Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection.
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ABSTRACT:
SUBMITTER: Molofsky AB
PROVIDER: S-EPMC1584282 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4(-/-) (Ipaf(-/-)), and caspase-1(-/-) derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7(-/-) macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.
| S-EPMC2657210 | biostudies-literature
Project description:Abstract Legionella pneumophila, the causative agent of Legionnaire’s disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of anti-apoptotic genes positively controlled by the transcriptional regulator NF-?B. Consistent with this finding, L. pneumophila triggered nuclear localization of NF-?B in human and mouse macrophage in a Dot/Icm-dependent manner. The mechanism of activation at low dose infections involved a signaling pathway that occurred independently of the TLR adaptor MyD88, and cytoplasmic sensor Nod1. In contrast, high MOI conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-?B. Inhibition of NF-?B translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one anti-apoptotic protein, PAI-2, host cell death increased in response to L. pneumophila infection, indicating that induction of anti-apoptotic genes is critical for host cell survival. Keywords: time course, dose response
2006-08-17 | GSE5551 | GEO
Project description:UnlabelledLegionella pneumophila is a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo-inositol (here termed inositol). To assess inositol metabolism by L. pneumophila, we constructed defined mutant strains lacking lpg1653 or lpg1652, which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection of Acanthamoeba castellanii, the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added to L. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ?iolT or ?iolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the Piol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila Finally, the parental strain and ?iolG mutant bacteria but not the ?iolT mutant strain accumulated [U-(14)C6]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen.ImportanceThe environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires' disease. The opportunistic pathogen replicates in protozoan and mammalian phagocytes in a unique vacuole. Amino acids are thought to represent the prime source of carbon and energy for L. pneumophila However, genome, transcriptome, and proteome studies indicate that the pathogen not only utilizes amino acids as carbon sources but possesses broader metabolic capacities. In this study, we analyzed the metabolism of inositol by extra- and intracellularly growing L. pneumophila By using genetic, biochemical, and cell biological approaches, we found that L. pneumophila accumulates and metabolizes inositol through the iol gene products, thus promoting the intracellular growth, virulence, and fitness of the pathogen. Our study significantly contributes to an understanding of the intracellular niche of a human pathogen.
| S-EPMC4968532 | biostudies-literature
Project description:A flagellin-independent caspase-1 activation pathway that does not require NAIP5 or NRLC4 is induced by the intracellular pathogen Legionella pneumophila. Here we demonstrate that this pathway requires caspase-11. Treatment of macrophages with LPS up-regulated the host components required for this caspase-11 activation pathway. Activation by Legionella differed from caspase-11 activation using previously described agonists in that Legionella caspase-11 activation was rapid and required bacteria with a functional type IV secretion system called Dot/Icm. Legionella activation of caspase-11 induced pyroptosis by a mechanism independent of the NAIP/NLRC4 and caspase-1 axis. Legionella activation of caspase-11 stimulated activation of caspase-1 through NLRP3 and ASC. Induction of caspase-11-dependent responses occurred in macrophages deficient in the adapter proteins TRIF or MyD88 but not in macrophages deficient in both signaling factors. Although caspase-11 was produced in macrophages deficient in the type-I IFN receptor, there was a severe defect in caspase-11-dependent pyroptosis in these cells. These data indicate that macrophages respond to microbial signatures to produce proteins that mediate a capsase-11 response and that the caspase-11 system provides an alternative pathway for rapid detection of an intracellular pathogen capable of evading the canonical caspase-1 activation system that responds to bacterial flagellin.
| S-EPMC3562791 | biostudies-literature
Project description:Legionella pneumophila, the etiological agent for Legionnaires' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding of L. pneumophila pathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmental L. pneumophila isolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmental L. pneumophila strains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratory L. pneumophila strains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding to L. pneumophila infection.
| S-EPMC3754228 | biostudies-literature
Project description:Legionella pneumophila is the causative agent of Legionnaires' disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.
| S-EPMC3128937 | biostudies-literature
Project description:UnlabelledNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 by Legionella pneumophila occurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages by L. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta following L. pneumophila infection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.ImportanceCaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacterium Legionella pneumophila induces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.
| S-EPMC3269931 | biostudies-literature
Project description:UNLABELLED:Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DATABASE:The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF. STRUCTURED DIGITAL ABSTRACT:LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].
| S-EPMC3982195 | biostudies-literature
Project description:BackgroundLegionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼ 300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs) to actively replicating L. pneumophila.Methodology/principal findingsHere, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling), anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression.Conclusion/significanceTaken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.
| S-EPMC4259488 | biostudies-literature
Project description:Legionella pneumophila (corby) infected blood derived macrophages are infected in a time course experiment (24& 48hours)
2019-01-25 | GSE125559 | GEO