (idiopathic, and termed primary RVVC), another group of women have RVVC as a result of being unable to avoid certain predisposing factors (secondary RVVC) (28). Historically, primary RVVC has been attributed to a putative local immune deficiency (reviewed in reference 11).Candida-specific cell-mediated immunity, acquired by exposure to Candida early in life, has been considered the predominant host defense mechanism against mucosal Candida infections. However, studies from a mouse model of vaginal candidiasis and many cross-sectional clinical studies evaluating women with primary RVVC over the past several decades have revealed a general lack of protection by local or systemic adaptive immunity (reviewed in reference 11). While data suggest that this may be the result of immunoregulatory mechanisms (31,33,34) and as such have provided important information, these studies provided few clues to the mechan...

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11⌬ mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11⌬ strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.

Yeast-hypha differentiation is believed to be necessary for the normal progression of Candida albicans infections. The emergence and extension of a germ tube from a parental yeast cell are accompanied by dynamic changes in vacuole size and morphology. Although vacuolar function is required during this process, it is unclear if it is vacuolar expansion or some other vacuolar function that is important. We previously described a C. albicans vps11Delta mutant which lacked a recognizable vacuole compartment and with defects in multiple vacuolar functions. These include sensitivities to stress, reduced proteolytic activities, and severe defects in filamentation. Herein we utilize a partially functional VPS11 allele (vps11hr) to help define which vacuolar functions are required for differentiation and which influence interaction with macrophages. Mutant strains harboring this allele are not osmotically or temperature sensitive and have normal levels of secreted aspartyl protease and carboxypeptidase Y activity but have a fragmented vacuole morphology. Moreover, this mutant is defective in filamentation, suggesting that the major role the vacuole plays in yeast-hypha differentiation may relate directly to its morphology. The results of this study support the hypothesis that vacuole expansion is required during germ tube emergence. Both vps11 mutants were severely attenuated in their ability to kill a macrophage cell line. The viability of the vps11delta mutant was significantly reduced during macrophage interaction compared to that in the control strains, while the vps11hr mutant was unaffected. This implies some vacuolar functions are required for Candida survival within the macrophage, while additional vacuolar functions are required to inflict injury on the macrophage.

The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21⌬ and ypt72⌬ mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21⌬ and ypt72⌬ mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.

Autophagy is a major cellular process that facilitates the bulk degradation of eukaryotic macromolecules and organelles, through degradation within the lysosomal/vacuole compartment. This has been demonstrated to influence a diverse array of eukaryotic cell functions including adaptation, differentiation and developmental programmes. For example, in Saccharomyces cerevisiae autophagy is required for sporulation and survival of nitrogen starvation. The opportunistic pathogen Candida albicans has the ability to colonize and cause disease within a diverse range of mammalian host sites. The ability to adapt and differentiate within the host is liable to be critical for host colonization and infection. Previous results indicated that the vacuole plays an important role in C. albicans adaptation to stress, differentiation, and survival within and injury of host cells. In this study the importance of vacuole-mediated degradation through the process of autophagy was investigated. This involved identification and deletion of ATG9, a C. albicans gene required for autophagy. The deletion strain was blocked in autophagy and the closely related cytoplasm to vacuole (cvt) trafficking pathway. This resulted in sensitivity to nitrogen starvation, but no defects in growth rate, vacuole morphology or resistance to other stresses. This indicates that the mutant has specific defects in autophagy/cvt trafficking. Given the importance of autophagy in the development and differentiation of other eukaryotes, it was surprising to find that the atg9D mutant was unaffected in either yeast-hypha or chlamydospore differentiation. Furthermore, the atg9D mutant survived within and killed a mouse macrophage-like cell line as efficiently as control strains. The data suggest that autophagy plays little or no role in C. albicans differentiation or during interaction with host cells.