The long range objective of the proposed study is to understand the molecular basis of opportunistic pathogenesis caused by Candida albicans in individuals immunocompromised by HIV infection. In particular, we will study the specific role of a secreted Candida aspartic acid protease (CAP), a putative virulence factor in candidiasis, in the manifestation of oral candidiasis in HIV-infected individuals. 1. The Molecular Characterization of the CAP Alleles of Candida species, in particular C. albicans, in order to address the regulatory mechanisms involved in protease gene transcription and expression and to determine the role of proteases in pathogenesis. After cloning, sequencing and characterizing the CAP genes, we will produce allele-specific probes to identify transcriptionally active members of the gene family. Promoter regions of transcriptionally active alleles will be defined by a combination of standard in vitro techniques and DNA transformation will be used to confirm promoter function in vivo. Promoters of functional CAP alleles will be coupled to reporters such as beta-galactosidease, chloramphenicol acetyl transferase (CAT) or luciferase to analyze CAP expression under a variety of in vitro and in vivo conditions. Regulation of protease expression with switch phenotype will be examined through molecular genetics in Candida and in other yeast. 2. The Correlation of CAP Gene Expression, Virulence and Pathogenesis. CAP expression will be monitored under a variety of growth conditions in a variety of laboratory strains. The effect of cytokines on the growth and virulence of Candida strains and CAP expression will be assessed. Clinical samples of Candida from symptomatic and asymptomatic HIV-infected individuals will be collected and these strains will be assayed for the levels of allele-specific CAP expression in cell extracts and cell supernatants. The presence and localization of CAP, as well as the presence of anti-CAP antibodies, will be determined in HIV-infected patients with candidiasis, as well as in animal models. Mice with anti- protease antibodies will be challenged with virulent Candida to determine if the antibody is protective. The mouse model will also be used to determine if strains of Candida with modified levels of protease have altered virulence. Finally, the effect of CAP as a virulence factor in immune compromised mice will be assayed. A study of the factors involved in CAP expression should provide a basis for understanding the regulatory pathways and gene products involved in pathogenicity and opportunism of Candida at a molecular and cellular level. Such insights will be relevant to the molecular characterization of factors which may trigger the expression of other opportunistic pathogens in HIV- disease.
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