CYTOPATHIC EFFECT OF THE BORRELIA BURGDORFERI EXOPROTEINS OMS28 AND ENOLASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。伯氏疏螺旋体产生的表面暴露蛋白被很好地研究并与发病机制有关，但对螺旋体分泌的胞外蛋白知之甚少。在以前的研究中，两种伯氏杆菌蛋白被释放到RPMI条件培养液中：外膜孔蛋白Oms28和BGP/PFS-2，BGP/PFS-2是一种双功能糖胺聚糖结合蛋白，也具有核苷酶活性。在这里，我们描述了菌株B31在RPMI或趋化介质中孵育后释放的另外12种胞外蛋白。浓缩条件培养液的一维SDS-PAGE分析显示，考马斯亮蓝染色显示15-20条蛋白条带，以牛血清白蛋白为主。除Oms28(BBA74)和BGP/PFS-2外，还鉴定了下列蛋白质：烯醇化酶(BB0337)、GroEL、PFS-1、一种功能未知的27 kDa蛋白质(BB0628)、OspA、OspC、HtrA周质丝氨酸蛋白酶同源物、一种氨基肽酶(BB0069)，该酶属于金属多肽酶M29家族成员、腺嘌呤脱氨酶、葡萄糖-6-磷酸异构酶和tRNA合成酶。我们假设其中一些外源蛋白是免疫原性的，并在宿主与病原体的相互作用中发挥作用。分泌型细菌孔蛋白，如Oms28，通常具有细胞溶解或细胞毒性作用。伴生放线杆菌GroEL是一种上皮细胞毒素，肺炎链球菌分泌的烯醇酶与纤溶酶原结合。免疫印迹实验表明，鞭毛蛋白也能分泌，并证实条件培养液中存在Oms28、GroEL和OspC。患者抗血清识别的条带大小分别为25、46和60 kDa。Bba74和bb0337已克隆到pMAL-c2x中，并以重组蛋白的形式表达。通过亲和层析和离子交换层析获得纯化的Oms28，并将其用于对马、兔和羊红细胞的溶血活性测试。重组烯醇化酶的生化特性和纤溶酶原结合活性正在计划中。本研究对伯氏杆菌外源蛋白的特性进行了研究，揭示了可能有助于毒力和提高莱姆病血清诊断水平的新因素。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Surface-exposed proteins produced by Borrelia burgdorferi are well studied and implicated in pathogenesis, but very little is known regarding extracellular protein secretion by the spirochete. In a previous study, two B. burgdorferi proteins were shown to be released into conditioned RPMI medium; Oms28, an outer-membrane porin, and Bgp/Pfs-2, a bifunctional glycosaminoglycan-binding protein that also has nucleosidase activity. Here, we describe 12 additional extracellular proteins released by strain B31 following incubation in either RPMI or chemotaxis medium. One-dimensional SDS-PAGE analysis of concentrated conditioned medium revealed 15 to 20 protein bands by Coomassie blue staining, with bovine serum albumin predominant. Select protein bands were excised and trypsin digests were sequenced by LC-MS/MS. In addition to Oms28 (BBA74) and Bgp/Pfs-2, the following proteins were identified: enolase (BB0337), GroEL, Pfs-1, a 27 kDa protein (BB0628) of unknown function, OspA, OspC, a HtrA periplasmic serine protease homolog, an aminopeptidase (BB0069) established by others as a member of the M29 family of metallopeptidases, adenine deaminase, glucose-6-phosphate isomerase, and tRNA synthetase. We hypothesize that some of these exoproteins are immunogenic and play a role in the host-pathogen interaction. Secreted bacterial porins such as Oms28 are often cytolytic or cytotoxic. Secreted Actinobacillus actinomycetemcomitans GroEL is an epithelial cell cytotoxin, and enolase secreted by Streptococcus pneumoniae binds plasminogen. Immunoblot experiments with specific antisera indicate that flagellin is also secreted, and confirmed the presence of Oms28, GroEL, and OspC in conditioned medium. Patient antisera recognized bands at 25, 46, and 60 kDa. bba74 and bb0337 have been cloned into pMAL-c2X and expressed as recombinant proteins. Purified Oms28 was obtained following affinity and ion exchange chromatography and will be tested for hemolytic activity against horse, rabbit, and sheep erythrocytes. Biochemical characterization and plasminogen-binding activity of the recombinant enolase is planned. This study characterizing B. burgdorferi exoproteins reveals new factors that may contribute to virulence and improve serodiagnosis of Lyme disease.