Protease production by Burkholderia pseudomallei and virulence in mice.
ABSTRACT The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6-6 x 10(5) CFU, and the LD50 was calculated after 30 days by the method of Reed and Muench. The lethal activity was studied for five strains of B. pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia. The three type strains appeared to be low protease producers (A405 = 0.11, 0.09 and 0.00, respectively) and avirulent. The two more virulent B. pseudomallei strains exhibited significantly different LD50, 3.5 x 10(2) (IPP 6068 VIR) versus 2.1 x 10(5) CFU/mouse (40/97), and protease activities (A405 = 0.046 and 0.79, respectively). Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A405 = 0.26 versus 0.046. These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B. pseudomallei is injected to mice via the intraperitoneal route.
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ABSTRACT: The environmental saphrophyte Burkholderia pseudomallei is the causative agent of melioidosis, a systemic, potentially life-threatening condition endemic to many parts of south-east Asia and northern Australia. We have used the soil nematode Caenorhabditis elegans as a model host to characterize the mechanisms by which this bacterium mounts a successful infection. We find that C. elegans is susceptible to a broad range of Burkholderia species, and that the virulence mechanisms used by this pathogen to kill nematodes may be similar to those used to infect mammals. We also find that the specific dynamics of the C. elegans–B. pseudomallei host–pathogen interaction can be highly influenced by environmental factors, and that nematode killing results at least in part from the presence of a diffusible toxin. Finally, by screening for bacterial mutants attenuated in their ability to kill C. elegans, we genetically identify several new potential virulence factors in B. pseudomallei. The use of C. elegans as a model host should greatly facilitate future investigations into how B. pseudomallei can interact with host organisms.Molecular Microbiology 05/2002; 44(5):1185 - 1197. · 5.03 Impact Factor
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ABSTRACT: Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in Southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalysing the formation of disulfide bonds in secreted and membrane associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A DsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a Balb/C mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family E. coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally, and determined that it is required for virulence of B. pseudomallei. Conclusion: These data establish a critical role for BpsDbsA in B. pseudomallei infection, which in combination with our structural characterisation of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug resistant organism.Antioxidants & Redox Signaling 07/2013; · 8.20 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6×109 colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2×106 to 2×109 CFU g−1. They were still 50–80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 102–104-fold less infected than the spleen, with loads ranging from 3×102 to 3×106 CFU g−1. The heart and the brain were sites of a delayed infection, with counts in a range from 103 to 107 times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8×103 to 6×104 CFU ml−1. Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.FEMS Immunology & Medical Microbiology 01/2001; 30(1):53 - 63. · 2.68 Impact Factor