Project 1. Azole resistance in Candida glabrata. A major and long term project of the laboratory has been to understand why azole resistance arises rapidly during treatment of Candida glabrata infections in humans, in contrast to other pathogenic yeasts. Transcriptional control of azole resistance: The association of azole resistance and the CgPDR1 mutations we reported last year was investigated in strains with the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. We wanted to identify the promoter sequence (pleiotropic drug response element or PDRE) of target genes to which CgPdr1 protein bound. Multiple Em (expectation-maximization algorithm) for Motif Elicitation (MEME) was used to analyze the upstream sequences of 18 genes which were upregulated in microarray analysis in the majority of the seven more resistant isolates. The motif analysis revealed that 17 out of 18 genes contained the putative PDRE motif (TCCAGCTGGA) in their 1kb upstream regions. The canonical sequence TCCACGGA appeared at the highest frequency and TCCGTGGA occurred the second most frequently. A motif comparison using the TOMTOM tool matched the motif with the PDRE motif of Pdr1p/Pdr3p in the S. cerevisiae promoter database (SCPD) (p value of 9.9 x 10-5), in which TCCGCGGA is the major PDRE motif. B. CgYTA12 and azole resistance A Candida glabrata mutant obtained via transposon mutagenesis showed greater than 8 folds increased resistance to fluconazole compared to its parental clinical isolate. Deletion of a gene, CAGL0J01353, was found to be responsible for the increased fluconazole resistance. The gene had the greatest homology with the S. cerevisiae gene YTA12, which codes for a component of the mitochondrial inner membrane m-AAA protease. This enzyme mediates degradation of misfolded or unassembled proteins and is also required for correct assembly of mitochondrial enzyme complexes. In, C. glabrata, deletion of CAGL0J01353 (CgYTA12) causes a marked decrease in the ability to utilize glycerol as a carbon source, a hallmark of mitochondrial deficiency. Mitochondria however are still present in these mutants based on MitoTracker red staining. Unlike the petite mutant of C. glabrata, which causes azole resistance by upregulating CgPDR1, the CgYTA12 had no effect on CgPDR1 expression but still upregulated the major CgPDR1 target, CgCDR1. Potential involvement of other pathway(s) in the upregulation of CgCDR1 will still need to be defined. Project 2. Voriconazole metabolism, genotype and toxicity. Now that this study has been completed,the data from last year can be updated as follows: Toxicity and 311 trough serum voriconazole concentrations were monitored in 93 patients. Genotype was assessed on 73 patients and metabolites measured on 164 samples from 49 patients. The following results were obtained: The major circulating metabolite of voriconazole is the fluoropyrimidine N-oxide, formed in vitro by the P450 2C19 and 3A forms. A 4-hydroxyl metabolite is known from a report of 6% of the drug appearing in urine as a glucuronide. Nothing is known about concentrations of these metabolites during treatment. N-oxide concentrations were a mean+/_SE of 2.35+/-0.11 (range 0.09-7.890), similar to the voriconazole serum concentrations on the same samples (2.42+/- 0.18). The mean+/-SE concentration of the 4-hydroxymetabolite was low at 68.5 +/ 6.5 ng/ml (range 0-464 ng/ml) B. Toxicity 1, Hepatotoxicity. Asymptomatic abnormal liver function occurred in six patients (6.2%), all beginning in the first week, reaching a peak within a week of stopping the drug and then resolving with no residual abnormality. Hepatitis recurred promptly in the only patient rechallenged with voriconazole but not in two later given fluconazole or one given posaconazole. None had elevated serum concentrations of voriconazole or the metabolites. 2. Hallucinations. Visual hallucinations occurred in 16 (16.7%) patients, accompanied by auditory hallucinations in 5. All but two occurred in the first 24 hours of treatment. In 6 patients, voriconazole was continued at a reduced dose and the hallucinations resolved. Trough voriconazole levels were measured 1-7 times in the first week of therapy in 15 of the 16 patients. Their mean level was 4.24 mcg/ml, range 2.0 to 12.3 mcg/ml, a significantly higher mean than the average for the entire study group (2.55;p=0.004), but overlap was extensive. Nine patients had the two metabolites measured during the first week of hallucinations with similar concentrations to other patients. 3. Visual changes and phototoxocity. Seventeen patients (17.7%) had visual changes, a sense of brighter light or more intense colors. Ten patients (10.5%) had photosensitivity. Blood concentrations of voriconazole and the metabolites were not higher in patients with visual changes or photosensitivity, even when patients only in outpatient status were included in the photosensitivity analysis. An unexplained finding was the concentrations of the 4-hydroxymetabolite were significantly lower in the phototoxic than nonphototoxic patients (0.0354 vs 0.0786 mcg/ml; p=0.003, Mann Whitney test). C. Effect of CYP450 genotype The 2C19 genotype was determined for 73 patients. In our largely Caucasian population, there was no discernable effect of the genotypes on the trough serum level of voriconazole or its metabolites. There were only two patients homozygous for the 2C19 slow metabolizers phenotype and only one of the two had metabolites measured. In agreement with studies on volunteers in Japan, those two patients had significantly higher voriconazole levels and one had lower levels of metabolites. This effect was not discernible in the 11 2C19 heterozygotes (2C19*1/*2.Our data found that the 2C9 and the 2C19 genotype, including the 806 up-stream promoter sequence, had no detectable impact on serum trough levels of voriconazole or its metabolites. Infectious disease training program project 3. infectious disease training program. Since 1976 the program has graduated 132 fellows, or an average of 4.4 fellows per year. Of the 132, 23 are currently on the NIH staff, 2 are in the Transitional Program and 6 are in an extended fellowship position. That is, 23.4% of our graduates are still at NIH. 12.8% are working for industry, 10.6% are in private practice and 53.2% are working for a university. Names and current positions of our graduates as well as the prior training of current fellows are posted on our web site, www.cc.nih.gov/training/gme/programs/infectious_diseases.html Our first time pass rate for the American Board of Internal Medicine is 100% for the past decade and over 95% since inception. THe program is providing a valuable source of young academicians, trained in infectious disease and clinically-related research.
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