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）：过去几十年来，真菌感染的发病率稳步上升，这主要是由于艾滋病的流行。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，并且仍然是主要在免疫功能低下患者中的主要临床问题。据报道，60% 至 80% 的 HIV 感染者在患病期间的某个时间会出现一种或多种真菌感染，其中最常见的一种是口咽念珠菌病 (1)。由念珠菌属引起的念珠菌病是美国医院感染性疾病 (NID) 的第四大常见原因，在世界范围内也有类似的趋势 (2-7)。由真菌引起的 NID 的严重程度反映在患者费用中。据一项估计，1998 年，美国每年约有 62,000 例侵袭性真菌感染的治疗费用为 26 亿美元，每位患者的平均归属费用为 31,200 美元 (5)。鉴于念珠菌病的临床重要性，迫切需要确定新的药物靶点以及新的抗真菌药物，以改进诊断测定并提供替代治疗选择，例如使用被动免疫抗念珠菌抗体或疫苗来治疗念珠菌病。高危患者的治疗。然而，由于与人类一样，真菌也是真核生物，因此抗真菌药物发现的靶点一定不能在人类中找到，而且还必须存在于所有真菌病原体中。已报道细菌和低等真核生物中存在双组分信号转导蛋白。迄今为止，它们尚未在动物中被发现，并且在人类基因组中不存在。先前的研究已经证实了双组分信号蛋白在血行播散性念珠菌病小鼠模型中白色念珠菌的发病机制、体外人中性粒细胞中的存活、对人食管组织的粘附、群体感应和对氧化剂的适应中的作用。这些特征使得双组分蛋白成为抗真菌药物发现的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。双组分磷酸中继系统包括膜结合的传感器组氨酸激酶 (HK) 蛋白，该蛋白在细菌中响应环境刺激而发生自磷酸化。发生磷酸转移至细胞质反应调节蛋白（RR），该蛋白反过来通过靶基因的直接转录激活或通过下游丝裂原激活蛋白激酶级联（MAPK）的激活来传递信号。白色念珠菌的基因组包括3个HK和2个RR，其中Ssk1p是一种反应调节因子，与酿酒酵母同源物在功能上不相关。我测试了双组分和下游 MAP 激酶突变体对可用抗真菌药物的敏感性。我的 MIC 肉汤微量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、氟康唑和伏立康唑过敏。有趣的是，ssk1突变体保留了野生型对其他抗真菌药物的敏感性，例如咪康唑和酮康唑（咪唑）、两性霉素B、卡泊芬净、5-FC以及各种其他抑制剂，例如刚果红、卡尔科弗洛白、潮霉素和尼可霉素Z. 因此，ssk1 突变体对三唑类药物的超敏反应似乎是非常特异的。此外，新数据表明，ssk1突变体对氟康唑的摄取水平升高，并在与氟康唑和伏立康唑一起孵育后失去活力，这表明在缺乏Ssk1p的情况下，氟康唑和伏立康唑对白色念珠菌的杀菌作用显着增强。我还通过微阵列分析鉴定了几种在 ssk1 突变体中上调的转运蛋白。基于这些初步数据，我假设 Ssk1 蛋白是一个有前途的治疗靶点，可用于开发或鉴定新的抗真菌药物。同样重要的是，我的研究将提供有关三唑类吸收的新观察结果，而文献中对此的信息很少。我还假设，包含三唑和一种针对 Ssk1p 或其调节蛋白质的药物的疗法可能使三唑具有杀真菌作用，而不是抑真菌作用。本研究提案有两个具体目标。在具体目标 1 A 部分中，我将描述 ssk1 突变体与 wt 细胞中氟康唑摄取的特征，以解释突变体的超敏性。在 B 部分中，我将确定负责氟康唑吸收的转运蛋白。在具体目标 2 中，将分析抗生素后效应 (PAE)，以解决 ssk1 突变体对三唑类药物的过敏是否也会在去除三唑类药物后发生的问题。众所周知，随着药物浓度降低，这种现象会导致微生物在患者体内持续存在。在具体目标 2 的 A 部分中，将比较 wt 和突变细胞的 PAE。在B部分中，将检查在药物暴露期间和暴露后的时间点上wt和ssk1突变体的基因转录。另一个实验室的最新数据表明，可以预测暴露后的分子事件，表明生物体已恢复。作为该子目标的一部分，我还将开始通过转录分析来分析下游效应蛋白，以识别与超敏反应表型相关的受 SSK1 调节的基因。这项研究的长期目标将集中于开发高通量测定法，以鉴定抑制 Ssk1p 或受 Ssk1p 调节的下游蛋白的化合物。公共卫生相关性 过去几十年来，真菌感染的发病率稳步上升，这主要是由于艾滋病的流行。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，并且仍然是主要在免疫功能低下患者中的主要临床问题。据报道，60% 至 80% 的 HIV 感染者在患病期间的某个时间会出现一种或多种真菌感染，最常见的一种是口咽念珠菌病 (1)。由念珠菌属引起的念珠菌病是美国医院感染性疾病 (NID) 的第四大常见原因，在世界范围内也有类似的趋势 (2-7)。由真菌引起的 NID 的严重程度反映在患者费用中。据一项估计，1998 年，美国每年约有 62,000 例侵袭性真菌感染的治疗费用为 26 亿美元，每位患者的平均归属费用为 31,200 美元 (5)。本研究旨在开发和鉴定可用于抗真菌药物发现的新抗真菌药物靶点。