Analysis of thePasteurella multocida outer membrane sub-proteome and its response to thein vivo environment of the natural host

Abstract

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.

Proteomic and Immunoblot Analyses of Bartonella quintana Total Membrane Proteins Identify Antigens Recognized by Sera from Infected Patients

“…One-quarter of the proteins that we identified are predicted to be membrane proteins, nearly one-third of the proteins are predicted to be cytoplasmic proteins, and the remainder have unknown localizations. As found in other gram-negative bacteria (6,16), including B. henselae (42), many of the proteins that fractionated with the TMP fraction are not membrane proteins. We performed a search of the B. quintana genome in the TIGR database for "membrane proteins" and identified 55 membrane-associated proteins.…”

“…Four of the immunodominant antigens were OMP (VompA, VompB, HbpE, and Ppi). The remainder were predicted to be cytoplasmic proteins, and many of these cytoplasmic proteins have been identified previously in the outer membrane fractions of other gram-negative organisms, including other Bartonella species (6,16,42). Each of the 24 immunoreactive antigens commonly recognized by patients' sera was labeled in the immunoblot shown in 3.…”

Pasteurella

“…However, while DNA microarray analyses comparing P. multocida gene expression during growth in iron -rich and iron -depleted medium identifi ed a large range of genes with altered expression; proteomic studies have revealed relatively few OMPs with altered expression in iron -depleted medium Boyce et al 2006 ).…”

Widespread Occurrence of Non-Enzymatic Deamidations of Asparagine Residues in Yersinia pestis Proteins Resulting from Alkaline pH Membrane Extraction Conditions

“…Spot trains of membraneassociated proteins were profiled in 2-DE gels after extraction/wash steps with Na 2 CO 3 at the pH of 11 in E. coli [27] and Pseudomonas aeruginosa [28]. Similar spot trains were also detected for proteins after extraction/wash steps with lauryl sarcosine at the pH of 7.5-7.8, in Helicobacter pylori [29] and Pasteurella multocida [30]. Two studies reported spot trains for membrane-associated proteins, following cell lysis at a pH of 7.5-8.0 without detergent or carbonate wash steps, in Borrelia burgdorferi [31] and E. coli [21].…”