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Triazole hypersensitivity in Candida albicans

白色念珠菌的三唑超敏反应

基本信息

批准号：
7567525
负责人：
NEERAJ CHAUHAN
金额：
$ 23.4万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-11-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7567525
关键词：
Acquired Immunodeficiency Syndrome Adherence Amphotericin B Animals Antibiotics Antibodies Antifungal Agents Bacteria Binding Biological Assay Candida Candida albicans Candidiasis Carrier Proteins Caspofungin Cells Clinical Communicable Diseases Congo Red Critiques Data Development Diagnostic Disseminated candidiasis Down-Regulation Drug Delivery Systems Drug Exposure Esophageal Tissue Eukaryota Event Excision Fluconazole Flucytosine Force Feedings Fungal Proteins Gene Targeting Generations Genes Genetic Transcription Genome Growth HIV Homologous Gene Human Human Genome Hypersensitivity Imidazole Immune Immunocompromised Host In Vitro Incidence Industrial fungicide Iron Ketoconazole Label Literature Membrane Methods Miconazole Mitogen-Activated Protein Kinases Molecular Mycoses OmpR protein Organism Oxidants Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phenotype Protein Microarray Assay Proteins Publishing Recovery Reporting Research Research Proposals Resistance Reverse Transcriptase Polymerase Chain Reaction Role Saccharomyces cerevisiae Signal Pathway Signal Transduction Signaling Protein Sorting - Cell Movement Stimulus Stream System Systemic infection Testing Therapeutic Time Transcript Transcriptional Activation Treatment Cost Triazoles Up-Regulation Vaccines Voriconazole base calcofluor white cost design drug development drug discovery drug sensitivity follow-up fungus high risk high throughput screening hygromycin A improved inhibitor/antagonist innovation interest mouse model mutant nikkomycin So(Z)oropharyngeal thrush pandemic disease pathogen public health relevance quorum sensing research study response sensor histidine kinase therapeutic target trend uptake

项目摘要

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）：在过去的几十年里，真菌感染的发病率稳步上升，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，可引起粘膜和全身感染，主要是免疫功能低下患者的主要临床问题。据报道，60%至80%的HIV感染患者在发病期间的某个时间会出现一种或多种真菌感染，最常见的是口咽念珠菌病(1)。念珠菌引起的念珠菌病是美国院内感染性疾病（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的费用上。根据一项估计，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，每位患者的平均可归属成本为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，从而确定新的抗真菌药物，改进诊断分析，并提供替代治疗方案，如使用被动免疫抗念珠菌抗体或疫苗治疗高危患者。然而，由于真菌和人类一样是真核生物，因此抗真菌药物的靶点不能在人类身上找到，而且必须存在于所有真菌病原体中。双组分信号转导蛋白已在细菌和低等真核生物中报道。迄今为止，它们尚未在动物身上被发现，在人类基因组中也不存在。先前的研究已经证实了双组分信号蛋白在小鼠血源性播散念珠菌病模型中白色念珠菌的发病机制、体外在人PMNs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等方面的作用。这些特征使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。双组分磷接力系统包括一种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自磷酸化。通过靶基因的直接转录激活或下游有丝分裂原激活的蛋白激酶级联（MAPK）的激活，发生向细胞质反应调节蛋白（RR）的磷酸化，进而传递信号。白色念珠菌的基因组包括3个hk和2个rr，其中Ssk1p是一个应答调节因子，与酿酒酵母菌同源物在功能上无关。我已经测试了双组分和下游MAP激酶突变体对现有抗真菌药物的敏感性。我的MIC肉汤微量稀释试验的初步结果表明，与野生型菌株CAF2-1相比，ssk1突变体对三唑类、氟康唑和伏立康唑过敏。有趣的是，ssk1突变体对其他抗真菌药物，如咪唑和酮康唑（咪唑）、两性霉素B、caspofungin、5-FC和各种其他抑制剂，如刚果红、钙氟白、湿霉素和尼克霉素z，保持了野生型的敏感性水平。因此，ssk1突变体对三唑类药物的超敏性似乎是非常特异性的。此外，新的数据表明，ssk1突变体与氟康唑和伏立康唑孵养后，氟康唑摄取水平升高，活力丧失，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真菌作用显著增强。我还通过微阵列分析确定了几种转运蛋白，它们在ssk1突变体中被上调。基于这些初步数据，我推测Ssk1蛋白是一个有希望的治疗靶点，可能在开发或鉴定新的抗真菌药物中被利用。同样重要的是，我的研究将提供关于三唑摄取的新观察，这方面的文献资料很少。我还假设，包括三唑和针对Ssk1p或其调节的蛋白质的药物的治疗可能使三唑成为杀真菌剂而不是抑真菌剂。本研究计划有两个具体目的。在具体目标1，A部分中，我将描述与wt细胞相比，ssk1突变体对氟康唑的摄取，以解释突变体的超敏性。在B部分，我将确定负责氟康唑摄取的转运蛋白。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超敏性是否也会在去除三唑类药物后发生的问题。众所周知，随着药物浓度的降低，这种现象会导致患者体内的有机体持续存在。在特异性目的2的A部分，我们将比较wt和突变细胞的PAE。在B部分，将检测wt和ssk1突变体在药物暴露期间和暴露后时间点的基因转录。另一个实验室最近的数据表明，在暴露后可以预测分子事件，表明生物体的恢复。作为本子目标的一部分，我还将开始通过转录谱分析下游效应蛋白，以鉴定与超敏表型相关的SSK1调节基因。本研究的长期目标将集中于开发高通量测定方法，以鉴定抑制Ssk1p或受Ssk1p调节的下游蛋白的化合物。在过去的几十年里，真菌感染的发病率稳步上升，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，可引起粘膜和全身感染，主要是免疫功能低下患者的主要临床问题。据报道，60 - 80%的HIV感染者在发病期间会出现一种或多种真菌感染，最常见的是口咽念珠菌病(1)。念珠菌引起的念珠菌病是美国院内感染性疾病（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的费用上。根据一项估计，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，每位患者的平均可归属成本为31,200美元(5)。本研究旨在开发和鉴定新的抗真菌药物靶点，这些靶点可能用于抗真菌药物的开发。