The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery

Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire’s disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host’s cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 Å resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365–524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter

The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (??loop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidD??loop phenocopied a L. pneumophila ??sidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae

Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of <it>Legionella </it>species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely <it>L. pneumophila </it>Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1) and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis.</p> <p>Results</p> <p>We show that <it>L. pneumophila </it>Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between <it>L. pneumophila </it>strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different <it>Legionella </it>species.</p> <p>Conclusion</p> <p>Horizontal gene transfer among bacteria and from eukaryotes to <it>L. pneumophila </it>as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time.</p

A global reference for caesarean section rates (C-Model): a multicountry cross-sectional study.

OBJECTIVE: To generate a global reference for caesarean section (CS) rates at health facilities. DESIGN: Cross-sectional study. SETTING: Health facilities from 43 countries. POPULATION/SAMPLE: Thirty eight thousand three hundred and twenty-four women giving birth from 22 countries for model building and 10,045,875 women giving birth from 43 countries for model testing. METHODS: We hypothesised that mathematical models could determine the relationship between clinical-obstetric characteristics and CS. These models generated probabilities of CS that could be compared with the observed CS rates. We devised a three-step approach to generate the global benchmark of CS rates at health facilities: creation of a multi-country reference population, building mathematical models, and testing these models. MAIN OUTCOME MEASURES: Area under the ROC curves, diagnostic odds ratio, expected CS rate, observed CS rate. RESULTS: According to the different versions of the model, areas under the ROC curves suggested a good discriminatory capacity of C-Model, with summary estimates ranging from 0.832 to 0.844. The C-Model was able to generate expected CS rates adjusted for the case-mix of the obstetric population. We have also prepared an e-calculator to facilitate use of C-Model (www.who.int/reproductivehealth/publications/maternal_perinatal_health/c-model/en/). CONCLUSIONS: This article describes the development of a global reference for CS rates. Based on maternal characteristics, this tool was able to generate an individualised expected CS rate for health facilities or groups of health facilities. With C-Model, obstetric teams, health system managers, health facilities, health insurance companies, and governments can produce a customised reference CS rate for assessing use (and overuse) of CS. TWEETABLE ABSTRACT: The C-Model provides a customized benchmark for caesarean section rates in health facilities and systems