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.
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