RESUMO
Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.
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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.
Assuntos
Burkholderia pseudomallei/genética , Caspase 6/genética , Interleucina-10/administração & dosagem , Interleucina-1beta/biossíntese , Melioidose/genética , Animais , Burkholderia pseudomallei/patogenicidade , Resistência Microbiana a Medicamentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/biossíntese , Interleucina-1beta/genética , Macrófagos/metabolismo , Macrófagos/patologia , Melioidose/microbiologia , Melioidose/patologia , Camundongos , Camundongos KnockoutRESUMO
Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91phox KO mice. Only infected gp91phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis.
Assuntos
Aggregatibacter actinomycetemcomitans , Resistência à Doença , NADPH Oxidases/metabolismo , Periodontite/metabolismo , Periodontite/microbiologia , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/patologia , Animais , Carga Bacteriana , Densidade Óssea , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoclastos/metabolismo , Periodontite/diagnóstico , Periodontite/genéticaRESUMO
Macrophages are essential components of the innate immune system and crucial for pathogen elimination in early stages of infection. We previously observed that bone marrow-derived macrophages (BMMs) from C57BL/6 mice exhibited increased killing activity against Burkholderia pseudomallei compared to BMMs from BALB/c mice. This effect was particularly pronounced when cells were treated with IFN-γ. To unravel mechanisms that could explain these distinct bactericidal effects, a comparative combined proteome and transcriptome analysis of untreated and IFN-γ treated BALB/c and C57BL/6 BMMs under standardized serum-free conditions was carried out. We found differences in gene expression/protein abundance belonging to cellular oxidative and antioxidative stress systems. Genes/proteins involved in the generation of oxidant molecules and the function of phagosomes (respiratory chain ATPase, lysosomal enzymes, cathepsins) were predominantly higher expressed/more abundant in C57BL/6 BMMs. Components involved in alleviation of oxidative stress (peroxiredoxin, mitochondrial superoxide dismutase) were more abundant in C57BL/6 BMMs as well. Thus, C57BL/6 BMMs seemed to be better equipped with cellular systems that may be advantageous in combating engulfed pathogens. Simultaneously, C57BL/6 BMMs were well protected from oxidative burst. We assume that these variations co-determine differences in resistance between BALB/c and C57BL/6 mice observed in many infection models. BIOLOGICAL SIGNIFICANCE: In this study we performed combined transcriptome and proteome analyses on BMMs derived from two inbred mouse strains that are frequently used for studies in the field of host-pathogen interaction research. Strain differences between BALB/c and C57BL/6 BMMs were found to originate mainly from different protein abundance levels rather than from different gene expression. Differences in abundance of respiratory chain complexes and lysosomal proteins as well as differential regulation of components belonging to various antioxidant stress systems help to explain long-known differences between the mouse strains concerning their different susceptibility in several infection models.
Assuntos
Interferon gama/farmacologia , Macrófagos/metabolismo , Animais , Antioxidantes/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Burkholderia pseudomallei/efeitos dos fármacos , Transporte de Elétrons , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteoma , TranscriptomaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Melioidose/microbiologia , Actinas/metabolismo , Animais , Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Feminino , Melioidose/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Organismos Livres de Patógenos Específicos , Virulência , Fatores de VirulênciaRESUMO
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.
Assuntos
Infecções por Burkholderia/imunologia , Burkholderia pseudomallei/imunologia , Inflamassomos/imunologia , Macrófagos/microbiologia , Transdução de Sinais/imunologia , Animais , Sistemas de Secreção Bacterianos/fisiologia , Western Blotting , Infecções por Burkholderia/metabolismo , Burkholderia pseudomallei/metabolismo , Caspase 1/metabolismo , Morte Celular/fisiologia , Modelos Animais de Doenças , Citometria de Fluxo , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , TransfecçãoRESUMO
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 × 10(2) 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) 1ß 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 IL1ß 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 IL1ß 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 IL1ß 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.
Assuntos
Anti-Inflamatórios/uso terapêutico , Burkholderia pseudomallei/efeitos dos fármacos , Glibureto/uso terapêutico , Melioidose/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Ceftazidima/uso terapêutico , Complicações do Diabetes , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Resultado do TratamentoRESUMO
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 (gp91phoxâ»/â» mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOSâ»/â») mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS-/- nor from gp91phoxâ»/â» 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.
Assuntos
NADPH Oxidases/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Pneumonia Estafilocócica/imunologia , Staphylococcus aureus/imunologia , Animais , Modelos Animais de Doenças , Feminino , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Doenças dos Roedores/imunologiaRESUMO
Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca(2+)-independent phospholipase A(2) activities. We have recently shown that exposure of murine bone marrow-derived macrophages to LPS and IFN-γ leads to induction of COX-2 expression and secretion of PGE(2), up-regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their ability to induce gene expression of Prxs, in particular Prx 6, and to determine the underlying regulatory mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD(2) or PGE(2). Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD(2)- or PGE(2)-dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and identified Nrf2 as a critical player mediating transcriptional induction.
Assuntos
Dinoprostona/metabolismo , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peroxirredoxina VI/metabolismo , Prostaglandina D2/metabolismo , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Dinoprostona/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Peroxirredoxina VI/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina D2/genética , Proteína Quinase C/metabolismo , Ativação Transcricional/genéticaRESUMO
Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/patogenicidade , Melioidose/microbiologia , Peptidilprolil Isomerase/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/genética , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Conformação Proteica , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Virulência , beta-Lactamas/farmacologiaRESUMO
BACKGROUND: Burkholderia 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. RESULTS: C57BL/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. CONCLUSION: Our 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.
Assuntos
Burkholderia pseudomallei/imunologia , Suscetibilidade a Doenças , Melioidose/imunologia , Óxido Nítrico/metabolismo , Animais , Burkholderia pseudomallei/patogenicidade , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Guanidina/administração & dosagem , Guanidina/análogos & derivados , Guanidina/farmacologia , Humanos , Imunidade Ativa , Ativação de Macrófagos/efeitos dos fármacos , Melioidose/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/genética , Especificidade da EspécieRESUMO
The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1ß, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.
RESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro , Macrófagos/citologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Burkholderia pseudomallei/imunologia , Quimiocina CCL2/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismoRESUMO
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-gamma) 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-gamma production is likely to contribute to resistance in murine melioidosis.
Assuntos
Burkholderia pseudomallei/patogenicidade , Caspase 1/metabolismo , Melioidose/imunologia , Animais , Apoptose , Células da Medula Óssea/citologia , Burkholderia pseudomallei/crescimento & desenvolvimento , Burkholderia pseudomallei/fisiologia , Regulação para Baixo , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-18/metabolismo , Macrófagos/citologia , Macrófagos/microbiologia , Masculino , Melioidose/microbiologia , Melioidose/mortalidade , Camundongos , Camundongos Endogâmicos C57BLRESUMO
CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4(-/-)) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4(-/-) mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4(-/-) animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4(-/-) mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.
Assuntos
Receptores CCR4/metabolismo , Sepse/imunologia , Animais , Apoptose/imunologia , Quimiocina CCL17/imunologia , Quimiocina CCL17/metabolismo , Quimiocina CCL22/imunologia , Quimiocina CCL22/metabolismo , Quimiocinas/imunologia , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/imunologia , Peritonite/metabolismo , Receptores CCR4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Sporadic cases of melioidosis have been reported from Vietnam for decades, but clinical and epidemiological data for the indigenous population are still scarce. In this study, we reviewed clinical and demographic data of patients with culture-proven melioidosis diagnosed at a single large referral hospital in Hanoi between November 1997 and December 2005. We found that the clinical manifestations of melioidosis (with fatal septicaemia as the most common presentation), a high rate of underlying diseases, and a peak of cases admitted during the wet season, were similar to studies from other endemic areas. The geographical origin of patients with melioidosis showed that melioidosis existed in at least 18 northern provinces. The characterization of clinical Burkholderia pseudomallei strains by multilocus sequence typing identified 17 different sequence types (STs), 11 of which have (as yet) not been found outside Vietnam. Several of these STs presumably were generated through recent evolutionary events in this rapidly diversifying bacterial species, and thus, restricted geographic distribution may be a consequence of limited time passed since emergence. To our knowledge, this is the first report on a series of cases describing clinical and epidemiological features of melioidosis and corresponding B. pseudomallei strains from northern Vietnam.
Assuntos
Burkholderia pseudomallei/isolamento & purificação , Melioidose/microbiologia , Estações do Ano , Sepse/microbiologia , Adolescente , Adulto , Idoso , Burkholderia pseudomallei/genética , Criança , Feminino , Humanos , Masculino , Melioidose/epidemiologia , Melioidose/genética , Pessoa de Meia-Idade , Sepse/epidemiologia , Sepse/genética , Fatores de Tempo , Vietnã/epidemiologia , Adulto JovemRESUMO
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.
Assuntos
Vacinas Bacterianas/imunologia , Burkholderia pseudomallei/imunologia , Genes Bacterianos/imunologia , Melioidose/prevenção & controle , Animais , Burkholderia pseudomallei/genética , Ciclo Celular , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/imunologia , Genes Bacterianos/genética , Melioidose/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Vacinas Atenuadas/imunologiaRESUMO
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-gamma) 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-gamma-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.
Assuntos
Burkholderia pseudomallei , Melioidose/enzimologia , Melioidose/imunologia , NADPH Oxidases/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Células Cultivadas , Feminino , Macrófagos/enzimologia , Macrófagos/microbiologia , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genéticaRESUMO
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.
Assuntos
Burkholderia pseudomallei/genética , Genes Bacterianos/fisiologia , Ácido 4-Aminobenzoico/metabolismo , Actinas/metabolismo , Animais , Proteínas de Bactérias/fisiologia , Burkholderia pseudomallei/crescimento & desenvolvimento , Burkholderia pseudomallei/patogenicidade , Elementos de DNA Transponíveis , Feminino , Histidina/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Purinas/biossíntese , Vacúolos/microbiologia , VirulênciaRESUMO
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 alpha-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 alpha-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.