The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia.

Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.

A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.

Characterization of a family I inorganic pyrophosphatase from Legionella pneumophila Philadelphia 1.

Inorganic pyrophosphate (PPi) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PPi must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PPi into two orthophosphates (Pi), preventing the toxic accumulation of the PPi byproduct in cells and making Pi available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Šresolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) ß-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.

Legionella para-effectors target chromatin and promote bacterial replication.

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a ∆lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout (∆lphD∆romA) can restore intracellular replication. Uniquely, we present evidence for "para-effectors", an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens has the potential to lead to the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.

Inflammasome activation and CCR2-mediated monocyte-derived dendritic cell recruitment restrict Legionella pneumophila infection.

Flagellin-induced NAIP/NLRC4 inflammasome activation and pyroptosis are critical events restricting Legionella pneumophila infection. However, the cellular and molecular dynamics of the in vivo responses against this bacterium are still unclear. We have found temporal coordination of two independent innate immunity pathways in controlling Legionella infection, the inflammasome activation and the CCR2-mediated Mo-DC recruitment. Inflammasome activation was an important player at the early stage of infection by lowering the numbers of bacteria for an efficient bacterial clearance conferred by the Mo-DC at the late stage of the infection. Mo-DC emergence highly depended on CCR2-signaling and dispensed inflammasome activation and pyroptosis. Also, Mo-DC compartment did not rely on the inflammasome machinery to deliver proper immune responses and was the most abundant cytokine-producing among the monocyte-derived cells in the infected lung. Importantly, when the CCR2- and NLRC4-dependent axes of response were simultaneously ablated, we observed an aggravated bacterial burden in the lung of infected mice. Taken together, we showed that inflammasome activation and CCR2-mediated immune response interplay in distinct pathways to restrict pulmonary bacterial infection. These findings extend our understanding of the in vivo integration and cooperation of different innate immunity arms in controlling infectious agents.

Structural basis for the acetylation mechanism of the Legionella effector VipF.

The pathogen Legionella pneumophila, which is the causative agent of Legionnaires' disease, secrets hundreds of effectors into host cells via its Dot/Icm secretion system to subvert host-cell pathways during pathogenesis. VipF, a conserved core effector among Legionella species, is a putative acetyltransferase, but its structure and catalytic mechanism remain unknown. Here, three crystal structures of VipF in complex with its cofactor acetyl-CoA and/or a substrate are reported. The two GNAT-like domains of VipF are connected as two wings by two ß-strands to form a U-shape. Both domains bind acetyl-CoA or CoA, but only in the C-terminal domain does the molecule extend to the bottom of the U-shaped groove as required for an active transferase reaction; the molecule in the N-terminal domain folds back on itself. Interestingly, when chloramphenicol, a putative substrate, binds in the pocket of the central U-shaped groove adjacent to the N-terminal domain, VipF remains in an open conformation. Moreover, mutations in the central U-shaped groove, including Glu129 and Asp251, largely impaired the acetyltransferase activity of VipF, suggesting a unique enzymatic mechanism for the Legionella effector VipF.

Ectopic Expression of Human Thymosin ß4 Confers Resistance to Legionella pneumophila during Pulmonary and Systemic Infection in Mice.

Thymosin beta-4 (Tß4) is an actin-sequestering peptide that plays important roles in regeneration and remodeling of injured tissues. However, its function in a naturally occurring pathogenic bacterial infection model has remained elusive. We adopted Tß4-overexpressing transgenic (Tg) mice to investigate the role of Tß4 in acute pulmonary infection and systemic sepsis caused by Legionella pneumophila Upon infection, Tß4-Tg mice demonstrated significantly lower bacterial loads in the lung, less hyaline membranes and necrotic abscess, with lower interstitial infiltration of neutrophils, CD4+, and CD8+ T cells. Bronchoalveolar lavage fluid of Tß4-Tg mice possessed higher bactericidal activity against exogenously added L. pneumophila, suggesting that constitutive expression of Tß4 could efficiently control L. pneumophila Furthermore, qPCR analysis of lung homogenates demonstrated significant reduction of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), which primarily originate from lung macrophages, in Tß4-Tg mice after pulmonary infection. Upon L. pneumophila challenge of bone marrow-derived macrophages (BMDM) in vitro, secretion of IL-1ß and TNF-α proteins was also reduced in Tß4-Tg macrophages, without affecting their survival. The anti-inflammatory effects of BMDM in Tß4-Tg mice on each cytokine were affected when triggering with tlr2, tlr4, tlr5, or tlr9 ligands, suggesting that anti-inflammatory effects of Tß4 are likely mediated by the reduced activation of Toll-like receptors (TLR). Finally, Tß4-Tg mice in a systemic sepsis model were protected from L. pneumophila-induced lethality compared to wild-type controls. Therefore, Tß4 confers effective resistance against L. pneumophila via two pathways, a bactericidal and an anti-inflammatory pathway, which can be harnessed to treat acute pneumonia and septic conditions caused by L. pneumophila in humans.

Toll-Interacting Protein in Pulmonary Diseases. Abiding by the Goldilocks Principle.

TOLLIP (Toll-interacting protein) is an intracellular adaptor protein with diverse actions throughout the body. In a context- and cell type-specific manner, TOLLIP can function as an inhibitor of inflammation and endoplasmic-reticulum stress, an activator of autophagy, or a critical regulator of intracellular vacuole trafficking. The distinct functions of this protein have been linked to innate immune responses and lung epithelial-cell apoptosis. TOLLIP genetic variants have been associated with a variety of chronic lung diseases, including idiopathic pulmonary fibrosis, asthma, and primary graft dysfunction after lung transplantation, and with infections, such as tuberculosis, Legionella pneumonia, and respiratory viruses. TOLLIP exists in a delicate homeostatic balance, with both positive and negative effects on the trajectory of pulmonary diseases. This translational review summarizes the genetic and molecular associations that link TOLLIP to the development and progression of noninfectious and infectious pulmonary diseases. We highlight current limitations of in vitro and in vivo models in assessing the role of TOLLIP in these conditions, and we describe future approaches that will enable a more nuanced exploration of the role of TOLLIP in pulmonary conditions. There has been a surge in recent research evaluating the role of this protein in human diseases, but critical mechanistic pathways require further exploration. By understanding its biologic functions in disease-specific contexts, we will be able to determine whether TOLLIP can be therapeutically modulated to treat pulmonary diseases.

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

The iron-regulated vacuolar Legionella pneumophila MavN protein is a transition-metal transporter.

Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.

TOLLIP deficiency is associated with increased resistance to Legionella pneumophila pneumonia.

Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires' disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip-/- mice demonstrated significantly fewer bacterial colony-forming unit and increased cytokine responses from BAL fluid. Tollip-/- macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16-0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.

Attenuated Legionella pneumophila Survives for a Long Period in an Environmental Water Site.

Legionella pneumophila is known as a human pathogen and is ubiquitous in natural and artificial aquatic environments. Many studies have revealed the virulence traits of L. pneumophila using clinical strains and a number of studies for characterizing environmental strains are also reported. However, the association between the virulence and survivability in the environment is unclear. In the present study, L. pneumophila was isolated from environmental water sites (Ashiyu foot spa, water fountain, and public bath), and the serogroups of isolated strains were determined by serological tests. Isolated strains were found to belong to serogroups SG1, SG2, SG3, SG4, SG5, SG8, SG9, and SG13. Untypeable strains were also obtained. Isolated strains were used for intracellular growth assay in a human monocytic cell line, THP-1. Among these strains, only an untypeable strain, named AY3, failed to replicate in THP-1. In addition, AY3 was maintained for a long period in an environmental water site, Ashiyu foot spa 2. Further, we compared the characteristics of several strains isolated from Ashiyu foot spa 2 and a clinical strain, Togus-1. AY3 failed to replicate in THP-1 cells but replicated in an amoeba model, Dictyostelium discoideum. Compared with Togus-1, the culturable cell number of environmental strains under stress conditions was higher. Moreover, biofilm formation was assessed, and AY3 showed the same degree of biofilm formation as Togus-1. Biofilm formation, replication in amoebae, and resistance against stress factors would explain the predominance of AY3 at one environmental site. Although the mechanism underlying the difference in the ability of AY3 to replicate in THP-1 cells or amoebae is still unclear, AY3 may abandon the ability to replicate in THP-1 cells to survive in one environment for a long period. Understanding the mechanisms of L. pneumophila in replication within different hosts should help in the control of Legionnaires' disease, but further study is necessary.

Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.

Legionella pneumophila translocated translation inhibitors are required for bacterial-induced host cell cycle arrest.

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.

The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Using an optimal set of features with a machine learning-based approach to predict effector proteins for Legionella pneumophila.

Type IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This study focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogen Legionella pneumophila strain Philadelphia-1, a cause of Legionnaires' disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 472 effector proteins that are deemed highly probable to be effectors and include 94% of known effectors. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.

A community outbreak of Legionnaires' disease in Geneva, Switzerland, June to September 2017.

PURPOSE: Eight confirmed cases of Legionnaires’ disease were identified at the Geneva University Hospitals between 28 July 2017 and 02 August 2017, leading to a detailed outbreak investigation. METHODS: Legionnaires’ disease cases were defined according to Swiss and European (ELDSNet) consensus guidelines. An outbreak investigation task force was put in place. Patients were interviewed, when feasible, with a standard questionnaire. A Legionella pneumophila urinary antigen test was performed in all cases. Lower respiratory tract (LRT) specimens were collected for culture, polymerase chain-reaction (PCR) assay, monoclonal antibody subtyping and sequenced-based typing (SBT). Multiple environmental samples were collected. Case geographical mapping was performed and local meteorological data were obtained. RESULTS: Thirty-four confirmed cases of Legionnaires’ disease were identified between 20 June 2017 and 16 September 2017, including 28 patients living in the Canton of Geneva and 6 cases in neighbouring cantons and France. The case fatality rate was 8.8%. The urinary antigen test was positive in 32/34 (94.1%) cases. Among the 17/34 (50%) cases with available LRT specimens, 8 (47.1%) were culture/PCR positive, 5 (29.4%) were PCR positive only, and 4 (23.5%) were culture/PCR negative. Monoclonal antibody subtyping and SBT on 12 samples allowed subtype identification of 8 samples, with a predominance of L. pneumophila serogroup-1 subtype-France/Allentown ST23 among clinical isolates. A specific city area was identified as a possible outbreak epicentre in 25/34 (73.5%) cases, although molecular analysis of clinical and environmental specimens revealed heterogeneous subtypes of L. pneumophila. CONCLUSIONS: In this largest documented outbreak of Legionnaires’ disease in Switzerland, we report prompt outbreak identification, leading to timely initiation of a detailed, well-orchestrated clinical and epidemiological investigation.

Legionella remodels the plasma membrane-derived vacuole by utilizing exocyst components as tethers.

During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.

Spatial structuring of a Legionella pneumophila population within the water system of a large occupational building.

The diversity of Legionella pneumophila populations within single water systems is not well understood, particularly in those unassociated with cases of Legionnaires' disease. Here, we performed genomic analysis of 235 L. pneumophila isolates obtained from 28 water samples in 13 locations within a large occupational building. Despite regular treatment, the water system of this building is thought to have been colonized by L. pneumophila for at least 30 years without evidence of association with Legionnaires' disease cases. All isolates belonged to one of three sequence types (STs), ST27 (n=81), ST68 (n=122) and ST87 (n=32), all three of which have been recovered from Legionnaires' disease patients previously. Pairwise single nucleotide polymorphism differences amongst isolates of the same ST were low, ranging from 0 to 19 in ST27, from 0 to 30 in ST68 and from 0 to 7 in ST87, and no homologous recombination was observed in any lineage. However, there was evidence of horizontal transfer of a plasmid, which was found in all ST87 isolates and only one ST68 isolate. A single ST was found in 10/13 sampled locations, and isolates of each ST were also more similar to those from the same location compared with those from different locations, demonstrating spatial structuring of the population within the water system. These findings provide the first insights into the diversity and genomic evolution of a L. pneumophila population within a complex water system not associated with disease.

The Legionella Effector Kinase LegK7 Hijacks the Host Hippo Pathway to Promote Infection.

The intracellular pathogen Legionella pneumophila encodes translocated effector proteins that modify host cell processes to support bacterial survival and growth. Here, we show that the L. pneumophila effector protein LegK7 hijacks the conserved Hippo signaling pathway by molecularly mimicking host Hippo kinase (MST1 in mammals), which is the key regulator of pathway activation. LegK7, like Hippo/MST1, phosphorylates the scaffolding protein MOB1, which triggers a signaling cascade resulting in the degradation of the transcriptional regulators TAZ and YAP1. Transcriptome analysis revealed that LegK7-mediated targeting of TAZ and YAP1 alters the transcriptional profile of mammalian macrophages, a key cellular target of L. pneumophila infection. Specifically, genes targeted by the transcription factor PPARγ, which is regulated by TAZ, displayed altered expression, and continuous interference with PPARγ activity rendered macrophages less permissive to L. pneumophila intracellular growth. Thus, a conserved L. pneumophila effector kinase exploits the Hippo pathway to promote bacterial growth and infection.