Legionella pneumophila cell surface RtxA release by LapD/LapG and its role in virulence.

BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacillus and is the causative agent of a severe form of pneumonia called Legionnaires' disease which accounts for 2-9% of cases of community acquired pneumonia. It produces an extremely large protein belonging to the RTX (Repeats in ToXin) family, called RtxA, and we previously reported that RtxA is transported by a dedicated type 1 secretion system (T1SS) to the cell surface. RTX proteins have been shown to participate in the virulence or biofilm formation of various bacteria, the most studied models being the pore forming hemolysin A (HlyA) of Escherichia coli and the biofilm associated protein LapA of P. fluorescens. LapA localization depends on the enzymatic release by LapD/LapG complex activity. This study aimed to elucidate the dual localization (cell surface associated or released state) of L. pneumophila RTX protein (RtxA) and whether this released versus sequestered state of RtxA plays a role in L. pneumophila virulence. RESULTS: The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (position 108-109). Consistently, a strain lacking LapG protease maintains RtxA on the cell surface, whereas a strain lacking the c-di-GMP receptor LapD does not exhibit cell surface RtxA because of its continuous cleavage and release, as in the LapA-D-G model of Pseudomonas fluorescens. Interestingly, our data point out a key role of RtxA in enhancing the infection process of amoeba cells, regardless of its location (embedded or released); therefore, this may be the result of a secondary role of this surface protein. CONCLUSIONS: This is the first experimental identification of the cleavage site within the RTX protein family. The primary role of RtxA in Legionella is still questionable as in many other bacterial species, hence it sounds reasonable to propose a major function in biofilm formation, promoting cell aggregation when RtxA is embedded in the outer membrane and facilitating biofilm dispersion in case of RtxA release. The role of RtxA in enhancing the infection process may be a result of its action on host cells (i.e., PDI interaction or pore-formation), and independently of its status (embedded or released).

Hyper-inflammatory profile and immunoparalysis in patients with severe Legionnaires' disease.

Introduction: Severe Legionnaires' disease (LD) can lead to multi-organ failure or death in 10%-30% of patients. Although hyper-inflammation and immunoparalysis are well described in sepsis and are associated with high disease severity, little is known about the immune response in LD. This study aimed to evaluate the immune status of patients with LD and its association with disease severity. Methods: A total of 92 hospitalized LD patients were included; 19 plasmatic cytokines and pulmonary Legionella DNA load were measured in 84 patients on the day of inclusion (day 0, D0). Immune functional assays (IFAs) were performed from whole blood samples collected at D2 and stimulated with concanavalin A [conA, n = 19 patients and n = 21 healthy volunteers (HV)] or lipopolysaccharide (LPS, n = 14 patients and n = 9 HV). A total of 19 cytokines (conA stimulation) and TNF-α (LPS stimulation) were quantified from the supernatants. The Sequential Organ Failure Assessment (SOFA) severity score was recorded at D0 and the mechanical ventilation (MV) status was recorded at D0 and D8. Results: Among the 84 patients, a higher secretion of plasmatic MCP-1, MIP1-ß, IL-6, IL-8, IFN-γ, TNF-α, and IL-17 was observed in the patients with D0 and D8 MV. Multiparametric analysis showed that these seven cytokines were positively associated with the SOFA score. Upon conA stimulation, LD patients had a lower secretion capacity for 16 of the 19 quantified cytokines and a higher release of IL-18 and MCP-1 compared to HV. IL-18 secretion was higher in D0 and D8 MV patients. TNF-α secretion, measured after ex vivo LPS stimulation, was significantly reduced in LD patients and was associated with D8 MV status. Discussion: The present findings describe a hyper-inflammatory phase at the initial phase of Legionella pneumonia that is more pronounced in patients with severe LD. These patients also present an immunoparalysis for a large number of cytokines, except IL-18 whose secretion is increased. An assessment of the immune response may be relevant to identify patients eligible for future innovative host-directed therapies.

Bacterial persistence in Legionella pneumophila clinical isolates from patients with recurring legionellosis.

Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue.

Intracellular persister: A stealth agent recalcitrant to antibiotics.

The bulk of bacteria transiently evading appropriate antibiotic regimes and recovered from non-resolutive infections are commonly refer to as persisters. In this mini-review, we discuss how antibiotic persisters stem from the interplay between the pathogen and the cellular defenses mechanisms and its underlying heterogeneity.

Chronic Exposure to Secondary Organic Aerosols Causes Lung Tissue Damage.

Recently, secondary organic aerosols (SOAs) emerged as a predominant component of fine particulate matter. However, the pathogenic mechanism(s) of SOAs are still poorly understood. Herein, we show that chronic exposure of mice to SOAs resulted in lung inflammation and tissue destruction. Histological analyses found lung airspace enlargement associated with massive inflammatory cell recruitment predominated by macrophages. Concomitant with such cell influx, our results found changes in the levels of a series of inflammatory mediators in response to SOA. Interestingly, we observed that the expression of the genes encoding for TNF-α and IL-6 increased significantly after one month of exposure to SOAs; mediators that have been largely documented to play a role in chronic pulmonary inflammatory pathologies. Cell culture studies confirmed these in vivo findings. Of importance as well, our study indicates increased matrix metalloproteinase proteolytic activity suggesting its contribution to lung tissue inflammation and degradation. Our work represents the first in vivo study, which reports that chronic exposure to SOAs leads to lung inflammation and tissue injury. Thus, we hope that these data will foster new studies to enhance our understanding of the underlying pathogenic mechanisms of SOAs and perhaps help in the design of therapeutic strategies against SOA-mediated lung injury.

TNF-α response in macrophages depends on clinical Legionella pneumophila isolates genotypes.

Legionnaires' Disease (LD) is a severe pneumonia mainly caused in Europe by Legionella pneumophila serogroup 1 (Lp1). Sequence-based typing methods reveal that some sequence types (ST) are overrepresented in clinical samples such as ST1 and ST47, suggesting that some strains are more fit for infection than others. In the present study, a collection of 108 Lp1 clinical isolates were used to evaluate the strain-dependent immune responses from human macrophages. Clinical Lp1 isolates induced differential TNFα secretion from macrophages. ST1 isolates induced a significantly higher TNF-α secretion than non-ST1, whereas ST47 isolates induced a significantly lower TNF-α secretion than non-ST47 isolates. ST1 isolates induced a significantly higher cell death than ST47 isolates evaluated by lactate dehydrogenase activity (cytotoxicity) and caspase-3 activity (apoptosis). Treatment of macrophages with anti-TNF-α antibodies significantly reduced the cell death in macrophages infected with ST1 or ST47 strains. The TNF-α secretion was neither explained by a differential bacterial replication nor by the number or type (bystander or infected) of TNF-α producing cells following infection but by a differential response from macrophages. The Paris ST1 reference strain elicited a significantly higher TNF-α gene transcription and a higher induction of NF-κB signaling pathway than the Lorraine ST47 reference strain.Clinical Lp1 isolates induce a diverse immune response and cell death, which could be related to the genotype. The two predominant sequence-types ST1 and ST47 trigger opposite inflammatory response that could be related to the host susceptibility.

Myosins, an Underestimated Player in the Infectious Cycle of Pathogenic Bacteria.

Myosins play a key role in many cellular processes such as cell migration, adhesion, intracellular trafficking and internalization processes, making them ideal targets for bacteria. Through selected examples, such as enteropathogenic E. coli (EPEC), Neisseria, Salmonella, Shigella, Listeria or Chlamydia, this review aims to illustrate how bacteria target and hijack host cell myosins in order to adhere to the cell, to enter the cell by triggering their internalization, to evade from the cytosolic autonomous cell defense, to promote the biogenesis of intracellular replicative niche, to disseminate in tissues by cell-to-cell spreading, to exit out the host cell, and also to evade from macrophage phagocytosis. It highlights the diversity and sophistication of the strategy evolved by bacteria to manipulate one of their privileged targets, the actin cytoskeleton.

Scar-Free Genome Editing in Legionella pneumophila.

Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.

The Importance of Revisiting Legionellales Diversity.

Bacteria of the order Legionellales, such as Legionella pneumophila, the agent of Legionnaires' disease, and Coxiella burnetii, the agent of Q fever, are widely recognized as human pathogens. While our view of the Legionellales is often limited to clinical isolates, ecological surveys are continually uncovering new members of the Legionellales that do not fall into the recognized pathogenic species. Here we emphasize that most of these Legionellales are nonpathogenic forms that have evolved symbiotic lifestyles with nonvertebrate hosts. The diversity of nonpathogenic forms remains, however, largely underexplored. We conjecture that its characterization, once contrasted with the data on pathogenic species, will reveal novel highlights on the mechanisms underlying lifestyle transitions of intracellular bacteria, including the emergence of pathogenesis and mutualism, transmission routes, and host specificity.

KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans-Translation Inhibition.

trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump.

Objectives: A previous study on 12 in vitro -selected azithromycin-resistant Legionella pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L. pneumophila . Methods: The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L. pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. Results: We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L. pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. Conclusions: LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.

Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors.

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity ( LeClaire et al., 2008 ). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits ( Vadlamudi et al., 2004 ; LeClaire et al., 2008 ; Narayanan et al., 2011 ; LeClaire et al., 2015 ). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation ( Narayanan et al., 2011 ; LeClaire et al., 2015 ). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm ( Michard et al., 2015 ). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling.

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.

The structure of Legionella pneumophila LegK4 type four secretion system (T4SS) effector reveals a novel dimeric eukaryotic-like kinase.

Bacterial pathogens subvert signalling pathways to promote invasion and/or replication into the host. LegK1-4 proteins are eukaryotic-like serine/threonine kinases that are translocated by the Dot/Icm type IV secretion system (T4SS) of several Legionella pneumophila strains. We present the crystal structures of an active fragment of the LegK4 protein in apo and substrate-bound states. The structure of LegK4(1-445) reveals a eukaryotic-like kinase domain flanked by a novel cap domain and a four-helix bundle. The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase. The helix αG is displaced compared to previous kinase structures, and its role in stabilization of the activation loop is taken on by the dimerisation interface. The apo-form of the protein has an open conformation with a disordered P-loop but a structured activation segment in absence of targeted phosphorylation. The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP. Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.

The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway.

UNLABELLED: Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. IMPORTANCE: Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

Post-translational modifications are key players of the Legionella pneumophila infection strategy.

Post-translational modifications (PTMs) are widely used by eukaryotes to control the enzymatic activity, localization or stability of their proteins. Traditionally, it was believed that the broad biochemical diversity of the PTMs is restricted to eukaryotic cells, which exploit it in extensive networks to fine-tune various and complex cellular functions. During the last decade, the advanced detection methods of PTMs and functional studies of the host-pathogen relationships highlight that bacteria have also developed a large arsenal of PTMs, particularly to subvert host cell pathways to their benefit. Legionella pneumophila, the etiological agent of the severe pneumonia legionellosis, is the paradigm of highly adapted intravacuolar pathogens that have set up sophisticated biochemical strategies. Among them, L. pneumophila has evolved eukaryotic-like and rare/novel PTMs to hijack host cell processes. Here, we review recent progress about the diversity of PTMs catalyzed by Legionella: ubiquitination, prenylation, phosphorylation, glycosylation, methylation, AMPylation, and de-AMPylation, phosphocholination, and de-phosphocholination. We focus on the host cell pathways targeted by the bacteria catalyzed PTMs and we stress the importance of the PTMs in the Legionella infection strategy. Finally, we highlight that the discovery of these PTMs undoubtedly made significant breakthroughs on the molecular basis of Legionella pathogenesis but also lead the way in improving our knowledge of the eukaryotic PTMs and complex cellular processes that are associated to.

Functional type 1 secretion system involved in Legionella pneumophila virulence.

Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.