Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei.

Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P < 0.001) and 9.0 (95% CI, 3.1 to 26.4; P < 0.001), respectively. High B. pseudomallei genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the B. pseudomallei detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomalleiIMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect significantly higher numbers of B. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent B. pseudomallei-specific qPCR targets further increased the detection rate of B. pseudomallei compared with that from single targets. Samples with a high molecular B. pseudomallei load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of B. pseudomallei infections in different parts of the world.

Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1ß and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1ß secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1ß, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.

BPSS1504, a cluster 1 type VI secretion gene, is involved in intracellular survival and virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (BpΔBPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The BpΔBPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

Caspase-6 mediates resistance against Burkholderia pseudomallei infection and influences the expression of detrimental cytokines.

Caspase-6 is a member of the executioner caspases and known to play a role in innate and adaptive immune processes. However, its role in infectious diseases has rarely been addressed yet. We here examined the impact of caspase-6 in an in vivo infection model using the Gram-negative rod Burkholderia pseudomallei, causing the infectious disease melioidosis that is endemic in tropical and subtropical areas around the world. Caspase-6-/- and C57BL/6 wild type mice were challenged with B. pseudomallei for comparing mortality, bacterial burden and inflammatory cytokine expression. Bone-marrow derived macrophages were used to analyse the bactericidal activity in absence of caspase-6. Caspase-6 deficiency was associated with higher mortality and bacterial burden in vivo after B. pseudomallei infection. The bactericidal activity of caspase-6-/- macrophages was impaired compared to wild type cells. Caspase-6-/- mice showed higher expression of the IL-1ß gene, known to be detrimental in murine melioidosis. Expression of the IL-10 gene was also increased in caspase-6-/- mice as early as 6 hours after infection. Treatment with exogenous IL-10 rendered mice more susceptible against B. pseudomallei challenge. Thus, caspase-6 seems to play a crucial role for determining resistance against the causative agent of melioidosis. To our knowledge this is the first report showing that caspase-6 is crucial for mediating resistance in an in vivo infection model. Caspase-6 influences the expression of detrimental cytokines and therefore seems to be important for achieving a well-balanced immune response that contributes for an efficient elimination of the pathogen.