Katrin Breitbach

Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

ABSTRACT The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

Role of Inducible Nitric Oxide Synthase and NADPH Oxidase in Early Control of Burkholderia pseudomallei Infection in Mice

ABSTRACT Infection with the soil bacterium Burkholderia pseudomallei can result in a variety of clinical outcomes, including asymptomatic infection. The initial immune defense mechanisms which might contribute to the various outcomes after environmental contact with B. pseudomallei are largely unknown. We have previously shown that relatively resistant C57BL/6 mice can restrict bacterial B. pseudomallei growth more efficiently within 1 day after infection than highly susceptible BALB/c mice. By using this model, our study aimed to investigate the role of macrophage-mediated effector mechanisms during early B. pseudomallei infection. Depletion of macrophages revealed an essential role of these cells in the early control of infection in BALB/c and C57BL/6 mice. Strikingly, the comparison of the anti-B. pseudomallei activity of bone marrow-derived macrophages (BMM) from C57BL/6 and BALB/c mice revealed an enhanced bactericidal activity of C57BL/6 BMM, particularly after gamma interferon (IFN-γ) stimulation. In vitro experiments with C57BL/6 gp91phox−/− BMM showed an impaired intracellular killing of B. pseudomallei compared to experiments with wild-type cells, although C57BL/6 gp91phox−/− cells still exhibited substantial killing activity. The anti-B. pseudomallei activity of C57BL/6 iNOS−/− BMM was not impaired. C57BL/6 gp91phox−/− mice lacking a functional NADPH oxidase were more susceptible to infection, whereas C57BL/6 mice lacking inducible nitric oxide synthase (iNOS) did not show increased susceptibility but were slightly more resistant during the early phase of infection. Thus, our data suggest that IFN-γ-mediated but iNOS-independent anti-B. pseudomallei mechanisms of macrophages might contribute to the enhanced resistance of C57BL/6 mice compared to that of BALB/c mice in the early phase of infection.

Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteins.

The facultative intracellular bacterium Burkholderia pseudomallei induces actin rearrangement within infected host cells leading to formation of actin tails and membrane protrusions. To investigate the underlying mechanism we analysed the contribution of cytoskeletal proteins to B. pseudomallei‐induced actin tail assembly. By using green fluorescent protein (GFP)‐fusion constructs, the recruitment of the Arp2/3 complex, vasodilator‐stimulated phosphoprotein (VASP), Neural Wiskott–Aldrich syndrome protein (N‐WASP), zyxin, vinculin, paxillin and α‐actinin to the surface of B. pseudomallei and into corresponding actin tails was studied. In addition, antibodies against the same panel of proteins were used for immunolocalization. Whereas the Arp2/3 complex and α‐actinin were incorporated into B. pseudomallei‐induced actin tails, none of the other proteins were detected in these structures. The overexpression of an Arp2/3 binding fragment of the Scar1 protein, shown previously to block actin‐based motility of Listeria, had no effect on B. pseudomallei tail formation. Infections of either N‐WASP‐ or Ena/VASP‐defective cells showed that these proteins are not essential for B. pseudomallei‐induced actin polymerization. In conclusion, our results suggest that B. pseudomallei induces actin polymerization through a mechanism that differs from those evolved by Listeria, Shigella, Rickettsia or vaccinia virus.

Induction of protective immunity against Burkholderia pseudomallei using attenuated mutants with defects in the intracellular life cycle

Melioidosis is a severe infectious disease caused by the Gram-negative rod Burkholderia pseudomallei. There is currently no vaccine available. We recently generated and characterized several highly attenuated transposon mutants with defects in the intracellular life cycle of B. pseudomallei. In the present study we examined the protective effects of six of these mutants: four harbouring knockouts in genes involved in several biosynthetic pathways (purN(-), purM(-), hisF(-), pabB(-)); a putative lipoate-protein ligase B; and a hypothetical protein. All live mutants conferred protection to some degree against wild-type challenge in susceptible BALB/c mice. Two mutants defective in distinct steps of the purine biosynthetic pathway were selected for further studies. Mutant 30:93 with a defect in the purN gene provided better protection against intraperitoneal challenge than mutant 56:65, which harboured a nonfunctional purM gene. Although mutant 30:93 conferred significant protection against acute fatal disease after intranasal and intraperitoneal challenge with B. pseudomallei, vaccination did not confer protection against chronic forms of melioidosis. Moreover, no protective effect could be seen against intravenous challenge. Further studies are required to analyze the precise nature of the immune response induced by the various live attenuated vaccines with different protective potential.

Caspase-1 Mediates Resistance in Murine Melioidosis

ABSTRACT The gram-negative rod Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease which is endemic in tropical and subtropical areas. The bacterium multiplies intracellularly within the cytosol, induces the formation of actin tails, and can spread directly from cell to cell. Recently, it has been shown that B. pseudomallei can induce caspase-1-dependent cell death in macrophages. The aim of the present study was to further elucidate the role of caspase-1 during B. pseudomallei infection. In vivo experiments with caspase-1−/− mice revealed a high susceptibility to B. pseudomallei challenge. This phenotype was associated with a significantly higher bacterial burden 2 days after infection and decreased gamma interferon (IFN-γ) and interleukin-18 cytokine levels 24 h after infection compared to control animals. caspase-1−/− bone marrow-derived macrophages (BMM) exhibited strong caspase-3 expression and reduced cell damage compared to wild-type (WT) cells during early B. pseudomallei infection, indicating “classical” apoptosis, whereas WT BMM showed signs of rapid caspase-1-dependent cell death. Moreover, we found that caspase-1−/− BMM had a strongly increased bacterial burden compared to WT cells 3 h after infection under conditions where no difference in cell death could be observed between both cell populations at this time point. We therefore suggest that caspase-1-dependent rapid cell death might contribute to resistance by reducing the intracellular niche for B. pseudomallei, but, in addition, caspase-1 might also have a role in controlling intracellular replication of B. pseudomallei in macrophages. Moreover, caspase-1-dependent IFN-γ production is likely to contribute to resistance in murine melioidosis.

Generation of murine bone marrow derived macrophages in a standardised serum-free cell culture system.

Murine bone marrow derived macrophages (BMM) are valuable tools to investigate macrophage functions such as cytokine production and bactericidal activities from different strains of mice. In most studies BMM are generated and characterised using cell culture systems with fetal calf serum (FCS) as an essential supplement. Since serum contains varying amounts of undefined components influencing the maturation and polarisation process of BMM there is a need for a more standardised methodology. The aim of the present study was to establish a cell culture system for the generation of murine BMM under standardised serum free conditions. The use of a newly developed compositionally defined serum supplement enabled us to gain mature BMM from BALB/c and C57BL/6 mice expressing the myeloid marker F4/80, CD11b and MOMA-2. Under these serum-free conditions LPS and IFN-gamma stimulated C57BL/6 BMM released more IL-12 and nitric oxide (NO) compared to BALB/c BMM whereas the latter cells produced higher levels of IL-10 and MCP-1 after LPS stimulation. Serum-free generated C57BL/6 BMM showed enhanced bactericidal activity against the Gram-negative rod Burkholderia pseudomallei compared to BALB/c BMM. In conclusion the serum-free generation of BMM described in this study will assure more standardised and reproducible conditions for the future characterisation of murine BMM.

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-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.

NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox⁻/⁻ mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS⁻/⁻) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS-/- nor from gp91phox⁻/⁻ mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.

Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis

Background Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis. Methods Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×102 cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM). Results Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion. Conclusions Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.

Distinct roles for nitric oxide in resistant C57BL/6 and susceptible BALB/c mice to control Burkholderia pseudomallei infection

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial infectious disease in tropical and subtropical areas. We recently showed that NADPH oxidase but not nitric oxide (NO) contributes to resistance in innately resistant C57BL/6 mice in a B. pseudomallei respiratory infection model. However, the function of NO for resistance was shown to differ among distinct strains of mice and proved also to be stage dependent in various infection models. The present study therefore aimed to examine the role of NO in a systemic infection model of melioidosis and to test whether the function of NO differs among innately resistant C57BL/6 and susceptible BALB/c mice after B. pseudomallei infection.ResultsC57BL/6 iNOS-/- mice that were intravenously infected with B. pseudomallei survived several weeks, whereas most of the wild type animals succumbed during this period. The bacterial burden in liver and spleen was significantly higher in wild type animals compared to iNOS-/- mice 13 days after challenge. In contrast, BALB/c mice that were treated with amminoguanidine to inhibit NO expression in vivo showed significantly enhanced mortality rates and higher bacterial loads in liver and spleen compared to control animals. The bactericidal function of IFN-γ stimulated C57BL/6 iNOS-/- macrophages were not altered after B. pseudomallei infection, but BALB/c macrophages exhibited reduced killing activity against the pathogen when NO was inhibited.ConclusionOur present data indicate a dual role of NO among resistant and susceptible mouse strains after B. pseudomallei infection. NO mediated mechanisms are an essential component to control the infection in susceptible BALB/c mice. In contrast, NO production in B. pseudomallei infected C57BL/6 mice rather harmed the host likely due to its detrimental effects.