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Infection and Immunity, October 2000, p. 5830-5838, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Disruption of the Gene Which Encodes a Serodiagnostic Antigen and Chitinase of the Human Fungal Pathogen Coccidioides immitis

Utz Reichard, Chiung-Yu Hung, Pei W. Thomas, and Garry T. Cole*

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614

Received 19 April 2000/Returned for modification 23 June 2000/Accepted 21 July 2000

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 Delta 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 Delta cts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Ave., Toledo, OH 43614-5806. Phone: (419) 383-5423. Fax: (419) 383-3002. E-mail: gtcole{at}mco.edu.

Infection and Immunity, October 2000, p. 5830-5838, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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