Molecular Genetics, Biochemistry And Therapy Of Pathogenic Fungi

National Institute Of Allergy And Infectious Diseases

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Abstract

Project I. Global Transcriptional Profiling of Candida glabrata during Phagocytosis by Neutrophils[unreadable] Human polymorphonuclear cells (PMN) are vital for host defense against infection by Candida glabrata (Cg). Gene expression by Cg during phagocytosis by PMN provides information on how PMN eliminate Cg from the host. In this study, we used Cg microarrays containing 70 mers of 5908 ORFs to compare the global transcriptional profiling of Cg phagocytized by PMN and Cg alone. Differentially expressed genes were defined by ratios of at least 2.5 or less than 0.4 in 4 of 6 arrays, comparing expression with and without PMN. A total of 650 genes were up-regulated and 439 down-regulated. Highly up-regulated genes were involved in methionine or other amino acid biosynthesis (STR3), gluconeogenesis (PCK1), glyoxylate cycle (MLS1), fatty acid degradation (FAA2) and oxidative stress response (IDP1) at 30 and 60 min incubation. Lysine biosynthesis genes were highly upregulated at 60 compared to 30 min. Multidrug resistance genes were also upregulated (PDR1, PDH1) along with the PKC signaling pathway, which is involved in Cg cell wall remodeling. Many of the down-regulated genes were involved in protein synthesis (ASC1), cell wall biogenesis (CIS3) and glycolysis (CDC19). Response of Cg to PMN attack reflects the lack of essential carbohydrate and amino acids and the presence of oxidative stress in the intracellular environment. Up-regulation of multidrug resistance genes even in the absence of drugs is an integral part of Cg response to PMN.[unreadable] [unreadable] Project 2: Study of CgPDR1 and its role in clinical azole resistance.[unreadable] Azole resistance frequently develops during prolonged treatment in Candida glabrata, a yeast with intrinsically low susceptibility to azoles. We have shown in our previous study that one of the resistance mechanisms was increased expression of the transcriptional regulator gene, CgPDR1, which upregulated the expression of CgCDR1, a multidrug transporter gene. Five putative functional domains (DNA-binding, inhibitory, fungal-specific transcriptional factor, nuclear localization, and activation) were identified in the CgPdr1p based on its similarity to Saccharomyces cerevisiae Pdr1p. We have analyzed a total of 10 pairs clinical susceptible and resistant isolates. Comparison of the CgPDR1 promoter and ORF deduced amino acid sequences within each pair revealed that each of the 10 resistant isolates had acquired a different single mutation. Six out of the total ten mutations occurred in the inhibitory domain with one in the activation domain and one in the fungal-specific transcriptional factor domain. Two mutations occurred in an area of undefined function, which is in the vicinity of the nuclear localization domain. CgPDR1 from one susceptible and five resistant isolates were introduced into a mutated cgpdr1 locus of an azole-susceptible laboratory strain via an integrative transformation. The transformants were subjected to gene expression and fluconazole susceptibility analyses. Fluconazole MIC analysis showed that the transformants which received the mutated CgPDR1 of resistant isolates had an increase in azole resistance compared to the laboratory wild-type strain as well as the strain which received the CgPDR1 of susceptible isolate, indicating that the mutations accounted for the fluconazole resistance. Real time PCR showed that the expression levels of CgPDR1 and CgCDR1 in all transformants carrying the mutated CgPDR1 genes were paralleled with their levels of fluconazole resistance suggesting that the CgPDR1 expression levels determined the levels of resistance to fluconazole. The mutated CgPDR1 genes also altered the expression of other transporters, PDH1, CgSNQ2, and CgYOR1. In contrast to CgCDR1, the increased PDH1 expression due to these CgPDR1 mutations was not correlated with the expression levels of CgPDR1. Also, only three out of five CgPDR1 mutations led to the increases in the CgYOR1 expression and four out of five CgPDR1 mutations led to the increases in CgSNQ2 expression. These data indicated that the impact of CgPDR1 mutations on the different transporters varied, probably driven by differences in the promoter complex. This result prompted our future interest in analyzing the components of the promoter complex[unreadable] [unreadable] Project 3: Analysis of voriconazole and its metabolites in patients blood[unreadable] Voriconazole is a novel azole antifungal metabolized mainly by CYP2C19 and to a lesser extent by CYP2C9 and CYP3A4. N-oxidation and 4-hydroxylation are important voriconazole metabolic pathways as shown in studies with human liver microsomes. In order to confirm these findings and to further understand voriconazole metabolism in human, we analyzed 171 serum samples in 62 predominantly Caucasian patients (range 1-17 samples/patient) treated with voriconazole between 2005 and 2007 in NIH. The patients CYP2C19 genotypes were also determined. There was no difference between median voriconazole and median n-oxide voriconazole serum levels in patients heterozygous for CYP2C19 mutations and the wild type (p>0.05). Only two patients were 2C19*2 homozygotes, too few to draw conclusions. There was a better correlation between serum n-oxide voriconazole levels and voriconazole dose (p=0.006, r=0.262) than between voriconazole levels and voriconazole dose (p=0.048, r=0.14). No correlation was found between n-oxide voriconazole and the voriconazole dose (p>0.05). Finally there was an inverse correlation of the voriconazole metabolic ratio (n-oxide voriconazole/voriconazole) and dose adjusted voriconazole, implying saturation of voriconazole metabolism in higher doses (p<0.001, r =0.48). Unlike the experience reported from Japan, the effect of CYP2C19 genotype on the levels of voriconazole and n-oxide voriconazole were modest and probably not clinically relevant in our population. There was such high variability between adult patients that voriconazole levels did not correlate well with the weight-adjusted dose[unreadable] [unreadable] Project 4: idiopathic CD4 lymphocytopenia (ICL)[unreadable] Thirty nine patients (17 men, 22 women) aged 25 to 85 years old with ICL referred to the NIH Clinical Center were evaluated between 1992 and 2006 and followed for a median of 49.5 months. Clinical data, T cell subsets, activation and turnover markers were studied. Cryptococcal and non-tuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In thirty two patients CD4+ T cell counts remained low throughout the study period and in seven they normalized after an average of 31 months. The follow up was significant for the incidence of human papilloma virus, dermatomal varicella zoster virus infections and autoimmune diseases. Patients without opportunistic infections at presentation remained asymptomatic. Seven patients died, four from ICL related opportunistic infections within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4+ T but not CD8+ T lymphocytes. CD8+ T lymphopenia (<180/mm3) and the degree of CD4+ T cell activation (measured by HLA-DR) at presentation were associated with an adverse outcome (ICL related death) (p=0.006 and 0.016 respectively). In conclusion, ICL is characterized by persistent CD4+ T cell lymphopenia and high CD4+ but not CD8+ T cell turnover. A CD8+ T cell count less that 180/mm3 and high CD4+ T cell activation at diagnosis were associated with a poor prognosis.