RESUMO
Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis-arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes-derived macrophages and D. melanogaster-derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella-containing phagosome matures to a late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod-derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster-derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster-derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its patho-adaptation to infect mammals.
Assuntos
Drosophila melanogaster/microbiologia , Francisella tularensis/patogenicidade , Fagossomos/microbiologia , Animais , Linhagem Celular , Drosophila melanogaster/citologia , Francisella tularensis/genética , Francisella tularensis/crescimento & desenvolvimento , Genes Bacterianos , Ilhas Genômicas , Humanos , Macrófagos/microbiologia , Tularemia/microbiologiaRESUMO
Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis ssp. novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants colocalize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape and bacterial proliferation within the cytosol is novel, complex and involves an unusually large portion of the genome of an intracellular pathogen.
Assuntos
Citosol/microbiologia , Francisella tularensis/patogenicidade , Macrófagos/microbiologia , Fagossomos/microbiologia , Linhagem Celular , Francisella tularensis/genética , Francisella tularensis/crescimento & desenvolvimento , Ilhas Genômicas , Humanos , Mutagênese Insercional , Tularemia/microbiologiaRESUMO
Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-kappaB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-kappaB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-kappaB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-kappaB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-kappaB by the wild type strain is abolished upon treatment with the NF-kappaB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-kappaB.
Assuntos
Apoptose , Francisella tularensis/patogenicidade , Macrófagos/microbiologia , NF-kappa B/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/metabolismo , Caspase 3/metabolismo , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/metabolismo , Transdução de SinaisRESUMO
The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), approximately 90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.
Assuntos
Citosol/microbiologia , Francisella tularensis/fisiologia , Macrófagos/microbiologia , Fagossomos/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Francisella tularensis/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Mutação , Fagossomos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Legionella pneumophila/metabolismo , Macrófagos/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Ativação Enzimática/imunologia , Humanos , Legionella pneumophila/genética , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.
Assuntos
Acanthamoeba/microbiologia , Proteínas de Bactérias/genética , Citosol/microbiologia , Legionella pneumophila/patogenicidade , Macrófagos/microbiologia , Mutação , Fagossomos/ultraestrutura , Acanthamoeba/crescimento & desenvolvimento , Acanthamoeba/ultraestrutura , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/fisiologia , Macrófagos/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Fagossomos/microbiologia , Fatores de Tempo , Células U937RESUMO
Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.
Assuntos
Legionella longbeachae/fisiologia , Fagossomos/microbiologia , Proteínas de Bactérias/metabolismo , Catepsina D/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Interferon gama/metabolismo , Legionella longbeachae/metabolismo , Legionella longbeachae/patogenicidade , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Ovalbumina/metabolismo , Receptor IGF Tipo 2/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Xantenos/metabolismoRESUMO
Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.
Assuntos
Caspases/metabolismo , Predisposição Genética para Doença , Legionella longbeachae/imunologia , Legionella pneumophila/imunologia , Legionelose/microbiologia , Doença dos Legionários/microbiologia , Macrófagos/microbiologia , Animais , Apoptose , Células Cultivadas , Contagem de Colônia Microbiana , Ativação Enzimática , Feminino , Expressão Gênica , Humanos , Imunidade Inata , Marcação In Situ das Extremidades Cortadas , Legionella longbeachae/crescimento & desenvolvimento , Legionella longbeachae/patogenicidade , Legionella pneumophila/crescimento & desenvolvimento , Legionella pneumophila/patogenicidade , Legionelose/imunologia , Doença dos Legionários/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Inibidora de Apoptose Neuronal/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase-3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase-3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers anti-apoptotic signalling within the infected human macrophages to halt caspase-3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila-infected human monocyte-derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm-dependent manner. Microarray analyses and real-time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti-apoptotic genes that are linked to activation of the nuclear transcription factor kappa-B (NF-kappaB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kappaB during exponential intracellular replication. Bacterial entry is essential both for the anti-apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65-/- and IKKalpha-/- beta-/- double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kappaB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kappaB requires the activity of IKKalpha and/or IKKbeta. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase-3 in human macrophages, it triggers a strong anti-apoptotic signalling cascade mediated, at least in part by NF-kappaB, which renders the cells refractory to external potent apoptotic stimuli.
Assuntos
Apoptose , Legionella pneumophila/metabolismo , Macrófagos/microbiologia , NF-kappa B/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Humanos , Quinase I-kappa B/genética , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Macrófagos/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição RelA/genética , Células U937 , Regulação para CimaRESUMO
The induction of virulence traits by Legionella pneumophila at the post-exponential phase has been proposed to be triggered by the stringent response mediated by RelA, which triggers RpoS. We show that L. pneumophila rpoS but not relA is required for early intracellular survival and replication within human monocyte-derived macrophages and Acanthamoeba polyphaga. In addition, L. pneumophila rpoS but not relA is required for expression of the pore-forming activity. We provide evidence that RpoS plays a role in the modulation of phagosome biogenesis and in adaptation to the phagosomal microenvironment. Thus, there is no functional link between the stringent response and RpoS in the pathogenesis of L. pneumophila.