Molecular genetics, biochemistry and therapy of pathogenic fungi

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Abstract

Candidiasis remains the most common deep mycosis, being the fourth most common cause of nosocomial bloodstream infections and occurring commonly in patients with neutropenia, prematurity and complicated abdominal surgery. Mucosal candidiasis is also the most common mycosis in AIDS patients. Broad use of fluconazole, particularly for prophylaxis and empiric therapy, has slowly increased resistance to fluconazole and other azole antifungals. Fluconazole resistance appears to rise rapidly in Candida glabrata, a species accounting for 15% of candidemias. In a series of 9 bone marrow transplant recipients who were persistently colonized by C. glabrata, minimum inhibitory concentrations of fluconazole rose in five patients from 16-32 mcg/ml to at least 128 mcg/ml during fluconazole prophylaxis. In all five patients, azole resistance arose in their original strain, rather than being due to acquisition of a resistant strain, as judged by computer- assisted analysis of macrodigested CHEF patterns. The major mechanism of fluconazole resistance in this species appears to be activation of drug efflux pumps. We have used Northern blotting with phosphoimager analysis to examine transcription of the ABC transporter gene, PDH1. Although disruption of this gene does not change fluconazole susceptibility, transcript abundance correlates with azole susceptibility, indicating coregulation with other transporters, at least one of which transports fluconazole. A wide range of apparently unrelated toxic substances increases abundance of the PDH1 transcript, including oligomycin, cycloheximide and rhodamine 6G. We extended our studies on the disruption of PDH1 to a second azole susceptible strain, confirming increased toxicity of oligomycin, cycloheximide, and cadmium, as well as increased intracellular concentrations of rhodamine. Increased estradiol uptake was also noted in this pdh1 mutant. No change in any of these parameters was observed when PDH1 was disrupted in one markedly azole resistant strain, probably because other transporters with overlapping specificity were activated and compensated for loss of pdh1p transport activity. Overlapping function of multidrug resistance genes and their transcriptional regulation may account for this species? rapid ability to overcome growth inhibition by azole antifungals.We have continued our study of azole resistant Candida albicans strains. The importance of the Y132H and I471T mutations in the gene coding for the azole target, C14demeythylase (ERG11) was confirmed by site directed mutagenesis. One copy of ERG11 gene in an azole susceptible strain of C. albicans was replaced with a C. albicans ERG11 gene that these mutations. Resistance was increased markedly by either mutation but was greatest with both mutations. To assist in identifying transmission of azole resistant strains, we compared three different molecular typing techniques and found that the macrodigested CHEF had the appropriate discimination index for our purpose. Both the CA3 and CARE2 probes of EcoR1 digested genomic DNA gave patterns that were so discriminating that microevolution within a single strain could not be distinguished from acquisition of a new strain. Cryptococcal meningitis in previously normal patients can respond poorly to all available antifungal agents. Cytokine therapy has not been evaluated clinically, largely because of concern that increased inflammation might worsen cerebral edema. In order to begin to understand how immunity may be altered favorably, we have begun study of experimental cryptococcosis. In IL12 knockout mice in a Balb/c background, death occurred much earlier than in Balb/c controls, confirming the importance of the Th1 arm of the immune system. In a clinical study, neurosurgical placement of a shunt in ten patients with hydrocephalus due to cryptococcal meningitis was found to restore neurologic function and not impair response to antifungal therapy by acting as a foreign body.Pneumonia due to respiratory syncytial virus (RSV) causes death in approximately 5% of allogeneic bone marrow transplant recipients. We have begun a study of early diagnosis and immunoprophylaxis of RSV in the NIH bone marrow transplant unit. - azole, antifungal, resistance, Candida, demethylase, transporter - Human Subjects