Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

BACKGROUND: Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae. RESULTS: The locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25°C compared with 37°C or 42°C but was mostly unaffected by pH or salinity, although at 25°C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients. CONCLUSION: The results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may assist our understanding of the reasons for this difference

Molecular Characterisation of the Interaction of Microbes with the Insulin Pathway

Exposure to microorganisms is considered an environmental factor which can contribute to diabetes mellitus via cytotoxicity or autoimmune responses against pancreatic cells. Firstly, the effects on rat insulinoma pancreatic β-cell line of secondary metabolites pyrrolnitrin (Burkholderia spp), phenazine compounds (Pseudomonas aeruginosa and Burkholderia spp) were investigated. Both compounds separately showed significant cytotoxicity after 24 h and at concentrations of 10 & 100 ng/ml potentiated insulin gene transcription, Ca2+ content and glucose-stimulated insulin secretion (GSIS). Furthermore, the outward membrane current was inhibited by phenazine (100 ng/ml) or pyrrolnitrin (10 or 100 ng/ml).Secondly, the capacity of 45 microbial species to bind insulin was screened in order to assess how common insulin binding was amongst microorganisms Burkholderia multivorans, B. cenocepacia and Aeromonas salmonicida bound insulin. A genomic library of B. multivorans was constructed in λ Zap Express and screened successfully for insulin binding recombinants. Recombinant phagemids p1 & p2 were excised, p1, encoded an insulin binding protein (IBP1 30 kDa) with homology to the iron complex siderophore receptor. For p2, two IBPs were detected at 20 & 30 kDa (IBP2 & IBP3), representing an intracellular and outer membrane peptide transporter. Comparison of IBP1 and human insulin receptor (HIR) produced 6 linear epitopes, and for IBP2 & IBP3 produced 3 epitopes.Thirdly, glutamic acid decarboxylase GAD65 is a major pancreatic autoantigen contributing to autoimmune diabetes. To assess the likelihood that microorganisms possess epitopes that mimic regions on GAD, 45 microbial species were tested for homology. This was facilitated by purifying recombinant GAD protein which was used to produce GAD antiserum. Four E. coli cross-reacting proteins were identified using mass spectrometry, outer-membrane protein A, formate dehydrogenase, superoxide dismutase and DNA starvation protein. Epitopes occurred at the C-terminal region of GAD65 (residues 419–565), a region previously reported to be targeted by autoantibodies.This study suggests that pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and B. multivorans IBPs linear epitopes may be diabetogenic, particularly in patients with cystic fibrosis related diabetes (CFRD) who suffer a long term infections with Pseudomonas and Burkholderia species. Furthermore, microbial GAD epitopes could potentially induce an autoimmune response leading to diabetes