Project I. Azole resistance in Candida glabrata? Second to Candida albicans, C. glabrata has emerged as the most common cause of bloodstream infection by fungi and exhibits intrinsically low susceptibility to fluconazole, the leading azole antifungal. This species is naturally about 8-fold more resistant to fluconazole than C. albicans and rapidly develops further resistance following prolonged therapy of patients with fluconazole. In prior work, we noted that doubling of the minimum inhibitory concentration (MIC) occurred on the average of every 31 days during fluconazole treatment. In a susceptible knockout mouse, even the most susceptible isolates of C. glabrata were resistant to fluconazole treatment. In work to be summarized below, we will describe two different approaches to analyzing the mechanism(s) by which this formidable fluconazole resistance occurs. First, we analyzed transposon insertion mutants selected for decreased fluconazole resistance. Secondly, we studied ten pairs of fluconazole susceptible and resistant isolates taken from patients receiving fluconazole. The isolates were selected as being from the same patient but at least four fold different in fluconazole MIC. The two members of the pair shared the same karyotype and the same nucleotide sequence in the open reading frame of ERG11. As reported previously, the more resistant isolate in each pair showed increased efflux of two compounds sharing the same drug transporters: radiolabeled fluconazole and the fluorescent dye, rhodamine 6G. We also noted that the more resistant isolate had increased transcription of two genes coding for drug transporters, CgCDR1 and PDH1. Deletion of these two transporters caused an 8-fold decrease in fluconazole susceptibility. Work to be reported this year includes further study using mutants with altered azole susceptibility and the paired clinical isolates? Mutations in CgPDR1 are a major mechanism of fluconazole resistance arising during therapy? The open reading frames and 2.5-kb 5!|flanking sequences from CgPDR1 were sequenced in 10 pairs of susceptible and resistant isolates. All the upstream sequences were identical within the pairs but the deduced amino acid sequences in the ORF!|s differed within the pairs by a single codon. Nine of the ten mutations that had occurred in the more resistant strains were in amino acids conserved between C. glabrata and the S. cerevisiae PDR1. All nine were in areas of Pdr1p in which gain-of-function mutations have been reported. Five of the ten amino acid changes in CgPdr1p were in areas corresponding to the !?inhibitory domain!? of Pdr1p, so named because at least six single amino acid changes have been described which increased transcriptional activity of Pdr1p. Four of the other five mutations in CgPDR1 were in areas corresponding to the !?middle homology domain!? of Pdr1p, a domain thought to influence substrate recognition and in which at least seven !?gain of function!? mutations have been described. The sequencing data indicated that the change in azole resistance during therapy might have been due to a mutation which conferred enhanced transcriptional activity. To determine whether this hypothesis was correct, two of the pairs had the sequence of the CgPDR1 ORF of the more susceptible isolate changed to that of the resistant isolate using integrative transformation. The transformants now became more resistant and increased expression of the CgPDR1 target gene, CgCDR1. Unexpectedly, transcript abundance of CgPDR1 was also increased, suggesting that the transcriptional regulator autoregulated itself. ? Comparison of the transcriptomes from susceptible and resistant isolate pairs by microarray identified common patterns of altered expression? Microarrays comparing the transcriptome of 7 of the ten pairs of susceptible and resistant isolates have been completed in quintuplicate using 70-mers from 5,908 putative open reading frames. Transcript abundance was found to be increased as expected in CgPDR1 and genes with known PDRE!|s, i.e., genes with PDR1 response elements in their promoter regions. Function of other upregulated gene products, based on homology with S. cerevisiae, included a putative flippase, RSB1, that may alter cytoplasmic membrane structure and function, four genes localized to mitochondia, and genes with putative function in cell cycle control, cell wall proteins, protein synthesis, stress response, and enzymatic house-keeping. A gene homologous to YDL212w, a putative ER chaperone, was down-regulated on the average of 4.8 fold. About a fourth of 70-mers with altered transcription were annotated to pseudogenes or genes of unknown function in S. cerevisiae. Transcript abundance was checked by RT-qPCR for 12 up-regulated genes and one down-regulated gene, with good agreement between microarray and RT-qPCR. We have deleted the single most upregulated gene, which has a deduced amino acid sequence similar to S. cerevisiae YIL077c. The function of YIL077c is unknown but its GFP fusion proteinwas shown to localize to the mitochondria. No difference in fluconazole resistance, morphology, or growth of the C. glabrata mutant has been observed so far.? ? Project II. Role of Candida albicans ERG3 in azole resistance? The Candida albicans ERG3 encodes a sterol C5-6 desaturase which is essential for synthesis of the fungal native sterol, ergosterol. Defective sterol C5-6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the !?Ura-blaster!? method, and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3?O-ol instead of ergosterol. For in vivo studies, a wild type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote, relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that a C. albicans erg3 mutant can be fluconazole susceptible in vivo regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism in C. albicans.? ? Project III Cryptococcosis in patients with idiopathic CD4 lymphocytopenia (ICL)? Major findings: Cryptococcosis in 53 patients with ICL had features in common with previously normal patients rather than patients with AIDS. ICL patients had a slight male predominance (1.2:1) and a median age of presentation of 41 years (range 4.5-85). Initial cerebrospinal fluid (CSF) findings showed glucose below 40 mg/dl in 60% of the patients, a median pleocytosis of 59 WBC/mm3 (range 0-884) and protein of 156 mg/dl (range 20-402mg/dl). The median CD4 count at diagnosis of ICL was 82 cells/mm3 (range 7-292) and at the last available measurement was 132 cells/mm3 (13-892) cells/mm3 respectively, for an average follow up period of 32 months. Unlike previously normal patients with cryptococcosis, those with ICL had an excess incidence of dermatomal zoster (7 episodes out of 46 ICL cases), even adjusting for age. Pneumocystis pneumonia was rare (one case), casting doubt on the necessity for prophylaxis in patients with ICL.
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