Affiliation:
1. Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614
Abstract
ABSTRACT
Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of
Coccidioides immitis
during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (
CTS1
and
CTS2
) of
C. immitis
have been cloned. Preliminary evidence has suggested that expression of
CTS1
is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-
Coccidioides
complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the
CTS1
gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of
C. immitis
. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the
CTS1
gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in
C. immitis
. However, loss of
CTS1
function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Δ
cts1
strains suggested that the absence of a functional
CTS1
gene can be compensated for by elevated expression of the
CTS2
gene. Current investigations are focused on disruption of
CTS2
in the Δ
cts1
host to further evaluate the significance of chitinase activity in the parasitic cycle of
C. immitis
.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
43 articles.
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