CYTOPATHIC EFFECT OF THE BORRELIA BURGDORFERI EXOPROTEINS OMS28 AND ENOLASE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。由伯氏疏螺旋体产生的表面暴露的蛋白质被很好地研究并且与发病机制有关，但是关于螺旋体的胞外蛋白质分泌知之甚少。在以前的研究中，两个B。Burgdorferi蛋白显示被释放到条件RPMI培养基中; Oms 28，一种外膜孔蛋白，和Bgp/Pfs-2，一种也具有核苷酶活性的双功能糖胺聚糖结合蛋白。在这里，我们描述了12个额外的细胞外蛋白释放菌株B31孵育后，无论是RPMI或趋化培养基。经考马斯亮蓝染色后，浓缩的条件培养基的一维SDS-PAGE分析显示15至20条蛋白带，以牛血清白蛋白为主。切下选择的蛋白条带，并通过LC-MS/MS对胰蛋白酶进行测序。除了Oms 28（BBA 74）和Bgp/Pfs-2之外，还鉴定了以下蛋白：烯醇化酶（BB 0337），GroEL，Pfs-1，一种功能未知的27 kDa蛋白（BB 0628），OspA，OspC，一种HtrA周质丝氨酸蛋白酶同系物，一种氨肽酶（BB 0069），由其他人确定为金属肽酶、腺嘌呤脱氨酶、葡萄糖-6-磷酸异构酶和tRNA合成酶的M29家族的成员。我们假设这些外蛋白中的一些是免疫原性的，并在宿主-病原体相互作用中发挥作用。分泌的细菌孔蛋白如Oms 28通常是溶细胞的或细胞毒性的。分泌型伴放线放线杆菌GroEL是一种上皮细胞毒素，肺炎链球菌分泌的烯醇化酶与纤溶酶原结合。用特异性抗血清进行的免疫印迹实验表明鞭毛蛋白也被分泌，并证实了条件培养基中Oms 28、GroEL和OspC的存在。患者抗血清识别25、46和60 kDa的条带。bba 74和bb 0337已被克隆到pMAL-c2 X中并作为重组蛋白表达。在亲和和离子交换层析后获得纯化的Oms 28，并将测试其对马、兔和绵羊红细胞的溶血活性。计划重组烯醇化酶的生化表征和纤溶酶原结合活性。本研究表征了B. burgdorferi exoproteins揭示了可能有助于莱姆病毒力和提高血清学诊断的新因素。

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.