The objective of this research is to gain an understanding of the mechanism(s) underlying drug resistance in the human parasite, Trichomonas vaginalis. Strains of T. vaginalis that are resistant to metronidazole, the only drug available in the U.S. for treatment, arise at a low frequency. Our preliminary studies show that four clinical isolates, refractory to treatment with metronidazole, have decreased intracellular levels of ferredoxin and its mRNA. Ferredoxin is a hydrogenosomal protein which is required to reduce metronidazole to its cytotoxic form. To our knowledge, this is the first demonstration of drug resistance arising due to the inability of the cell to chemically modify a drug to its active form. Additionally, we have shown that ferredoxin gene transcription is reduced in two of these resistant isolates. This indicates that regulation of gene expression is the basis of resistance.
The specific aims of the proposed studies are: 1) to examine the levels of ferredoxin, as well as other hydrogenosomal proteins which are involved in drug activation, in additional sensitive and resistant lines. Resistant cell lines which have decreased intracellular levels of ferredoxin will be examined to determine if ferredoxin mRNA and/or gene transcription is altered. 2)to examine the 5' and 3' sequences flanking the ferredoxin gene in resistant and sensitive strains for differences which might account for altered transcription of the gene in resistant lines. DNA sequence motifs, and the proteins which bind to such putative transcriptional regulatory elements, will be studied. In addition to examining the mechanism of drug resistance, these studies provide a framework for exploring gene expression in this primitive eukaryote. Thus, the proposed investigation addresses both medically and fundamentally important aspects of the biology of this human pathogen. A better knowledge of the mechanisms underlying resistance will be critical for designing effective treatment regimens for cases refractory to treatment. In addition to treating trichomoniasis, metronidazole is commonly used in treatment of infections caused by other anaerobic organisms, such as Entamoeba, Giardia, Bacteroides, and Clostridium. Isolates of these organisms which are relatively resistant to the drug have been reported, but virtually nothing is known regarding the mechanism of resistance. Thus our studies on metronidazole resistance in T. vaginalis may possibly shed light on the basis of resistance in a number of pathogenic bacteria and protozoa.
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