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 TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds

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

International audiencePost-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

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

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

Molecular mimicry and original biochemical strategies for the biogenesis of a Legionella pneumophila replicative niche in phagocytic cells.

International audienceLegionella pneumophila is a paradigm of highly adapted intravacuolar pathogens that acquired the rare ability to replicate within a phagocytic cell. Here, we review recent progress about the role of Type 4 secretion system effectors involved in the biogenesis of the replicative niche, the Legionella containing vacuole

The Importance of Revisiting Legionellales Diversity

International audienceHighlights : Members of the order Legionellales are best known as major intracellular pathogens, including the agent of Legionnaires’ disease and the agent of Q fever. Most of our knowledge is currently biased toward the pathogenic species of the Legionellales.Recent ecological surveys have uncovered an unexpected diversity of Legionellales associated with nonvertebrate hosts. Some of these novel Legionellales use alternative strategies to persist in nonvertebrates, including a complex mutualistic lifestyle.This diversity makes Legionellales particularly interesting for studying the mechanisms underlying bacterial lifestyle transition, including the emergence of pathogenicity and mutualism, host specificity, and transmission. Investigations on nonpathogenic Legionellales are now needed to identify specific molecular mechanisms driving adaptations to different hosts and lifestyles

Functional Type 1 Secretion System Involved in Legionella pneumophila Virulence

International audienceLegionella pneumophilais a Gram-negative pathogen found mainly in water, either in a free-living form or within infected pro-tozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severeform of pneumonia called legionellosis or Legionnaires’ disease. The involvement of type II and IV secretion systems in the viru-lence ofL. pneumophilais 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 beenestablished. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle ofL.pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secretedthrough an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed thatthis T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these resultsunderline the active participation ofL. pneumophila, via its T1SS, in its internalization into host cells

A 'Secreted Army' for the Invasion and Survival of Legionella pneumophila Within Host Cells

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

International audienceBacterial 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