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is a gram-negative bacterium that inhabits freshwater ecosystems, where it is present in biofilm or as planktonic form. is mainly found associated with protozoa, which serve as protection from hostile environments and as replication niche. If inhaled within aerosols, is also able to infect and replicate in human alveolar macrophages, eventually causing the Legionnaires' disease. The transition between intracellular and extracellular environments triggers a differentiation program in which metabolic as well as morphogenetic changes occur. We here describe the current knowledge on how the different developmental states of this bacterium are regulated, with a particular emphasis on the stringent response activated during the transition from the replicative phase to the infectious phase and the metabolic features going in hand. We propose that the cellular differentiation of this intracellular pathogen is closely associated to key metabolic changes in the bacterium and the host cell, which together have a crucial role in the regulation of virulence.

IFN receptor signaling induces cell-autonomous immunity to infections with intracellular bacterial pathogens. Here, we demonstrate that IFN-inducible guanylate binding protein (Gbp) proteins stimulate caspase-11–dependent, cell-autonomous immunity in response to cytoplasmic LPS. Caspase-11–dependent pyroptosis is triggered in IFN-activated macrophages infected with the Gram-negative bacterial pathogen Legionella pneumophila. The rapid induction of pyroptosis in IFN-activated macrophages required a cluster of IFN-inducible Gbp proteins encoded on mouse chromosome 3 (Gbp ᶜʰʳ³). Induction of pyroptosis in naive macrophages by infections with the cytosol-invading Δ sdhA L. pneumophila mutant was similarly dependent on Gbp ᶜʰʳ³, suggesting that these Gbp proteins play a role in the detection of bacteria accessing the cytosol. Cytoplasmic LPS derived from Salmonella ssp. or Escherichia coli has recently been shown to trigger caspase-11 activation and pyroptosis, but the cytoplasmic sensor for LPS and components of the caspase-11 inflammasome are not yet defined. We found that the induction of caspase-11–dependent pyroptosis by cytoplasmic L. pneumophila -derived LPS required Gbp ᶜʰʳ³ proteins. Similarly, pyroptosis induced by cytoplasmic LPS isolated from Salmonella was diminished in Gbp ᶜʰʳ³-deficient macrophages. These data suggest a role for Gbp ᶜʰʳ³ proteins in the detection of cytoplasmic LPS and the activation of the noncanonical inflammasome.

Key Points Legionella pneumophila is a Gram-negative intracellular pathogen of both amoebae and humans that grows in lung macrophages. The intracellular replication strategy of this bacterium, which involves growth in a membrane-bound compartment called a vacuole, seems to be similar in all cell types in which it grows. A protein translocation apparatus that encodes more than 20 proteins, called Dot/Icm, is required for formation of the replication vacuole and intracellular growth. Proteins move through the apparatus across membranes in contact with the bacterium, and are thought to manipulate host cell proteins that are involved in host cell secretory traffic. Eighty five translocated protein substrates of Dot/Icm have been identified, but there are probably many more. The targets of several have been identified, and they include proteins that modulate the activation state of Arf1 and Rab1 and are involved in vesicle trafficking in host cells, as well as proteins that antagonize host cell death pathways. Mutations in single translocated substrates rarely result in strong defects in intracellular growth. This has led to the model that there is considerable functional redundancy among the substrates. A model is provided to attempt to explain how L. pneumophila acquired such a diverse set of translocated substrates. In this Review, the authors evaluate the strategies that the intracellular pathogen Legionella pneumophila uses to establish growth inside cells and probe why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted to host cells. 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.

Abstract Legionella pneumophila is an endosymbiotic bacterial species able to infect and reproduce in various protist and human hosts. Upon entry into human lungs, they may infect lung macrophages, causing Legionnaires' disease (LD), an atypical pneumonia, using similar mechanisms as in their protozoan hosts, despite the 2 hosts being separated by a billion years of evolution. In this study, we used experimental evolution to identify genes conferring host specificity to L. pneumophila. To this end, we passaged L. pneumophila in 2 different hosts—Acanthamoeba castellanii and the human macrophage-like cells U937—separately and by switching between the hosts twice a week for a year. In total, we identified 1,518 mutations present in at least 5% of the population at the time of sampling. Forty-nine mutations were fixed in the 18 populations at the end of the experiment. Two interesting groups of mutations included (i) mutations in 4 different strain-specific genes involved in lipopolysaccharide (LPS) synthesis, found only in the lineages passaged with A. castellanii and (ii) mutations in the gene coding for LerC, a key regulator of protein effector expression, which was independently mutated in 6 lineages grown in presence of the macrophage cells. We propose that the mutations degrading the function of the regulator LerC improve the fitness of L. pneumophila in human-derived cells and that modifications in the LPS are beneficial for growth in A. castellanii. This study is a first step in further investigating determinants of host specificity in L. pneumophila.

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.

Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexisting risk factors and frequently require hospitalization. The objectives of this report are to alert professionals of the impact of OPPPs, the fact that 30% of the population may be exposed to OPPPs, and the need to develop means to reduce OPPP exposure. We herein present a review of the epidemiology and ecology of these three bacterial OPPPs, specifically to identify common and unique features. A Water Research Foundation-sponsored workshop gathered experts from across the United States to review the characteristics of OPPPs, identify problems, and develop a list of research priorities to address critical knowledge gaps with respect to increasing OPPP-associated disease. OPPPs share the common characteristics of disinfectant resistance and growth in biofilms in water distribution systems or premise plumbing. Thus, they share a number of habitats with humans (e.g., showers) that can lead to exposure and infection. The frequency of OPPP-infected individuals is rising and will likely continue to rise as the number of at-risk individuals is increasing. Improved reporting of OPPP disease and increased understanding of the genetic, physiologic, and structural characteristics governing the persistence and growth of OPPPs in drinking water distribution systems and premise plumbing is needed. Because broadly effective community-level engineering interventions for the control of OPPPs have yet to be identified, and because the number of at-risk individuals will continue to rise, it is likely that OPPP-related infections will continue to increase. However, it is possible that individuals can take measures (e.g., raise hot water heater temperatures and filter water) to reduce home exposures.

Pathogen specificity in innate immunity The inflammasomes are multiprotein complexes involved in innate immunity, and induce an immune response to pathogenic microbes by activating the caspase 1 protease. Two groups now report that the intracellular receptors known as NAIPs (NLR family, apoptosis inhibitory proteins), previously thought to have an auxiliary role in recognizing microbial proteins, are in fact central to the process. Eric Kofoed and Russell Vance, and Feng Shao and colleagues, show that different members of the NAIP family bind to different bacterial ligands, including bacterial flagellin and a conserved bacterial type III secretion system rod protein. Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death 1 , 2 . The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component 3 , 4 , 5 . How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages 6 , is a universal component of the flagellin–NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5–NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.

The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire’s Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.