Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

Bast, Antje; Schmidt, Imke H. E.; Brauner, Paul; Brix, Bettina; Breitbach, Katrin; Steinmetz, Ivo

doi:10.3389/fmicb.2011.00277

Cited by 13 publications

(13 citation statements)

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“…To further demonstrate that cytosolic presence of the pathogen is essential for caspase-1 activation, we included a B. pseudomallei E8 T3SS3 mutant that harbours a defect in the bsaU gene (BPSS1539). We previously found a transposon mutant of BsaU to be trapped within lysosomal-associated membrane protein-1 (LAMP-1) positive lysosomes after B. pseudomallei E8 infection and with a delayed escape into the cytosol [31], [32]. In the present study infection of macrophages with a deletion mutant of the putative needle length control protein BsaU (ΔBsaU) did not result in any processing of caspases or PARP in the early phase (Figure 6).…”

Section: Resultssupporting

confidence: 49%

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

et al. 2014

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The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β 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-1β 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-1β, 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.

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confidence: 49%

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

et al. 2014

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“…5C), a B. pseudomallei mutant with a defect in the bsaU gene (Bp⌬bsaU) belonging to the apparatus of the T3SS cluster 3 was included as a control. The Bp⌬bsaU strain was previously found to be trapped within LAMP-1-positive lysosomes after infection and showed delayed escape into the cytosol (7,35). In addition, the T3SS3 bsa locus was reported not to be directly required for MNGC formation (36).…”

Section: Resultsmentioning

confidence: 99%

BPSS1504 , a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

et al. 2014

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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 (Bp⌬BPSS1504) 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 Bp⌬BPSS1504 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.

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“…There is also a known role for reactive oxygen species (ROS) responses due to IFN-␥ stimulation in controlling early infection with B. pseudomallei (16,18). While the effects of IFN-␥ on infected macrophages have been studied in great detail (18)(19)(20)(21)(22), there are few studies that look at the combination of immune stimulation with antibiotic treatment, even though this scenario is realistically encountered in patients treated for melioidosis. Our lab has studied these immuno-antimicrobial interactions and has previously shown that treatment with IFN-␥ can synergistically enhance the effect of ceftazidime treatment in controlling B. pseudomallei infection, both in vitro and in vivo, by significantly reducing intracellular bacterial burden (23).…”

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Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Mosovsky

Silva

Troyer

et al. 2014

Antimicrob Agents Chemother

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Burkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-␥) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-␥-and ceftazidime-mediated killing, which led to our hypothesis that IFN-␥-induced ROS interacted with ceftazidime to synergistically kill Burkholderia bacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-␥ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-␥ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 g/ml. We investigated the exact role of IFN-␥-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated with Burkholderia bacteria in IFN-␥-treated macrophages, which led to our finding that IFN-␥-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-␥-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity against Burkholderia bacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

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Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

Cited by 13 publications

References 51 publications

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

BPSS1504 , a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

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