AIDS Related Candidiasis: Dynamic Expression of Drug Resistant Genes

描述（由申请方提供）：在致病性酵母菌白色念珠菌（Ca）中，通常分析临床分离株对生长反应梯度的生长反应。这些生长模式是细胞内基因表达模式的结果。在耐药分离株中，已知四种基因组成型过表达，包括靶酶ERG 11和三种外排泵，CDR 1、CDR 2和MDR 1。在易感的Ca中，当暴露于药物时，这些基因可以被转录诱导。然而，这些基因对唑类药物的反应还没有在耐药菌株中进行分析。这一提议是基于这样的假设，即细胞对药物的反应可能是已知和未知抗性基因表达改变的结果，包括在没有药物的情况下的组成型过表达和在药物存在下的诱导基因表达。本提案的总体目标是定义和表征一系列临床分离株对抗真菌药物的反应，并研究这些分离株的基因诱导改变作为除组成性过表达外的耐药机制。具体目的如下：目的1 -确定真菌细胞对唑类化合物的反应谱。临床分离株将根据其对唑类药物的生长反应进行聚类。然后将在随后的基因表达目标中分析来自这些集群的8个代表性分离株。目的2 -使用表达谱鉴定独特的基因诱导模式。将使用微阵列对目标1中聚类分析的代表性菌株进行表达谱分析，以鉴定和表征响应于7种药物浓度梯度的基因诱导模式。目的3 -表征独特的唑诱导的基因表达模式。QRT-PCR将用于详细表征特定基因的诱导模式。目的4 -确定和表征与表达模式改变相关的分子机制。临床分离株中的突变将通过测序进行鉴定，然后表征为实验室菌株的重建。该提案的重点是唑类如何改变细胞的生长和唑类如何改变基因诱导的模式，定义真菌对唑类药物的动态反应。这具有临床意义，因为患者中的真菌从未简单地暴露于单一的高药物浓度。这项研究的结果将对开发新的治疗方法和新的耐药性诊断方法以及重组治疗方法以最大限度地利用当前的抗真菌治疗产生影响。公共卫生相关性：致病性酵母菌白色念珠菌是艾滋病患者最常见的机会性感染，发生在90%以上的患者。念珠菌对唑类药物的反应和念珠菌耐药性的先前分析使用了敏感和耐药临床分离株。这些分离株的基因表达分析集中在不存在药物的情况下的组成型基因表达，以及在单一药物浓度下的基因表达。目前的建议将集中在基因诱导在一个选择的条件谱，以了解真菌唑类药物的动态响应。这具有临床意义，因为患者中的真菌从未简单地暴露于一组定义的药物/真菌条件。

DESCRIPTION (provided by applicant): In the pathogenic yeast Candida albicans (Ca), clinical isolates are commonly analyzed for their growth response to a gradient of growth responses. These growth patterns are the result of gene expression patterns within the cell. In resistant isolates, four genes are known to be constitutively overexpressed include the target enzyme ERG11, and three efflux pumps, CDR1, CDR2, and MDR1. In susceptible Ca, these genes can be transcriptionally induced when exposed to drugs. However, the response to azole by these genes has not been analyzed in resistant strains. This proposal is based on the hypothesis that the cell's response to drug may be the result of altered expression of known and unknown resistance genes that includes constitutive overexpression in the absence of drug and induced gene expression in the presence of drug. The overall goals of this proposal are to define and characterize the response to antifungal drug for a spectrum of clinical isolates, and to investigate those isolates for altered gene induction as a mechanism of resistance apart from constitutive overexpression. The Specific Aims of the proposal as follows: Aim 1 - To define the spectrum of responses of fungal cells to azoles. Clinical isolates will be clustered based on their growth responses to azole drugs. Eight representative isolates from these clusters will then be analyzed in subsequent aims for gene expression. Aim 2 - To identify unique gene induction patterns using expression profiling. Representative strains from the cluster analysis in Aim 1 will be expression profiled using microarrays to identify and characterize the gene induction patterns in response to a gradient of seven drug concentrations. Aim 3 - To characterize unique azole-induced gene expression patterns. QRT-PCR will be used to characterize the induction patterns of specific genes in detail. Aim 4 - To identify and characterize the molecular mechanisms associated with altered expression patterns. Mutations in the clinical isolates will be identified by sequencing and then characterized as reconstructions in laboratory strains. This proposal focuses on how azoles alter the cell's growth and how azoles alter the patterns of gene induction, defining a dynamic response of fungi to azole drugs. This has clinical significance, as fungi in a patient are never simply exposed to a single high drug concentration. The results from this study will have implications for the development of new therapies and new diagnostics of resistance, and for restructuring therapies to maximize current antifungal treatment. PUBLIC HEALTH RELEVANCE: The pathogenic yeast Candida albicans is the most common opportunistic infection in AIDS patients, occurring in over 90% of patients. Previous analyses of Candida's response to azole drugs and Candida drug resistance have used susceptible and resistance clinical isolates. Gene expression analysis in these isolates has focused on constitutive gene expression in the absence of drug, and on gene expression at a single drug concentration. The current proposal will focus on gene induction over a select spectrum of conditions to understand the dynamic response of fungi to azole drugs. This has clinical significance, as fungi in a patient are never simply exposed to one defined set of drug/fungus conditions.