The Legionella collagen-like protein employs a distinct binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual trimer arrangement with a positively charged external surface and negatively charged solvent exposed internal cavity. Through molecular dynamics simulations, we show how the glycosaminoglycan chondroitin-4-sulphate associates with the Lcl-CTD surface via distinct binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate-binding mechanism.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila.

Legionella pneumophila-mediated host posttranslational modifications.

Legionella pneumophila is a Gram-negative bacterium ubiquitously present in freshwater environments and causes a serious type of pneumonia called Legionnaires' disease. During infections, L. pneumophila releases over 300 effector proteins into host cells through an Icm/Dot type IV secretion system to manipulate the host defense system for survival within the host. Notably, certain effector proteins mediate posttranslational modifications (PTMs), serving as useful approaches exploited by L. pneumophila to modify host proteins. Some effectors catalyze the addition of host protein PTMs, while others mediate the removal of PTMs from host proteins. In this review, we summarize L. pneumophila effector-mediated PTMs of host proteins, including phosphorylation, ubiquitination, glycosylation, AMPylation, phosphocholination, methylation, and ADP-ribosylation, as well as dephosphorylation, deubiquitination, deAMPylation, deADP-ribosylation, dephosphocholination, and delipidation. We describe their molecular mechanisms and biological functions in the regulation of bacterial growth and Legionella-containing vacuole biosynthesis and in the disruption of host immune and defense machinery.

Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium.

The causative agent of a severe pneumonia termed "Legionnaires' disease", Legionella pneumophila, replicates within protozoan and mammalian phagocytes in a specialized intracellular compartment called the Legionella-containing vacuole (LCV). This compartment does not fuse with bactericidal lysosomes but communicates extensively with several cellular vesicle trafficking pathways and eventually associates tightly with the endoplasmic reticulum. In order to comprehend in detail the complex process of LCV formation, the identification and kinetic analysis of cellular trafficking pathway markers on the pathogen vacuole are crucial. This chapter describes imaging flow cytometry (IFC)-based methods for the objective, quantitative and high-throughput analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we use the haploid amoeba Dictyostelium discoideum as an infection model for L. pneumophila, to analyze either fixed intact infected host cells or LCVs from homogenized amoebae. Parental strains and isogenic mutant amoebae are compared in order to determine the contribution of a specific host factor to LCV formation. The amoebae simultaneously produce two different fluorescently tagged probes enabling tandem quantification of two LCV markers in intact amoebae or the identification of LCVs using one probe and quantification of the other probe in host cell homogenates. The IFC approach allows rapid generation of statistically robust data from thousands of pathogen vacuoles and can be applied to other infection models.

Rapid adaptations of Legionella pneumophila to the human host.

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.

Legionellapneumophila induces methylomic changes in ten-eleven translocation to ensure bacterial reproduction in human lung epithelial cells.

Introduction. Legionella pneumophila is a Gram-negative flagellated bacteria that can infect human lungs and cause a severe form of pneumonia named Legionnaires' disease.Hypothesis. We hypothesize that L. pneumophila infection induces methylomic changes in methylcytosine dioxygenases, ten-eleven translocation (TET) genes, and controls DNA methylation following infection.Aim. In the current research, we sought to further investigate DNA methylation changes in human lung epithelial cells upon L. pneumophila infection and determine how methylation inhibitor agents disturb L. pneumophila reproduction.Methodology. A549 cell line was used in L. pneumophila infection and inhibitors' treatment, including 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG).Results. Interestingly, DNA methylation analysis of infected A549 using sodium bisulfite PCR and the methylation-sensitive HpaII enzyme showed potential methylation activity within the promoter regions of ten-eleven translocation (TET) genes located on CpG/397-8 and CpG/385-6 of TET1 and TET3, respectively. Such methylation changes in TET effectors decreased their expression profile following infection, indicated by quantitative real-time PCR (RT-qPCR), immunoblotting and flow cytometry. Furthermore, pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased the bacterial reproduction characterized by the expression of L. pneumophila 16S ribosomal RNA and the c.f.u. ml-1 of bacterial particles. Moreover, both methylation inhibitors showed potent inhibition of methionine synthase (MS) expression, which was further confirmed by the docking analysis of inhibitor ligands and crystal structure of MS protein.Conclusion. These data provide evidence for the methylomic changes in the promoter region of TET1 and TET3 by L. pneumophila infection in the A549 cell line and suggest the anti-bacterial properties of 5-AZA and EGCG, as methylation inhibitors, are due to targeting the epigenetic effector methionine synthase.

Cigarette smoke depletes alveolar macrophages and delays clearance of Legionella pneumophila.

Legionella pneumophila is the main etiological agent of Legionnaires' disease, a severe bacterial pneumonia. L. pneumophila is initially engulfed by alveolar macrophages (AMs) and subvert normal cellular functions to establish a replicative vacuole. Cigarette smokers are particularly susceptible to developing Legionnaires' disease and other pulmonary infections; however, little is known about the cellular mechanisms underlying this susceptibility. To investigate this, we used a mouse model of acute cigarette smoke exposure to examine the immune response to cigarette smoke and subsequent L. pneumophila infection. Contrary to previous reports, we show that cigarette smoke exposure alone causes a significant depletion of AMs using enzymatic digestion to extract cells, or via imaging intact lung lobes by light-sheet microscopy. Furthermore, treatment of mice deficient in specific types of cell death with smoke suggests that NLRP3-driven pyroptosis is a contributor to smoke-induced death of AMs. After infection, smoke-exposed mice displayed increased pulmonary L. pneumophila loads and developed more severe disease compared with air-exposed controls. We tested if depletion of AMs was related to this phenotype by directly depleting them with clodronate liposomes and found that this also resulted in increased L. pneumophila loads. In summary, our results showed that cigarette smoke depleted AMs from the lung and that this likely contributed to more severe Legionnaires' disease. Furthermore, the role of AMs in L. pneumophila infection is more nuanced than simply providing a replicative niche, and our studies suggest they play a major role in bacterial clearance.

Characterization of a Novel Regulator of Biofilm Formation in the Pathogen Legionella pneumophila.

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen that causes severe pneumonia known as Legionnaires' disease. The bacterium causes disease when contaminated water is aerosolized and subsequently inhaled by individuals, which allows the bacteria to gain access to the lungs, where they infect alveolar macrophages. L. pneumophila is ubiquitous in the environment, where it survives by growing in biofilms, intracellularly within protozoa, and planktonically. Biofilms are a major concern for public health because they provide a protective niche that allows for the continuous leaching of bacteria into the water supply. In addition, biofilms enhance the survival of the bacteria by increasing resistance to temperature fluctuations and antimicrobial agents. Currently, there is little known about biofilm formation and regulation by L. pneumophila. Here, we present evidence of a specific gene, bffA, which appears to be involved in the regulation of motility, biofilm formation, cellular replication, and virulence of L. pneumophila. A strain lacking bffA has an enhanced biofilm formation phenotype, forming biofilms that are both faster and thicker than wild type. Additionally, the knockout strain has significantly reduced motility, enhanced uptake into amoebae, and altered growth kinetics on solid media. Our data suggest a potential role for bffA in signaling pathways that govern changes in growth rate and motility in response to environmental conditions.

Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases.

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila.

Non-canonical activation of the ER stress sensor ATF6 by Legionella pneumophila effectors.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ∼330 effector proteins into the host cell to sculpt an ER-derived replicative niche. We previously reported five L.p. effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset of L.p. toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation, and target gene transcription. In a deviation from the conventional model, this L.p-dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control that L.p exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways.

The unity of opposites: Strategic interplay between bacterial effectors to regulate cellular homeostasis.

Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.

Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER.

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.

Activation of the Legionella pneumophila LegK7 Effector Kinase by the Host MOB1 Protein.

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique ß-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.

CD8+ T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection.

Legionella pneumophila, an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8+ T cells are directly associated with protection against L. pneumophila, with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila, they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8+ CTLs. Of the 13 PAL peptides tested, PAL92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL92-100-specific CD8+ T cells play a central role in the host defense response against L. pneumophila.

Performance of the ImmuView and BinaxNOW assays for the detection of urine and cerebrospinal fluid Streptococcus pneumoniae and Legionella pneumophila serogroup 1 antigen in patients with Legionnaires' disease or pneumococcal pneumonia and meningitis.

The performances of the ImmuView Streptococcus pneumoniae (Sp) and Legionella pneumophila (Lp) urinary antigen test were compared to that of the BinaxNOW Sp and Lp assays, using frozen urine from 166 patients with Legionnaires' disease (LD) and 59 patients with pneumococcal pneumonia. Thirty Sp-positive or contrived cerebrospinal fluids (CSF) were also tested. Test specimens were collected and tested at different sites, with each site testing unique specimens by technologists blinded to expected results. No significant differences in test concordances were detected for the ImmuView and BinaxNOW assays for the Sp or Lp targets for urine from patients with pneumococcal pneumonia or LD when performance from both sites were combined. At one of two test sites the ImmuView Lp assay was more sensitive than the BinaxNOW assay, with no correlation between test performance and Lp serogroup 1 monoclonal type. Urines from six of seven patients with LD caused by Legionella spp. bacteria other than Lp serogroup 1 were negative in both assays. Both tests had equivalent performance for Sp-positive CSF. The clinical sensitivities for pneumococcal pneumonia were 88.1 and 94.4% for the ImmuView and Binax assays, and 87.6 and 84.2% for the Lp assays, respectively. Test specificities for pneumococcal pneumonia were 96.2 and 97.0% for the ImmuView and Binax assays, and 99.6 and 99.1% for the Lp assays. Both assays were highly specific for Sp in pediatric urines from children with nasopharyngeal colonization by the bacterium. ImmuView and BinaxNOW assay performance was equivalent in these studies.

Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila.

Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires' disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0-108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.

Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Crystal structure of the metaeffector MesI (Lpg2505) from Legionella pneumophila.

Persistence and replication of the gram-negative bacterium Legionella pneumophila in the human host cell depend on so-called effector proteins that target diverse cellular functions and modulate them in favor of the pathogen. We solved the crystal structure of the L. pneumophila effector protein MesI de novo to a resolution of 2.2 Å. The 34 kDa polypeptide chain folds into two distinct α-helical domains. The larger C-terminal domain shows similarity to tetratricopeptide repeat proteins. Using size-exclusion chromatography, we confirmed that MesI binds tightly to full-length SidI and that deletion of either the N- or the C-terminus weakens the interaction. Based on the three-dimensional structure of MesI we suggest a possible binding mode for SidI and identified two homologs of MesI within the proteome of L. pneumophila that do not bind to SidI, but may act as specific inhibitors of other yet to be identified effectors.