Abstract
Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.
Highlights
Burkholderia pseudomallei is a Gram-negative soil dwelling saprophyte, and an opportunistic pathogen responsible for the severe tropical disease melioidosis [1]
Mice infected with B. pseudomallei disulfide bond forming protein A (DsbA) knockouts have an increased rate of survival compared with mice infected with wild type B. pseudomallei [34, 35]. These findings suggest that many B. pseudomallei virulence factors are substrates of DsbA, as is observed in Escherichia coli [36, 37], Klebsiella pneumoniae [38], Salmonella enterica [39], Francisella tularensis [40] and many more [30, 31, 41]
Our analysis found that B. pseudomallei encodes homologues of 15 DsbA substrates (Table 3)
Summary
Burkholderia pseudomallei is a Gram-negative soil dwelling saprophyte, and an opportunistic pathogen responsible for the severe tropical disease melioidosis [1]. B. pseudomallei infections are difficult to treat [2,3,4] and are intrinsically resistant to almost all available antibiotics [5,6,7,8]. Predominant resistance factors utilised by B. pseudomallei include a thick, impermeable cell. Identification of potential B. pseudomallei DsbA substrates involved in virulence study design, data collection and analysis, decision to publish, or preparation of the manuscript
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