Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche.
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ABSTRACT:
SUBMITTER: Escoll P
PROVIDER: S-EPMC8718111 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.
| S-EPMC2631402 | biostudies-literature
Project description:Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.
| S-EPMC5179073 | biostudies-literature
Project description:Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.
| S-EPMC2841630 | biostudies-literature
Project description:Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells1-5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot-Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot-Icm apparatus was solved in situ by cryo-electron tomography at 3.5?nm resolution and the cytoplasmic complex was solved at 3.0?nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot-Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.
| S-EPMC5970066 | biostudies-literature
Project description:The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-?B signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-?B, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.
| S-EPMC4841580 | biostudies-literature
Project description:Legionella pneumophila grows within cells ranging from environmental amoebae to human macrophages. In spite of this conserved strategy of pathogenesis, identification of host factors that restrict L. pneumophila intracellular replication has not been extended outside components of the mammalian innate immune response. We performed a double-stranded RNA (dsRNA) screen against more than 50% of the Drosophila melanogaster annotated open reading frames (ORFs) to identify host cell factors that restrict L. pneumophila The majority of analyzed dsRNAs that stimulated L. pneumophila intracellular replication were directed against host proteins involved in protein synthesis or cell cycle control. Consistent with disruption of the cell cycle stimulating intracellular replication, proteins involved in translation initiation also resulted in G1 arrest. Stimulation of replication was dependent on the stage of cell cycle arrest, as dsRNAs causing arrest during S phase had an inhibitory effect on intracellular replication. The inhibitory effects of S phase arrest could be recapitulated in a human cell line, indicating that cell cycle control of L. pneumophila replication is evolutionarily conserved. Synchronized HeLa cell populations in S phase and challenged with L. pneumophila failed to progress through the cell cycle and were depressed for supporting intracellular replication. Poor bacterial replication in S phase was associated with loss of the vacuole membrane barrier, resulting in exposure of bacteria to the cytosol and their eventual degradation. These results are consistent with the model that S phase is inhibitory for L. pneumophila intracellular survival as a consequence of failure to maintain the integrity of the membrane surrounding intracellular bacteria.IMPORTANCELegionella pneumophila has the ability to replicate within human macrophages and amoebal hosts. Here, we report that the host cell cycle influences L. pneumophila intracellular replication. Our data demonstrate that the G1 and G2/M phases of the host cell cycle are permissive for bacterial replication, while S phase is toxic for the bacterium. L. pneumophila replicates poorly within host cells present in S phase. The inability of L. pneumophila to replicate relies on its failure to control the integrity of its vacuole, leading to cytosolic exposure of the bacteria and eventual degradation. The data presented here argue that growth-arrested host cells that are encountered by L. pneumophila in either the environment or within human hosts are ideal targets for intracellular replication because their transit through S phase is blocked.
| S-EPMC5565972 | biostudies-other
Project description:We established a new Brucella neotomaein vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitroBrucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.
| S-EPMC5400850 | biostudies-literature
Project description:The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts.
| S-EPMC7108532 | biostudies-literature
Project description:Legionella pneumophila invades protozoa with an "accidental" ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.
| S-EPMC5910436 | biostudies-literature
Project description:Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages.IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.
| S-EPMC7157531 | biostudies-literature