U. Reichard et al., Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis, INFEC IMMUN, 68(10), 2000, pp. 5830-5838
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.