The aim of this proposal is to elucidate the extracellular acid proteinase of Candida spp. (CAP) as a constitutive pathogenic factor in Candida infections by demonstrating: 1) CAP participation in invasion of the skin by Candida in experimental rodent models of cutaneous candidiasis; 2) CAP participation in protecting Candida blastospores from uptake and killing by phagocytic cells; 3) the prevalence of CAP both phenotypically and genomically, in pathogenic and commensal isolates of Candida species, and its correlation with pathogenic and virulent behavior. The role of CAP in cutaneous infections will be performed by studying CAP producing and non-producing isogenic isolates and strains of Candida in experimental rodent models of cutaneous candidiasis. Their ability to invade the epidermis, dermis and fat layers of skin will be assessed by light and electron microscopy for disruption or dissolution of the major proteins of skin, keratin and collagen, which are CAP substrates. CAP inhibition by pepstatin and neutralizing antibody, as well as supplementation with purified CAP, will be studied in these infections. The enzyme will be localized in infected tissue by immunofluorescent microscopy. The role of CAP protecting blastospores from phagocyte uptake and killing will be assessed by standard phagocytosis and killing assays of CAP-producing and non-producing isogenic isolates and species of Candida, in the presence and absence of pepstatin or neutralizing antibody, or supplemental active purified CAP. Direct cytotoxicity of CAP will be assessed by 51Cr-release assays of labelled monocyte, macrophages, and neutrophils. The prevalence of CAP in pathogenic and commensal isolates of Candida will be determined phenotypically by enzyme production in restrictive cultures, and genomically by Southern blot hybridization of restriction endonuclease digests of Candida DNA with a cDNA probe for the CAP encoding gene. Virulent behavior of the isolates will be confirmed in the experimental rodent model of cutaneous candidiasis. This study will characterize the participation of CAP in cutaneous invasion and phagocyte interactions of Candida organisms, demonstrating facilitation of tissue invasion and protection against host inflammatory response. This will be the first study to characterize a virulence factor in Candida species at the genetic level through molecular biology. Data may allow discrimination of commensal and pathogenic isolates for diagnostic testing while revealing critical steps in pathogenesis amenable to therapeutic intervention.
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