Role for the Burkholderia pseudomallei type three secretion system cluster 1 bpscN gene in virulence

Abstract

Burkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. The B. pseudomallei genome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved in B. pseudomallei virulence in a hamster infection model. We have characterized a B. pseudomallei mutant lacking expression of the predicted TTSS1 ATPase encoded by bpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analyses in vitro showed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1 bpscN gene product plays an important role in the intracellular survival of B. pseudomallei and the pathogenesis of murine infection.

Fig. 1.

Kaplan-Meier survival curves for mice infected with either 2 × 10⁷ CFU (A) or 2 × 10⁵ CFU (B) of wild-type or bpscN mutant B. pseudomallei.

Fig. 2.

Bacterial survival and replication within RAW264.7 macrophage-like cells infected at an MOI of 6 with wild-type B. pseudomallei, the bpscN mutant, the bpscN mutant expressing the empty pBHR1 plasmid, or the bpscN mutant complemented (comp) with an intact bpscN gene. Error bars indicate the standard error of the mean of nine biological replicates. *, P < 0.05; **, P < 0.001.

Fig. 3.

TEM analysis of the intracellular locations of wild-type and bpscN mutant B. pseudomallei bacteria in infected RAW264.7 macrophage-like cells. Bacteria were scored as free in the cytoplasm or encapsulated in a single membrane. At least 100 bacteria were scored for both strains in triplicate experiments. Error bars represent the standard error of the mean of biological triplicates.

Fig. 4.

Colocalization of wild-type and bpscN mutant bacteria with LC3-GFP. RAW264.7 macrophage-like cells stably expressing LC3-GFP were infected with wild-type or bpscN mutant B. pseudomallei bacteria and viewed at 2, 4, and 6 h p.i. (A) Bacterial colocalization with LC3-GFP was defined by the presence of labeled bacteria (red) (yellow fluorescence) which were fully overlaid by intense green or fully contained within a green ring. Scale bars, 5 μm. (B) Quantitative analysis of bacterial colocalization with LC3-GFP. The experiment was performed in biological triplicates, and data are presented as the mean ± the standard error of the mean. *, P < 0.01.