AIDS Related Candidiasis: Dynamic Expression of Drug Resistant Genes

艾滋病相关念珠菌病：耐药基因的动态表达

• 批准号: 7857989
• 负责人: Theodore C. White
• 金额: $ 44.02万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7857989
• 关键词: ABCB1 gene; Acquired Immunodeficiency Syndrome; Antifungal Agents; Azoles; Biochemistry; CDR1 gene; Candida; Candida albicans; Candidiasis; Cells; Clinical; Cluster Analysis; Collection; Development; Drug resistance; Drug usage; Enzymes; Ergosterol; Exhibits; Gene Expression; Gene Expression Profiling; Genes; Goals; Growth; Kinetics; Laboratories; Measures; Metabolic; Microbiology; Minimum Inhibitory Concentration measurement; Molecular; Molecular Profiling; Mutation; Opportunistic Infections; Patients; Pattern; Pharmaceutical Preparations; Predisposition; Resistance; Sorting - Cell Movement; Test Result; Testing; Time; Yeasts; base; cell growth; clinically significant; efflux pump; fungus; gene induction; novel diagnostics; overexpression; promoter; public health relevance; reconstruction; resistance mechanism; resistant strain; response; transcription factor

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.

描述（由申请人提供）：在病原性酵母白色念珠菌（CA）中，通常分析其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。在抗性分离株中，已知四个基因的组成性过表达包括靶酶ERG11和三个外排泵，即CDR1，CDR2和MDR1。在敏感的CA中，这些基因暴露于药物时可以转录诱导。但是，这些基因对硫唑的反应尚未在抗性菌株中分析。该建议基于以下假设：细胞对药物的反应可能是由于缺乏药物和在药物存在下诱导基因表达的已知和未知抗性基因表达改变的结果。该提案的总体目标是定义和表征各种临床分离株对抗真菌药物的反应，并研究这些分离株是否改变了基因诱导的改变，这是除构型过表达外的抗药性机制。该提案的具体目的如下：目标1-定义真菌细胞对唑的响应的光谱。临床分离株将根据其对硫唑药物的生长反应聚类。然后，将在随后的基因表达目标中分析这些簇中的八种代表性分离物。 AIM 2-使用表达分析确定独特的基因诱导模式。 AIM 1中群集分析的代表性菌株将使用微阵列进行表达，以识别和表征响应7种药物浓度的梯度的基因诱导模式。 AIM 3-表征独特的Azole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。目标4-识别和表征与改变表达模式相关的分子机制。临床分离株中的突变将通过测序鉴定，然后将其表征为实验室菌株中的重建。该提案的重点是硫唑如何改变细胞的生长以及硫唑如何改变基因诱导的模式，从而定义了真菌对偶氮药物的动态反应。这具有临床意义，因为患者中的真菌从未仅仅暴露于单一的高药物浓度。这项研究的结果将对新疗法的发展和抗药性的新诊断和重组疗法具有影响，以最大程度地提高当前抗真菌治疗。公共卫生相关性：病原性酵母白色念珠菌是艾滋病患者中最常见的机会性感染，发生在90％以上的患者中。先前对Candida对唑药和耐药性耐药性的反应的分析已经使用了易感性和耐药性临床分离株。这些分离株中的基因表达分析集中在缺乏药物的情况下，以及在单个药物浓度下的基因表达。当前的建议将集中在一系列条件上的基因诱导上，以了解真菌对硫唑药物的动态反应。这具有临床意义，因为患者中的真菌从未仅仅暴露于一套定义的药物/真菌疾病中。