scholarly journals Mouse Macrophages Are Permissive to Motile Legionella Species That Fail To Trigger Pyroptosis

2009 ◽  
Vol 78 (1) ◽  
pp. 423-432 ◽  
Author(s):  
Natalie N. Whitfield ◽  
Brenda G. Byrne ◽  
Michele S. Swanson
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Defense Mechanisms ◽  
Opportunistic Pathogen ◽  
Pathogenic Potential ◽  
Innate Defense ◽  
Transplant Patients ◽  
Type Iv ◽  
Mouse Macrophages ◽  
Motile Species

ABSTRACT Legionella pneumophila, a motile opportunistic pathogen of humans, is restricted from replicating in the lungs of C57BL/6 mice. Resistance of mouse macrophages to L. pneumophila depends on recognition of cytosolic flagellin. Once detected by the NOD-like receptors Naip5 and Ipaf (Nlrc4), flagellin triggers pyroptosis, a proinflammatory cell death. In contrast, motile strains of L. parisiensis and L. tucsonensis replicate profusely within C57BL/6 macrophages, similar to flagellin-deficient L. pneumophila. To gain insight into how motile species escape innate defense mechanisms of mice, we compared their impacts on macrophages. L. parisiensis and L. tucsonensis do not induce proinflammatory cell death, as measured by lactate dehydrogenase (LDH) release and interleukin-1β (IL-1β) secretion. However, flagellin isolated from L. parisiensis and L. tucsonensis triggers cell death and IL-1β secretion when transfected into the cytosol of macrophages. Neither strain displays three characteristics of the canonical L. pneumophila Dot/Icm type IV secretion system: sodium sensitivity, LAMP-1 evasion, and pore formation. Therefore, we postulate that when L. parisiensis and L. tucsonensis invade a mouse macrophage, flagellin is confined to the phagosome, protecting the bacteria from recognition by the cytosolic surveillance system and allowing Legionella to replicate. Despite their superior capacity to multiply in mouse macrophages, L. parisiensis and L. tucsonensis have been associated with only two cases of disease, both in renal transplant patients. These results point to the complexity of disease, a product of the pathogenic potential of the microbe, as defined in the laboratory, and the capacity of the host to mount a measured defense.

scholarly journals Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection

2006 ◽  
Vol 203 (4) ◽  
pp. 1093-1104 ◽  
Author(s):  
Ari B. Molofsky ◽  
Brenda G. Byrne ◽  
Natalie N. Whitfield ◽  
Cressida A. Madigan ◽  
Etsu T. Fuse ◽  
...  
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Type Iv Secretion ◽  
Listeriolysin O ◽  
Nuclear Condensation ◽  
Macrophage Response ◽  
Caspase 1 ◽  
Type Iv ◽  
Mouse Macrophages ◽  
Bacterial Replication

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

scholarly journals Assessment of Safety of Lactobacillus Strains Based on Resistance to Host Innate Defense Mechanisms

2003 ◽  
Vol 10 (1) ◽  
pp. 169-173 ◽  
Author(s):  
Takashi Asahara ◽  
Masatoshi Takahashi ◽  
Koji Nomoto ◽  
Hiroo Takayama ◽  
Masaharu Onoue ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Safety Assessment ◽  
Intracellular Killing ◽  
Pathogenic Potential ◽  
Innate Defense ◽  
Defense Systems ◽  
In Vitro Sensitivity ◽  
Mouse Macrophages ◽  
Type Strains

ABSTRACT Seven Lactobacillus strains belonging to four species were evaluated for pathogenicity as well as for in vitro sensitivity to the bactericidal mechanisms of macrophages in a rabbit infective endocarditis (IE) model. Two bacteremia-associated strains, L. rhamnosus PHLS A103/70 and L. casei PHLS A357/84, as well as the L. rhamnosus type strain and the probiotic L. rhamnosus strain ATCC 53103, showed moderate infectivity, and the virulence of the probiotic L. casei strain Shirota and type strains such as L. acidophilus ATCC 4356T and L. gasseri DSM 20243T in the model was negligible. The strains that showed pathogenic potential in the rabbit IE model (PHLS A357/84, PHLS A103/70, and ATCC 53103) were more resistant than strain Shirota to intracellular killing activity by mouse macrophages in vitro and also to bactericidal nitrogen intermediates, such as nitric oxide and NO2 − ions. These results suggest that resistance to host innate defense systems, which would function at inflammatory lesions, should be considered in the safety assessment of Lactobacillus strains.

scholarly journals Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

2021 ◽  
Author(s):  
Michael J Sheedlo ◽  
Clarissa L Durie ◽  
Jeong Min Chung ◽  
Louise Chang ◽  
Michele Swanson ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Genetic Locus ◽  
Opportunistic Pathogen ◽  
Effector Proteins ◽  
Large Set ◽  
Type Iv ◽  
Specific Proteins ◽  
Multiple Conformations ◽  
Membrane Spanning ◽  
Species Specific

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires’ Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure. We hypothesize this conformational flexibility is likely to facilitate the Dot/Icm T4SS’s ability to translocate a remarkably large set of ~300 putative substrates across the inner and outer membranes of the bacterial cell.

scholarly journals Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael J Sheedlo ◽  
Clarissa L Durie ◽  
Jeong Min Chung ◽  
Louise Chang ◽  
Jacquelyn Roberts ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Genetic Locus ◽  
Opportunistic Pathogen ◽  
Effector Proteins ◽  
Type Iv ◽  
Specific Proteins ◽  
Legionnaires Disease ◽  
Multiple Conformations ◽  
Membrane Spanning ◽  
Species Specific

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

scholarly journals Insertion sequences drive the emergence of a highly adapted human pathogen

2020 ◽  
Vol 6 (9) ◽  
Author(s):  
Erwin Sentausa ◽  
Pauline Basso ◽  
Alice Berry ◽  
Annie Adrait ◽  
Gwendoline Bellement ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Opportunistic Pathogen ◽  
Insertion Sequences ◽  
Pathogenic Potential ◽  
Highly Pathogenic ◽  
Type Iv ◽  
Genome Wide ◽  
Outer Membrane Porin ◽  
Specific Antigens ◽  
Different Levels

Pseudomonas aeruginosa is a highly adaptive opportunistic pathogen that can have serious health consequences in patients with lung disorders. Taxonomic outliers of P. aeruginosa of environmental origin have recently emerged as infectious for humans. Here, we present the first genome-wide analysis of an isolate that caused fatal haemorrhagic pneumonia. In two clones, CLJ1 and CLJ3, sequentially recovered from a patient with chronic pulmonary disease, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to the antibiotics used to combat the infection. Comparative genome, proteome and transcriptome analyses revealed that this ISL3-family insertion sequence disrupted the genes for flagellar components, type IV pili, O-specific antigens, translesion polymerase and enzymes producing hydrogen cyanide. Seven-fold more insertions were detected in the later isolate, CLJ3, than in CLJ1, some of which modified strain susceptibility to antibiotics by disrupting the genes for the outer-membrane porin OprD and the regulator of β-lactamase expression AmpD. In the Galleria mellonella larvae model, the two strains displayed different levels of virulence, with CLJ1 being highly pathogenic. This study revealed insertion sequences to be major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both its virulence and its resistance to antimicrobials, and explains how this bacterium adapts from the environment to a human host.

scholarly journals Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

2013 ◽  
Vol 81 (9) ◽  
pp. 3077-3088 ◽  
Author(s):  
Lili Tao ◽  
Wenhan Zhu ◽  
Bi-Jie Hu ◽  
Jie-Ming Qu ◽  
Zhao-Qing Luo
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Immune Surveillance ◽  
Human Macrophage ◽  
Aqueous Environment ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Extensive Cell Death ◽  
Extensive Cell

ABSTRACTLegionella 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 ofL. pneumophilapathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmentalL. pneumophilaisolates 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 environmentalL. pneumophilastrains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratoryL. pneumophilastrains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding toL. pneumophilainfection.

scholarly journals Potentiation of Cytokine-Mediated Restriction ofLegionellaIntracellular Replication by a Dot/Icm-Translocated Effector

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Alex J. Hydock ◽  
Sandhya Ganesan ◽  
Stephanie R. Shames
Keyword(s):  
Legionella Pneumophila ◽  
Defense Mechanisms ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Host Restriction ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Ifn Γ ◽  
Necrosis Factor

ABSTRACTLegionella pneumophilais ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However,L. pneumophilais also an accidental human pathogen that can cause Legionnaires’ disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes,L. pneumophilautilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction ofL. pneumophila. We found previously that the effector LegC4 is important forL. pneumophilareplication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires’ disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuatesL. pneumophilareplication. We found that LegC4 enhanced restriction ofL. pneumophilareplication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression oflegC4was sufficient to restrictLegionella longbeachaereplication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes toL. pneumophilaclearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionellaspp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controlsLegionellainfection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction ofLegionellaby macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

scholarly journals Membrane Vesicles Shed by Legionella pneumophila Inhibit Fusion of Phagosomes with Lysosomes

2006 ◽  
Vol 74 (6) ◽  
pp. 3285-3295 ◽  
Author(s):  
Esteban Fernandez-Moreira ◽  
Juergen H. Helbig ◽  
Michele S. Swanson
Keyword(s):  
Legionella Pneumophila ◽  
Surface Composition ◽  
Membrane Vesicles ◽  
Type Iv Secretion ◽  
Phagosome Maturation ◽  
Developmentally Regulated ◽  
E Coli ◽  
Type Iv ◽  
Mouse Macrophages ◽  
Legionnaires Disease

ABSTRACT When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only ∼25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas ∼75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered ∼72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.

scholarly journals The Dot/Icm-translocated effector LegC4 potentiates cytokine-mediated restriction of Legionella within accidental hosts.

2018 ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Alex J Hydock ◽  
Sandhya Ganesan ◽  
Stephanie Rochelle Shames
Keyword(s):  
Legionella Pneumophila ◽  
Defense Mechanisms ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Innate Immune ◽  
Host Cells ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Ifn Γ ◽  
Necrosis Factor

Legionella pneumophila is ubiquitous in freshwater environments where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires’ Disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires’ Disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon (IFN)-γ. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.

scholarly journals Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Brenda G. Byrne ◽  
Jean-Francois Dubuisson ◽  
Amrita D. Joshi ◽  
Jenny J. Persson ◽  
Michele S. Swanson
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Mouse Genetics ◽  
Leucine Rich Repeat ◽  
Nucleotide Binding Domain ◽  
Caspase 1 ◽  
Primary Mouse ◽  
Mouse Macrophages

ABSTRACTWhen microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellateLegionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K+efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death.IMPORTANCEAn exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.

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