Paralogous genes encoding membrane proteins of brachyspira hyodysenteriae
Creators: name: Witchell, Timothy Douglas
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: The spirochetal outer membrane forms a physical barrier between the cellular contents and the external environment and is the first point of contact with the host. Proteins of the outer membrane are of major interest for research in bacterial pathogenesis and vaccine development fields. For Brachyspira hyodysenteriae, the etiological agent of swine dysentery, only a small number of membrane proteins has been identified to date, and none of these has yet been assigned experimentally-confirmed functions. Initially, work was undertaken to characterize the V sp proteins. These are the most abundant proteins of the B. hyodysenteriae outer membrane and are exposed on the bacterial cell surface. Eight highly related genes arranged in two loci of four genes have been identified in B. hyodysenteriae strain B204. In this study, it was shown that several of these genes were transcribed in vitro, with v5pF and v5pH the most highly expressed. The protein products of both of these genes were also detected by mass spectrometry. New sequencing technologies have significantly increased the speed at which proteins of interest may be selected for further study. The genome of B. hyodysenteriae strain X576, a highly virulent Australian isolate, was determined by pyrosequencing and subsequent analysis was undertaken to identify membrane proteins for further investigation. Only five of the previously identified vsp genes were present in the X576 genome. However, two additional genes encoding V sp-like proteins, here named v5pJ and vspJ, were identified. These newly discovered genes were also found to be present in the genomes of strains B204 and WAl. B. hyodysenteriae membrane proteins separated under semi-denaturing conditions were found to contain protein complexes that resolved into 40 kDa proteins in subsequent denaturing SDS-PAGE gels. The complexes consisted primarily of V spF; however, V spE, V spI and V spD were also identified. No other proteins were co-purified with the Vsp complexes. The Vsp proteins had previously been suggested to be antigenic. Work undertaken in this study was unable to verify this claim using a similar method. However, Vsp protein complexes are recognized by convalescent pig sera, but not after heat denaturation, indicating the importance of conformational epitopes in the antigenicity of V sp proteins. BlpA, also reported as BmpB, is a 28 kDa lipoprotein of B. hyodysenteriae and is encoded within a locus containing three paralogs of blpA, namely blp£, blpF and blpG. Prior to this study, only BlpA has been shown to be expressed by B. hyodysenteriae cells and has been one of the most studied membrane proteins of this organism. No function has been experimentally confirmed for these proteins; however, they share limited sequence similarity with periplasmic components of methionine uptake systems in other organisms. In this study, alterations in the methionine and cysteine content of the growth medium were found to induce changes in the expression of genes from the blpGFEA locus, with the first three genes expressed more highly when sulfur-containing amino acid levels were not added to the growth medium. Sequence comparisons with Tp32, a methionine-binding protein of Treponema pallidum, indicated that BlpE and BlpF have a number of amino acid differences in sites crucial to binding methionine. This suggests that BlpE and BlpF are duplicated from either BlpG or BlpA and have been adapted for the uptake of other amino acids or related molecules when levels of these are low in the surrounding environment.