RESUMO
PPE37 is a member of the Mycobacterium tuberculosis proline-proline-glutamic acid (PPE) multigene family. Its expression is upregulated in bacteria that are phagocytosed by macrophages and is enhanced even more in bacteria isolated from the lungs of infected mice. This raises the possibility that PPE37 may play a role in the virulence of M. tuberculosis and led to this investigation of the function of PPE37. Recombinant bacterial strains, one expressing the M. tuberculosis PPE37 protein (Ms_ppe37) and another harbouring the vector alone (Ms_vec) were generated from the non-pathogenic Mycobacterium smegmatis. These bacterial strains were used to infect peritoneal exudate and bone marrow-derived macrophages. It was found that, despite the comparable intracellular survival between the two recombinant M. smegmatis strains, Ms_ppe37 induced a significantly lower level of tumour necrosis factor alpha and interleukin 6 in the infected macrophages compared with Ms_vec. Western blot analyses revealed that the activation levels of nuclear factor kappa B, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase and MAPK/p38 were lower in macrophages infected with Ms_ppe37 than in macrophages infected with Ms_vec. These results suggest that PPE37 may have a potential role in interfering with the pro-inflammatory cytokine response of infected macrophages.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Animais , Variação Antigênica , Sequência de Bases , Morte Celular , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Vetores Genéticos , Interleucina-6/biossíntese , Interleucina-6/genética , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Recombinação Genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Virulência/genética , Virulência/imunologiaRESUMO
Listeriolysin O (LLO), an hly-encoded cytolysin of Listeria monocytogenes, plays an essential role in the entry of L. monocytogenes into the host cell cytoplasm. L. monocytogenes-infected macrophages produce various proinflammatory cytokines, including interleukin-1 alpha (IL-1 alpha), that contribute to the host immune response. In this study, we have examined IL-1 alpha production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Delta hly). Expression of IL-1 alpha mRNA and accumulation of pro-IL-1 alpha in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1 alpha from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Delta hly mutant. A recovery of the ability to induce IL-1 alpha secretion was shown in a mutant strain complemented with the hly gene. The Toll-like receptor (TLR)/MyD88 signaling pathway was exclusively required for the expression of pro-IL-1 alpha, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1 alpha was abolished by addition of calcium chelators, and only LLO-producing L. monocytogenes strains were able to induce elevation of the intracellular calcium level in infected macrophages. A calcium-dependent protease, calpain, was implicated in the maturation and secretion of IL-1 alpha induced by LLO-producing L. monocytogenes strains based on the effect of calpain inhibitor. Functional activation of calpain was detected in macrophages infected with LLO-producing L. monocytogenes strains but not with a mutant strain lacking LLO. These results clearly indicated that LLO-mediated cytoplasmic entry of bacteria could induce the activation of intracellular calcium signaling, which is essential for maturation and secretion of IL-1 alpha in macrophages during L. monocytogenes infection through activation of a calcium-dependent calpain protease. In addition, recombinant LLO, when added to macrophages infected with the Delta hly strain, could induce calcium influx and IL-1 alpha secretion at doses exhibiting cytolytic activity, suggesting that LLO produced by intracellular L. monocytogenes may be implicated in induction of calcium influx through pore formation.
Assuntos
Toxinas Bacterianas/metabolismo , Calpaína/biossíntese , Citoplasma/microbiologia , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Interleucina-1alfa/metabolismo , Listeria monocytogenes/patogenicidade , Macrófagos/microbiologia , Fatores de Virulência/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes , Teste de Complementação Genética , Proteínas de Choque Térmico/deficiência , Proteínas Hemolisinas/deficiência , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A genomic locus called "region of difference 1" (RD1) in Mycobacterium tuberculosis has been shown to contribute to the generation of host protective immunity as well as to the virulence of the bacterium. To gain insight into the molecular mechanism, we investigated the difference in the cytokine-inducing ability between H37Rv and a mutant strain deficient for RD1 (DeltaRD1). We found that RD1 is implicated in the production of caspase-1-dependent cytokines, interleukin-18 (IL-18) and IL-1beta, from infected macrophages. The expression of these cytokines was similarly induced after infection with H37Rv and DeltaRD1. However, the activation of caspase-1 was observed only in H37Rv-infected macrophages. The cytokine production and caspase-1 activation were induced independently of type I interferon receptor signaling events. We also found that the activation of caspase-1 was markedly inhibited with increasing concentrations of extracellular KCl. Furthermore, the production of IL-18 and IL-1beta and caspase-1 activation were induced independently of a P2X7 purinergic receptor, and the inability of DeltaRD1 in caspase-1 activation was compensated for by nigericin, an agent inducing the potassium ion efflux. Based on these results, we concluded that RD1 participates in caspase-1-dependent cytokine production via induction of the potassium ion efflux in infected macrophages.
Assuntos
Caspase 1/metabolismo , Genoma Bacteriano , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Potássio/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/fisiologia , Ativação Enzimática , Feminino , Interferon beta/fisiologia , Interleucina-18/fisiologia , Interleucina-1beta/fisiologia , Interleucina-6/biossíntese , Transporte de Íons , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nigericina/farmacologia , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X7 , Fator de Necrose Tumoral alfa/biossínteseRESUMO
It was shown that virulent Mycobacterium tuberculosis H37Rv induces necrosis of infected RAW264 cells at 24 h post infection while avirulent H37Ra and an attenuated H37Rv mutant that is deficient for RD1 region (H37RvDeltaRD1) cause less necrosis of the infected cells. While H37Rv caused damage of the mitochondrial inner membrane and decreased the level of intracellular ATP, H37RvDeltaRD1 did not exhibit these harmful effects in infected cells. On the other hand, there was no difference in the level of intracellular reactive oxygen species after infection with H37Rv or H37RvDeltaRD1, and the intracellular bacterial numbers of H37RvDeltaRD1 and H37Ra were comparable to that of H37Rv. These results suggested that some virulence factors of H37Rv may contribute to the necrosis of infected cells through induction of mitochondrial dysfunction and depletion of intracellular ATP. RD1 appeared to encode some components possibly playing a central role in the induction of host cell necrosis after M. tuberculosis infection.