This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Background: Candida species are the most frequent opportunistic pathogens afflicting humans, with C. albicans the most prevalent. C. albicans can cause a diverse array of mucosal disease including oral and esophageal thrush, and denture stomatitis. More than 90% of HIV+ patients will incur oral candidiasis and these infections are highly recurrent. This has necessitated long term treatment with antifungals in these patients, with the consequent emergence of antifungal resistant strains. Candida can also cause life threatening systemic fungal infections, and new therapeutic strategies are badly needed to combat these infections. The capacity of C. albicans to reversibly switch between yeast and hyphal growth forms is crucial for pathogenicity. The highly polarized hyphal form is thought to facilitate tissue invasion, while the smaller, rounded yeast cells may aid dissemination. During the transition from yeast to hyphal growth, cytoplasmic material is maintained in the apical compartment while the fungal vacuole undergoes a dramatic expansion to generate highly 'vacuolated' sub-apical compartments. This represents a novel mechanism of differentiation.Objectives: 1). To establish the molecular mechanism of hyphal cell vacuolation in C. albicans. This will be achieved through the functional analysis of proteins Vps21p and Ypt7p which are key regulators of pre-vacuole compartment (PVC) and vacuole biogenesis. 2). To determine the therapeutic potential of disrupting the function of the fungal vacuole. This will be established though testing mutant strains deficient in PVC (vps21D) and vacuolar biogenesis (ypt7D) in in vitro 'virulence assays', models of tissue invasion, and animal based models of infection.Results: We have generated Green Fluorescent Protein (GFP) tagged versions of Vps21p and Ypt7p. Localization analysis of GFP-Vps21p and GFP-Ypt7p has demonstrated these proteins localize to the PVC and vacuole compartments as predicted. Western blot analysis has also revealed that Ypt7p is expressed at a higher level during hyphal than yeast growth. Furthermore, while Vps21p expression levels are similar, a second form of increased molecular weight was detected specifically during hyphal growth. This may reflect a post-translational modification which modulates Vps21p activity during hyphal growth. We have also demonstrated that a vps11D mutant (defective in PVC and vacuole biogenesis) is incapable of oral tissue invasion using an in vitro model system. Furthermore, mutants deficient in PVC (vps21D) or vacuole biogenesis (ypt7D) are highly attenuated in a mouse model of systemic disease. Discussion: These data suggest that the activity of Vps21p and Ypt7p, which are key regulators of vacuole biogenesis, are both modulated during the transition to the tissue invasive hyphal form. Moreover, that defects in either PVC or vacuole biogenesis lead to the loss of virulence in this pathogen.
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