Dataset Information

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

ABSTRACT: The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

SUBMITTER: Gilling DH

PROVIDER: S-EPMC4897507 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

Gilling Damian H DH Luna Vicki Ann VA Pflugradt Cori C

International scholarly research notices 20141002

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively t ...[more]

PMID: 27350960

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Project description:Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells.The boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.

Project description:BackgroundBurkholderia 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.MethodsThe 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.ResultsProteome 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.ConclusionsAnalyzing 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.

Dataset Information

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

Publications

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

Similar Datasets

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