Molecular Genetics, Biochemistry And Therapy Of Pathogen

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Abstract

Project 1. Azole resistance in Candida glabrata. (1) Sequencing was completed in CgAP1, a transcriptional activator which we cloned from C. glabrata. Strong similarity was found to the Saccharomyces cerevisiae gene,YAP1, and the Candida albicans gene, CAP1, including presence of a basic, leucine-rich amino terminal sequence and cysteines in conserved positions at the carboxy-terminal end. Expression of the gene in Saccharomyces cerevisiae markedly decreased susceptibility to a diverse group of drugs, including fluconazole, diamide, 4-nitroquinolone oxide and cycloheximide, indicating that this gene regulates one or more drug efflux pumps and has functional similarity to CAP1. Northern analysis was performed on resistant and susceptible C. glabrata isolates from the same patient and having the same CHEF pattern. Whereas the ABC transporters, CgDR1 and PDH1 showed increased transcript abundance of both genes in the resistant isolate, CgAP1 transcript abundance was the same. This raises the possibility that alterations in the cysteine-rich domain may account for increased regulatory activity, rather than increased expression. (2) Studies with C. glabrata were completed in a murine infection model using p47-phox (-/-) knockout mice. Fluconazole 80 mg/kg daily, the maximum dose that can be administered parenterally, was not effective in treating mice infected with C. glabrata whereas amphotericin B and caspofungin acetate were effective. Susceptibility of the isolates to the three drugs was nearly identical in vitro and blood concentrations of fluconazole were far in excess of those achieved with the other drugs. Project 2. Studies in hematopoietic stem cell transplant recipients: A cluster of 12 cases of human parainfluenza virus 3 (HPIV3) occurred in April-July, 2000 among 64 patients who had received an allogeneic hematopoietic stem cell transplant in the NHLBI bone marrow transplant unit. Upper respiratory symptoms predominated but three patients had pneumonia. Exposure history and molecular analysis of the HPIV3 isolates found that both community acquired and nosocomial transmission had occurred. A chain of transmission was identified among 4 outpatients which extended to 2 hospitalized patients, one of whom died. Institution of control measures ended the outbreak after 11 weeks.