Triazole hypersensitivity in Candida albicans

白色念珠菌的三唑超敏反应

• 批准号: 7749806
• 负责人: NEERAJ CHAUHAN
• 金额: $ 12.56万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-11-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749806
• 关键词: Acquired Immunodeficiency Syndrome; Adherence; Animals; Antibiotics; Antibodies; Antifungal Agents; Bacteria; Binding; Biological Assay; Candida; Candida albicans; Candidiasis; Cells; Clinical; Communicable Diseases; Data; Development; Diagnostic; Disseminated candidiasis; Drug Delivery Systems; Drug Exposure; Drug effect disorder; Esophageal Tissue; Eukaryota; Eukaryotic Cell; Event; Excision; Fluconazole; Gene Targeting; Genes; Genetic Transcription; Genome; HIV; Homologous Gene; Human; Human Genome; Hypersensitivity; Immune; Immunocompromised Host; In Vitro; Incidence; Invasive; Membrane; Mitogen-Activated Protein Kinases; Molecular; Mycoses; OmpR protein; Organism; Oxidants; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Proteins; Recovery; Reporting; Research; Research Proposals; Resistance; Risk; Role; Saccharomyces cerevisiae; Signal Transduction; Signal Transduction Pathway; Stimulus; Stream; System; Systemic infection; Test Result; Testing; Therapeutic; Time; Transcriptional Activation; Treatment Cost; Triazoles; Vaccines; Voriconazole; Western Blotting; base; cell type; cost; drug discovery; fungus; high throughput screening; improved; interest; mouse model; mutant; oropharyngeal thrush; pandemic disease; pathogen; quorum sensing; response; sensor histidine kinase; trend

DESCRIPTION (provided by applicant): There has been a steady increase in the incidence of fungal infections over the past few decades, primarily due to the AIDS pandemic. Candida albicans is the most common human fungal pathogen, causing both mucosal and systemic infections and remains a major clinical problem primarily in immunocompromised patients. It has been reported that 60 to 80% of HIV infected patients develop one or more fungal infections at some time during their illness, the most frequent one being oropharyngeal candidiasis (1). Candidiasis caused by Candida spp is the fourth most common cause of nosocomial infectious disease (NID) in the US, and there is a similar trend world-wide (2-7).The magnitude of NID caused by fungi is reflected in patient costs. According to one estimate, during 1998, the cost of treatment of invasive fungal infections, estimated to be approximately 62,000 cases per year in the US, was $2.6 billion, and the average per-patient attributable cost was $31,200 (5). Given the clinical importance of candidiasis, there is an urgent need to identify new drug targets and therefore new anti-fungal drugs, to improve diagnostic assays and to provide alternative therapeutic options such as the use of passive immune anti-Candida antibodies or vaccines for the treatment of high-risk patients. However, since like humans, fungi are eukaryotes, targets for anti-fungal drug discovery must not be found in humans and, in addition, present in all fungal pathogens. Two-component signal transduction proteins have been reported in bacteria and lower eukaryotes. To date they have not yet been identified in animals and are absent in the human genome. Previous studies have confirmed the role of the two-component signal proteins in the pathogenesis of C. albicans in a mouse model of hematogenously disseminated candidiasis, survival in human PMNs in vitro, adherence to human esophageal tissue, quorum sensing and adaptation to oxidants. These features make two-component proteins very attractive targets for antifungal drug discovery since they have important functions and are not found in humans. Two-component phosphorelay systems include a membrane-bound, sensor histidine kinase (HK) protein, which in bacteria, is autophosphorylated in response to an environmental stimulus. Phosphotransfer to a cytoplasmic response regulator (RR) protein occurs which in turn transmits the signal, either through direct transcriptional activation of target genes, or by activation of a down stream mitogen-activated protein kinase cascade (MAPK). The genome of Candida albicans includes three HKs and two RRs, of which Ssk1p is a response regulator that is not functionally related to the Saccharomyces cerevisiae homologue. I have tested two-component and downstream MAP kinase mutants for sensitivity to available antifungal drugs. My preliminary results of MIC broth micro dilution assays indicate that the ssk1 mutant is hypersensitive to the triazoles, fluconazole and voriconazole compared to the wild type strain CAF2-1. Interestingly, the ssk1 mutant retains the wild type levels of sensitivity to other antifungals such as miconazole and ketoconazole (imidazoles), amphotericin B, caspofungin, 5-FC, and a variety of other inhibitors such as Congo red, calcofluor white, hygromycin and nikkomycin Z. So the hypersensitivity of the ssk1 mutant to triazoles appears to be very specific. Further, new data indicate that the ssk1 mutant has elevated levels of fluconazole uptake and lost viability upon incubation with fluconazole and voriconazole, suggesting that in the absence of Ssk1p, fluconazole and voriconazole have significantly increased fungicidal effect on C. albicans. I have also identified several transport proteins by microarray analysis that are up-regulated in ssk1 mutant. Based on this preliminary data, I hypothesize that the Ssk1 protein is a promising therapeutic target and may be exploited in the development or identification of new antifungal drugs. Equally important, my studies will provide new observations on the uptake of triazoles, of which there is very little information in the literature. I also hypothesize that therapy which includes a triazole and a drug which targets Ssk1p or proteins it regulates may enable triazoles to be fungicidal rather than fungistatic. There are two specific aims in this research proposal. In Specific aim 1, part A, I will characterize fluconazole uptake in the ssk1 mutant compared to wt cells in order to explain the hypersensitivity of the mutant. In part B, I will identify the transporter(s) responsible for fluconazole uptake. In Specific aim 2, post antibiotic effects (PAE) will be analyzed to resolve the question as to whether the hypersensitivity of the ssk1 mutant to triazoles also occurs following removal of triazoles. This phenomenon notoriously results in persistence of the organism in the patient as the drug concentrations decrease. In part A of specific aim 2, the PAE of wt and mutant cells will be compared. In part B, gene transcription of wt and the ssk1 mutant at time points during exposure of the drugs and post-exposure will be examined. Recent data by another lab indicate that molecular events can be predicted during post-exposure that indicate recovery of the organism. As part of this sub aim, I will also begin to analyze downstream effector proteins by transcriptional profiling to identify genes regulated by SSK1 that are associated with the hypersensitivity phenotype. Long-term objectives of this research will focus upon the development of high throughput assays to identify compounds that inhibit Ssk1p or downstream proteins that are regulated by Ssk1p. PUBLIC HEALTH RELEVANCE There has been a steady increase in the incidence of fungal infections over the past few decades, primarily due to the AIDS pandemic. Candida albicans is the most common human fungal pathogen, causing both mucosal and systemic infections and remains a major clinical problem primarily in immunocompromised patients. It has been reported that 60 to 80% HIV infected patients develop one or more fungal infection at some time during their illness, the most frequent one being oropharyngeal candidiasis (1). Candidiasis caused by Candida spp is the fourth most common cause of nosocomial infectious disease (NID) in the US, and there is a similar trend world-wide (2-7).The magnitude of NID caused by fungi is reflected in patient costs. According to one estimate, during 1998, the cost of treatment of invasive fungal infections, estimated to be approximately 62,000 cases per year in the US, was $2.6 billion, and the average per-patient attributable cost was $31,200 (5). The present study is aimed at the development and identification of newer antifungal drug targets which may be exploited in anti-fungal drug discovery.

描述（由申请人提供）：在过去的几十年中，真菌感染的发生率一直在稳步上升，这主要是由于艾滋病大流行。白色念珠菌是最常见的人类真菌病原体，引起粘膜和全身感染，并且主要是免疫功能低下的患者的主要临床问题。据报道，有60％至80％的艾滋病毒感染患者在患病期间的某个时候会出现一种或多种真菌感染，最常见的是口咽念珠菌病（1）。由念珠菌引起的念珠菌病是美国医院传染病（NID）的第四大原因，在世界范围内也存在类似的趋势（2-7）。由真菌引起的NID幅度反映在患者成本上。根据一项估计，在1998年，在美国，侵入性真菌感染的治疗费用约为每年62,000例，为26亿美元，平均每位患者归因成本为31,200美元（5）。鉴于念珠菌症的临床重要性，迫切需要鉴定新的药物靶标，因此需要新的抗真菌药物，以改善诊断测定法，并提供替代性治疗方法，例如使用被动免疫抗Candida抗体或疫苗来治疗高风险患者。但是，由于像人类一样，真菌是真核生物，因此在人类中不能发现抗真菌药物发现的靶标，而且在所有真菌病原体中都存在。已经报道了细菌和较低的真核生物中的两个组分信号转导蛋白。迄今为止，它们尚未在动物中鉴定出来，并且在人类基因组中不存在。先前的研究已经证实了两分量信号蛋白在白色念珠菌的发病机理中的作用，在血源性传播的小鼠模型中，体外人类PMN中的存活，对人类食管组织的依从性，法定人数感应和适应氧化剂。这些特征使两组分蛋白具有抗真菌药物的发现非常有吸引力的靶标，因为它们具有重要的功能，并且在人类中没有发现。两个组分的磷化系统包括膜结合的传感器组氨酸激酶（HK）蛋白，该蛋白在细菌中被响应于环境刺激而自磷酸化。向细胞质反应调节剂（RR）蛋白的磷酸转移会发生，从而通过靶基因的直接转录激活或通过向下流丝分裂原子激活的蛋白激活蛋白激酶级联（MAPK）激活来传递信号。白色念珠菌的基因组包括三个HK和两个RR，其中SSK1P是一个反应调节剂，与酿酒酵母同源物在功能上无关。我已经测试了两组分和下游MAP激酶突变体，以敏感可用的抗真菌药物。我的MIC肉汤微稀释测定的初步结果表明，与野生型菌株CAF2-1相比，SSK1突变体对三唑，氟康唑和伏立康唑高度敏感。 Interestingly, the ssk1 mutant retains the wild type levels of sensitivity to other antifungals such as miconazole and ketoconazole (imidazoles), amphotericin B, caspofungin, 5-FC, and a variety of other inhibitors such as Congo red, calcofluor white, hygromycin and nikkomycin Z. So the hypersensitivity of the ssk1 mutant to三唑似乎非常具体。此外，新数据表明，与氟康唑和伏立康唑孵育后，SSK1突变体的氟康唑摄取水平升高，生存力降低，这表明在没有SSK1P，氟康唑，氟康唑和伏立康唑的情况下，对C. albicans的杀真菌性效应显着提高。我还通过微阵列分析确定了几种在SSK1突变体中上调的转运蛋白。基于此初步数据，我假设SSK1蛋白是一个有前途的治疗靶标，可以在开发或鉴定新的抗真菌药物中被利用。同样重要的是，我的研究将提供有关三龙组吸收的新观察，其中文献中的信息很少。我还假设该疗法包括三唑和针对SSK1P或蛋白质的药物，它可以调节的蛋白可能使三唑可以杀真菌而不是燃料。这项研究建议有两个具体的目标。在特定的目标1（A部分）中，我将与WT细胞相比，表征SSK1突变体中氟康唑的摄取，以解释突变体的过敏性。在B部分中，我将确定负责氟康唑摄取的转运蛋白。在特定的目标2中，将分析抗生素效应（PAE），以解决有关在去除三唑后，SSK1突变体对三唑的超敏反应是否也会发生的问题。众所周知，随着药物浓度的降低，这种现象会导致患者中生物体的持续存在。在特定目标2的A部分中，将比较WT和突变细胞的PAE。在B部分中，将检查WT和SSK1突变体的基因转录在药物暴露期间的时间点和暴露后的时间点。另一个实验室的最新数据表明，可以在暴露后表明恢复生物体的情况下预测分子事件。作为该子目标的一部分，我还将通过转录分析开始分析下游效应蛋白，以鉴定由SSK1调节的基因，这些基因与超敏表型相关。这项研究的长期目标将着重于开发高通量测定法，以识别抑制SSK1P调节的SSK1P或下游蛋白的化合物。在过去的几十年中，真菌感染的发生率一直在稳步增长，这主要是由于艾滋病大流行。白色念珠菌是最常见的人类真菌病原体，引起粘膜和全身感染，并且主要是免疫功能低下的患者的主要临床问题。据报道，有60％至80％的艾滋病毒感染患者在患病期间的某个时候会出现一种或多种真菌感染，最常见的是口咽念珠菌病（1）。由念珠菌引起的念珠菌病是美国医院传染病（NID）的第四大原因，在世界范围内也存在类似的趋势（2-7）。由真菌引起的NID幅度反映在患者成本上。根据一项估计，在1998年，在美国，侵入性真菌感染的治疗费用约为每年62,000例，为26亿美元，平均每位患者归因成本为31,200美元（5）。本研究旨在开发和鉴定较新的抗真菌药物靶标，这些抗真菌药物可能在抗真菌药物发现中被利用。