Molecular Studies Of Human Pneumocystis Carinii

人卡氏肺孢子虫的分子研究

基本信息

批准号：
7733534
负责人：
JOSEPH A KOVACS
金额：
$ 8.24万
依托单位：
CLINICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7733534
关键词：
Area Biological Assay Clinical Collaborations Detection Diagnosis Diagnostic Diagnostic Procedure Dihydrofolate Reductase Dihydropteroate Synthase Enzyme Kinetics Enzymes Epidemiology Evaluation Failure Gene Family Gene Targeting Genes Genetic Recombination Genome Highly Active Antiretroviral Therapy Human Human Cloning Introns Invasive Membrane Glycoproteins Membrane Proteins Methods Microbiology Molecular Mutation Numbers Oral Organism Patients Pneumocystis Pneumocystis Infections Pneumocystis carinii Pneumocystis carinii Pneumonia Polymerase Chain Reaction Prophylactic treatment Recombinants Research Personnel Resistance Sampling Screening procedure Single-Stranded Conformational Polymorphism Site Specimen Sulfamethoxazole System Tandem Repeat Sequences Techniques Therapeutic Agents Treatment Protocols Trimethoprim Yeasts base cohort improved insight prophylactic rapid technique therapy resistant

项目摘要

To try to develop highly sensitive, non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human Pneumocystis. This is a family of genes that are closely related and that encode an important surface protein of Pneumocystis. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at approximately equal to 50-100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department and investigators at SFGH using oral washes as the clinical specimen. Because human Pneumocystis cannot be cultured, we cannot directly determine if resistance to commonly used therapeutic agents is developing. However, molecular techniques can be used to identify mutations that may confer resistance in genes that are targets of therapeutic agents. The most commonly used agent to treat Pneumocystis pneumonia is the combination of trimethoprim, which targets dihydrofolate reductase (DHFR), and sulfamethoxazole, which targets dihydropteroate synthase (DHPS). We have cloned the human Pneumocystis DHFR gene, and have examined (by PCR and sequencing) the Pneumocystis DHFR and DHPS genes of a variety of human isolates from patients with Pneumocystis pneumonia. DHPS mutations were found in about one-third of patients, while no mutations have been found to date in the DHFR gene. We have also expressed recombinant human Pneumocystis DHFR, characterized the kinetics of this enzyme, and developed a rapid screening assay for agents which target DHFR, by expressing the enzyme in a yeast system. We have also developed a rapid method for detection of DHPS mutations using SSCP (single strand conformational polymorphisms), and have examined a large number of samples for DHPS mutations, including organisms obtained from an Italian cohort. We have also developed and evaluated a new typing technique using tandem repeats that occur in an intron of the MSG gene. Further studies of a region of the MSG expression site are ongoing; we have been able to demonstrate that recombination occurs in human Pneumocystis. These studies should provide improved diagnostic methods for PCP, help to better understand the epidemiology of Pneumocystis infection, and insights into the reasons for therapy or prophylaxis failures.

为了开发高度敏感的非侵入性诊断方法，我们一直在使用基于人肺细胞的主要表面糖蛋白（MSG）基因的引物来评估聚合酶链反应（PCR）。这是一个密切相关的基因家族，并编码了肺炎胸膜的重要表面蛋白。使用基于该基因的引物的PCR可能是一种高度敏感的方法，因为这是一个多拷贝基因（估计约为50-100拷贝/基因组）。我们一直在使用基因家族的保守区域评估诊断潜力。我们的研究表明，基于MSG的引物的敏感性大于先前使用的引物的敏感性。目前，我们正在使用口腔洗涤作为临床标本与微生物部门和SFGH的研究人员进行前瞻性评估这些引物。由于无法培养人类肺炎，因此我们无法直接确定对常用治疗剂的抗性是否正在发展。但是，分子技术可用于识别可能赋予治疗剂靶标的抗性的突变。最常用的治疗肺炎藻肺炎的药物是甲氧苄啶的组合，它靶向二氢叶酸还原酶（DHFR）和磺胺甲恶唑，靶向二氢替代蛋白酸酯合成酸酯（DHPS）。我们已经克隆了人类肺炎藻DHFR基因，并检查了（通过PCR和测序）来自肺炎肺炎肺炎患者的多种人分离株的肺炎藻DHFR和DHPS基因。在大约三分之一的患者中发现了DHP突变，而在DHFR基因中尚未发现突变。我们还表达了重组人肺炎藻DHFR，表征了该酶的动力学，并通过在酵母菌系统中表达酶来对靶向DHFR的药物进行快速筛选测定。我们还开发了一种使用SSCP（单链构象多态性）检测DHP突变的快速方法，并检查了大量用于DHPS突变的样品，包括从意大利人群中获得的生物体。我们还使用在MSG基因内含子中发生的串联重复序列开发并评估了一种新的键入技术。对MSG表达位点区域的进一步研究正在进行中；我们已经能够证明重组发生在人的肺囊肿中。这些研究应为PCP提供改进的诊断方法，有助于更好地了解肺炎胸膜感染的流行病学以及对治疗或预防失败原因的见解。