The Characterization of Pneumocystis Surface Antigens

肺孢子虫表面抗原的表征

• 批准号: 9549422
• 负责人: JOSEPH A KOVACS
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 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/9549422
• 关键词: Amino Acid Sequence; Animals; Antibody Response; Antibody titer measurement; Antigens; CDKN1C gene; Characteristics; Data; Disease; Enzyme-Linked Immunosorbent Assay; Epidemiology; Genes; Genome; Geographic Locations; Glycine decarboxylase; Goals; HIV Infections; High Pressure Liquid Chromatography; Human; Immune response; Immunocompromised Host; Individual; Length; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular; Mus; Organism; Pathogenesis; Patients; Plasmids; Pneumocystis; Pneumocystis Infections; Pneumocystis carinii Pneumonia; Rattus; Recombinants; Recording of previous events; Serum; Site; Source; Subgroup; Surface Antigens; Tandem Repeat Sequences; Variant; Western Blotting; base; cDNA Library; next generation sequencing; pathogen; prevent; protein expression

We have an ongoing project to characterize the antigens of mouse, rat, and human Pneumocystis. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use Pneumocystis from both sources because the organisms are different species. Subsequently, we identified a number of clones from a cDNA library of rat Pneumocystis that contain genes encoding for the MSG. These clones were clearly related but not identical, demonstrating that multiple genes encode the MSG. We have continued studies to characterize potential antigens of Pneumocystis. We have cloned a number of human Pneumocystis MSG genes and have expressed a full-length MSG in two fragments. We developed an ELISA to examine antibody responses to these antigens, and have utilized it to examine sera from patients with or without HIV infection, and with or without a history of PCP, as well as sera from a variety of healthy controls. In about 15% of healthy patients followed serially we have been able to document changes in antibody titers, suggesting that these individuals have developed reinfection or reactivation of Pneumocystis infection. We will continue these studies to better understand the epidemiology of Pneumocystis infection in humans. We have also identified the unique expression site of MSG in human Pneumocystis, and can now identify the MSG variants that are expressed in a patient with PCP. Within this expression site we have identified a region of tandem repeats that varies among different Pneumocystis isolates, and thus provides a method for typing human Pneumocystis. We have sequenced MSG variants from a single human pneumocystis isolate and have shown that the isolates can be divided, into 2 subgroups that are genetically distinct. We have cloned and sequenced MSG variants from additional patients from around the world to see if this will provide information about the distribution of different Pneumocystis strains. To date we have found substantial variability in MSG variants from different parts of the world, but no clustering by geographic location. To better examine the MSG repertoire and MSG variability, we have utilized next generation sequencing. By 454 sequencing, we were unable to reconstruct the original sequences using a mixture of plasmids, in large part because of the conserved regions in MSG variants. PacBio sequencing, which allows potentially longer reads, appears to be able to overcome the assembly problem. We are continuing to evaluate results generated by PacBio sequencing using mouse and human isolates of Pneumocystis. We have also expressed a p57 protein of P. murina that is predicted to be a surface protein based on a predicted GPI-type anchor. Based on western blot studies this is an antigen recognized by mouse serum from exposed or infected animals. We are conducting studies to characterize expression of this protein by Pneumocystis. We have recently sequenced the genomes of 3 Pneumocystis species, and will be using the data from the genomes to identify potential surface proteins that may be antigens, based on the predicted amino acid sequence characteristics. The goal of this study is to better understand the pathogenesis of Pneumocystis pneumonia with the hope that we can use this information to control or prevent this disease.

我们正在进行一个项目来表征小鼠、大鼠和人类肺孢子虫的抗原。 我们之前已经使用 HPLC 纯化了大鼠和人肺孢子虫的主要表面糖蛋白 (MSG)。 有必要使用两个来源的肺孢子虫，因为这些生物体是不同的物种。 随后，我们从大鼠肺孢子虫的 cDNA 文库中鉴定出许多克隆，其中含有编码 MSG 的基因。 这些克隆明显相关但不相同，表明多个基因编码味精。 我们继续研究来表征肺孢子虫的潜在抗原。 我们克隆了许多人肺孢子虫 MSG 基因，并以两个片段表达了全长 MSG。 我们开发了一种 ELISA 来检查抗体对这些抗原的反应，并用它来检查来自有或没有 HIV 感染、有或没有 PCP 病史的患者的血清，以及来自各种健康对照的血清。 在约 15% 的健康患者中，我们连续随访，记录了抗体滴度的变化，表明这些个体已发生肺孢子虫感染的再感染或再激活。 我们将继续这些研究，以更好地了解人类肺孢子虫感染的流行病学。 我们还鉴定了人肺孢子虫中 MSG 的独特表达位点，现在可以鉴定 PCP 患者中表达的 MSG 变异体。 在该表达位点内，我们鉴定了不同肺孢子虫分离株之间存在差异的串联重复区域，从而提供了对人类肺孢子虫进行分型的方法。 我们对单个人类肺孢子虫分离株的 MSG 变异体进行了测序，并表明这些分离株可分为 2 个遗传上不同的亚组。 我们对来自世界各地的其他患者的味精变异进行了克隆和测序，看看这是否能提供有关不同肺孢子虫菌株分布的信息。 迄今为止，我们发现世界不同地区的味精变体存在很大差异，但没有按地理位置进行聚类。 为了更好地检查味精库和味精变异性，我们利用了下一代测序。通过 454 测序，我们无法使用质粒混合物重建原始序列，这在很大程度上是因为 MSG 变体中的保守区域。 PacBio 测序可能允许更长的读取，似乎能够克服组装问题。 我们正在继续评估使用小鼠和人类肺孢子菌分离株进行 PacBio 测序所产生的结果。 我们还表达了 P. murina 的 p57 蛋白，根据预测的 GPI 型锚定，该蛋白被预测为表面蛋白。 根据蛋白质印迹研究，这是一种被暴露或感染动物的小鼠血清识别的抗原。 我们正在进行研究来表征肺孢子虫表达这种蛋白质的特征。我们最近对 3 个肺孢子菌物种的基因组进行了测序，并将根据预测的氨基酸序列特征，使用基因组数据来识别可能是抗原的潜在表面蛋白。 本研究的目的是更好地了解肺孢子菌肺炎的发病机制，希望我们能够利用这些信息来控制或预防这种疾病。