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Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen.

Lee SH, Ooi SK, Mahadi NM, Tan MW, Nathan S - PLoS ONE (2011)

Bottom Line: Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.A combination of genetic and environmental factors affects virulence.In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, UKM Bangi, Selangor, Malaysia.

ABSTRACT

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/principal findings: In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/significance: A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.

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Related in: MedlinePlus

B. pseudomallei diffusible toxins alone are not sufficient to mediate full killing effect.(A and B) One-day old Glp worms were transferred to individual B. pseudomallei free culture, Human PMC2000 (open diamonds), Human D286 (open triangles), Human R15 (crosses), Human H10 (open circles), Rabbit 2514 (closed squares), Sheep 4523 (closed triangles), Ostrich 9166 (closed circles) and E. coli OP50 (open squares). Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (A) Conditioned NG medium with grown B. pseudomallei on a 0.22 µm pore size sterilized nitrocellulose filter. (B) Filtered culture supernatant of B. pseudomallei supplemented with S-basal medium.
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pone-0016707-g002: B. pseudomallei diffusible toxins alone are not sufficient to mediate full killing effect.(A and B) One-day old Glp worms were transferred to individual B. pseudomallei free culture, Human PMC2000 (open diamonds), Human D286 (open triangles), Human R15 (crosses), Human H10 (open circles), Rabbit 2514 (closed squares), Sheep 4523 (closed triangles), Ostrich 9166 (closed circles) and E. coli OP50 (open squares). Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (A) Conditioned NG medium with grown B. pseudomallei on a 0.22 µm pore size sterilized nitrocellulose filter. (B) Filtered culture supernatant of B. pseudomallei supplemented with S-basal medium.

Mentions: To determine if diffusible secreted factors contribute to B. pseudomallei-mediated killing of C. elegans, we assayed the survival of C. elegans exposed to culture filtrates alone. This was achieved by growing B. pseudomallei isolates on a 0.22 µm nitrocellulose filter placed over the media surface. As the filter pore size permits passage of secreted molecules but not the bacteria, any observed killing of the nematode population upon removal of the filter can be ascribed to the presence of diffusible toxin(s). When the filter assay was carried out on NG medium, with the exception of Human PMC2000, killing by the other isolates was negligible (Figure 2A). To validate the effect of B. pseudomallei secreted factors on worm mortality, we used bacterial cell-free culture filtrate and repeated the killing assay. Filtrates from all the isolates had negligible effects on worm mortality up to 76 hours of exposure (Figure 2B). Data obtained from both the filter and bacteria cell-free culture filtrate assays above demonstrated that, for all the isolates tested, secreted molecules alone were insufficient to mediate full killing. In addition, heat-killed B. pseudomallei also failed to cause worm mortality (data not shown). We therefore conclude that the lethal effects of B. pseudomallei require direct and prolonged contact between the viable bacteria and C. elegans.


Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen.

Lee SH, Ooi SK, Mahadi NM, Tan MW, Nathan S - PLoS ONE (2011)

B. pseudomallei diffusible toxins alone are not sufficient to mediate full killing effect.(A and B) One-day old Glp worms were transferred to individual B. pseudomallei free culture, Human PMC2000 (open diamonds), Human D286 (open triangles), Human R15 (crosses), Human H10 (open circles), Rabbit 2514 (closed squares), Sheep 4523 (closed triangles), Ostrich 9166 (closed circles) and E. coli OP50 (open squares). Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (A) Conditioned NG medium with grown B. pseudomallei on a 0.22 µm pore size sterilized nitrocellulose filter. (B) Filtered culture supernatant of B. pseudomallei supplemented with S-basal medium.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049773&req=5

pone-0016707-g002: B. pseudomallei diffusible toxins alone are not sufficient to mediate full killing effect.(A and B) One-day old Glp worms were transferred to individual B. pseudomallei free culture, Human PMC2000 (open diamonds), Human D286 (open triangles), Human R15 (crosses), Human H10 (open circles), Rabbit 2514 (closed squares), Sheep 4523 (closed triangles), Ostrich 9166 (closed circles) and E. coli OP50 (open squares). Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (A) Conditioned NG medium with grown B. pseudomallei on a 0.22 µm pore size sterilized nitrocellulose filter. (B) Filtered culture supernatant of B. pseudomallei supplemented with S-basal medium.
Mentions: To determine if diffusible secreted factors contribute to B. pseudomallei-mediated killing of C. elegans, we assayed the survival of C. elegans exposed to culture filtrates alone. This was achieved by growing B. pseudomallei isolates on a 0.22 µm nitrocellulose filter placed over the media surface. As the filter pore size permits passage of secreted molecules but not the bacteria, any observed killing of the nematode population upon removal of the filter can be ascribed to the presence of diffusible toxin(s). When the filter assay was carried out on NG medium, with the exception of Human PMC2000, killing by the other isolates was negligible (Figure 2A). To validate the effect of B. pseudomallei secreted factors on worm mortality, we used bacterial cell-free culture filtrate and repeated the killing assay. Filtrates from all the isolates had negligible effects on worm mortality up to 76 hours of exposure (Figure 2B). Data obtained from both the filter and bacteria cell-free culture filtrate assays above demonstrated that, for all the isolates tested, secreted molecules alone were insufficient to mediate full killing. In addition, heat-killed B. pseudomallei also failed to cause worm mortality (data not shown). We therefore conclude that the lethal effects of B. pseudomallei require direct and prolonged contact between the viable bacteria and C. elegans.

Bottom Line: Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.A combination of genetic and environmental factors affects virulence.In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, UKM Bangi, Selangor, Malaysia.

ABSTRACT

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/principal findings: In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/significance: A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.

Show MeSH
Related in: MedlinePlus