Complete Killing of Caenorhabditis elegans by Burkholderia pseudomallei Is Dependent on Prolonged Direct Association with the Viable Pathogen

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

<i>B. pseudomallei</i> diffusible toxins alone are not sufficient to mediate full killing effect.

<p>(A and B) One-day old Glp worms were transferred to individual <i>B. pseudomallei</i> 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 <i>E. coli</i> 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 <i>B. pseudomallei</i> on a 0.22 µm pore size sterilized nitrocellulose filter. (B) Filtered culture supernatant of <i>B. pseudomallei</i> supplemented with S-basal medium.</p

Distinct isolates of <i>B. pseudomallei</i> kill <i>C. elegans</i> with different kinetics.

<p>(A and B) One-day old Glp worms were transferred to individual <i>B. pseudomallei</i> isolates, 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 <i>E. coli</i> OP50 (open squares). Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (A) <i>B. pseudomallei</i> grown on NG medium. (B) <i>B. pseudomallei</i> grown on PG medium.</p

Calculated TD_mean for <i>B. pseudomallei</i>-infected <i>C. elegans</i> on NG, PG and PGS medium.

1<p>Not done.</p

Description of <i>B. pseudomallei</i> isolates utilized in this study.

<p>Description of <i>B. pseudomallei</i> isolates utilized in this study.</p

Calculated TD_mean for <i>B. pseudomallei</i> isolates in infected <i>C. elegans</i> and mice.

1<p>Data not available.</p

<i>B. pseudomallei</i> does not persistently accumulate within the <i>C. elegans</i> gut.

<p>(A) One-day old Glp worms fed with <i>B. pseudomallei</i> Human R15 (straight line) and <i>E. coli</i> OP50 (dashed line) for 4 hours (open triangles), 12 hours (open circles) and 20 hours (open squares) then shifted to <i>E. coli</i> OP50. Graph shows the mean ± SD of three replicates (30 worms/replicate) from a representative of 3 independent experiments. (B and C) Representative N2 worm infected with R15-GFP. These merged images show bacterial green fluorescence at the anterior intestinal lumen (black arrow head). (B) 4 hours post-infection. (C) 24 hours post-infection. (D–E) Representative <i>tnt-3(aj3)</i> mutant worm infected with R15-GFP for 6 hours and transferred to <i>E. coli</i> OP50. These merged images show bacterial fluorescence (green channel) (black arrow head) and the auto-fluorescence (red channel) (white arrow). (D) 2 hours post-shifting at the anterior intestine (E) 2 hours post-shifting at the posterior intestine. (F) Representative fully colonized <i>tnt-3(aj3)</i> mutant worm infected with R15-GFP. These merged images show bacterial green fluorescence at the anterior intestinal lumen (black arrow head). (G) Ingested R15-GFP was eradicated from the worm gut via defecation within a short period of time upon transfer to OP50 lawn. Columns represent mean ± SEM from two independent experiments.</p