Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

Background: Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. Methods: The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. Results: Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB–OprA, BpeAB–OprB, BpeEF–OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB–OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. Conclusions: Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei. Author summary: Meliodosis and glanders are both potential biological threat agents. Glanders is endemic in Southeast Asia and Northern Australia but occasionally found in other countries. Meliodosis is caused by Burkholderia pseudomallei, while glanders is caused by Burkholderia mallei. The diagnosis of these diseases is difficult because the clinical picture varies and the causative agents are difficult to identify. There is no vaccine available and antimicrobial therapy is lengthy and with an uncertain outcome. This study was conducted to determine whether certain characteristics are or aren't expressed by multiple isolates from the same species. Therefore, the proteins expressed by 5 B. mallei and 6 B. pseudomallei were characterized using LC-HRMS/MS. Results of this study demonstrated; The homogeneity in B. mallei isolates and the diversity in B. pseudomallei isolates. That proteomic analysis can be used to identify B. pseudomallei and B. mallei. The need to study multiple isolates of a pathogen to determine whether a particular virulence factor or antimicrobial related protein is important for the species to be pathogenic or confer antimicrobial resistance. The expression of multiple virulence factors, proteins of several PKS/NRPS clusters and antimicrobial related proteins was demonstrated. No relation between phenotype and antimicrobial proteins could be made. The demonstrated expression of virulence factors in all isolates can be used for the development of new diagnostic assays or drug development. Proteome analysis of infectious bacteria can be used to identify the species but also to characterize the presence of important factors that putatively contribute to the pathogenesis of the species. [ABSTRACT FROM AUTHOR]