Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis

Disruption of genes in medically important fungi has proved to be a powerfu l tool for evaluation of putative virulence factors and identification of p otential protein targets for novel antifungal drugs. Chitinase has been sug gested to play a pivotal role in autolysis of the parasitic cell wall of Co ccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immiti s have been cloned. Preliminary evidence has suggested that expression of C TS1 is markedly increased during endospore formation. The secreted CTS1 chi tinase has also been shown to react with patient anti-Coccidioides compleme nt-fixing (CF) antibody and is a valuable aid in the serodiagnosis of cocci dioidomycosis. To examine the role of CTS1 in the morphogenesis of parasiti c cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construc t that contained a 1.1-kb fragment of the chitinase gene into the chromosom al DNA of C, immitis. Results of Southern hybridizations, immunoblot analys es of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion t est for CF antigenicity, and substrate gel electrophoresis assays of chitin ase activity confirmed that the CTS1 gene was disrupted and nonfunctional, This is the first report of a successful targeted gene disruption in C. imm itis. However, Loss of CTSI function had no effect on virulence or endospor ulation. Comparative assays of chitinase activity in the parental and Delta cts1 strains suggested that the absence of a functional CTSI gene can be c ompensated fur by elevated expression of the CTS2 gene. Current investigati ons are focused on disruption of CTS2 in the Delta cts1 host to further eva luate the significance of chitinase activity in the parasitic cycle of C. i mmitis.