The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome-and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.Coxiella burnetii | host-pathogen interactions | phosphoinositides

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling.(2016) Biofouling, Open Archive TOULOUSE Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of some Toulouse researchers and makes it freely available over the web where possible. This is an author's version published in : http://oatao.univ-toulouse.fr/19753Official URL : https://doi.org/10. 1080/08927014.2016.1212988 Any correspondence concerning this service should be sent to the repository administrator : tech-oatao@listes-diff.inp-toulouse.fr The waterborne pathogen Legionella pneumophila grows as a bio lm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in bio lm 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 bio lm regulation, the consequences on bio lm 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 di erent aspects of bio lm formation, while two proteins with dual activity (DGC/PDE) inhibit bio lm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that speci c c-di-GMP pathways control L. pneumophila bio lm 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 bio lm in response to nitric oxide, a signal for bio lm dispersion in many other species. New insights into Legionella pneumophila bio lm regulation by c-di-GMP signaling

The Q fever agentCoxiella burnetiiuses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigatingC. burnetiieffector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to theC. burnetii nopA::Tn or a Dot/Icm-defectivedotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-typeC. burnetiior the correspondingnopAcomplemented strain. Thus, NopA is an important regulator of the innate immune response allowingCoxiellato behave as a stealth pathogen.

g Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/ Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over-and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. C yclic-di-GMP (c-di-GMP) is a nearly ubiquitous bacterial second messenger involved in signaling pathways that control lifestyle transitions, among which are the transition from the motile to the sessile state and, for pathogenic bacteria, the transition from pathogenic to environmental lifestyles or from acute/planktonic infections to chronic/biofilm infections (1-4). c-di-GMP is produced from GTP by diguanylate cyclases (DGCs), is degraded by phosphodiesterases (PDEs), and acts through various effector proteins or RNA riboswitches (5-8). The catalytic site of DGCs is associated with the GGDEF domain, while PDE activity involves either the EAL or the HD-GYP domains. Genes encoding these proteins have been identified in all major bacterial phyla and are particularly expanded in gammaproteobacteria, with a median of 22 genes per genome (9). Consistently, the genomes of gammaproteobacterium Legionella pneumophila strains encode from 22 to 24 GGDEF/EAL proteins (10, 11). Few are specific to each strain, but most of them are highly conserved in L. pneumophila strains (11). c-di-GMP seems to be strongly involved in Legionella cell signaling, since the 22 GGDEF/EAL proteins represent 13.5% of proteins contain...

2020): Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence, Autophagy, ABSTRACT Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiellacontaining vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host–pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

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Among the bacterial secretion systems, the Type III, IV, and VI secretion systems enable bacteria to secrete proteins directly into a target cell. This specific form of secretion, referred to as translocation, is essential for a number of pathogens to alter or kill targeted cells. The translocated proteins, called effector proteins, can directly interfere with the normal processes of the targeted cells, preventing elimination of pathogens and promoting their multiplication. The function of effector proteins varies greatly depending on the considered pathogen and the targeted cell. In addition, there is often no magic bullet, and the number of effector proteins can range from a handful to hundreds, with, for instance, a substrate of over 300 effector proteins of the Icm/Dot Type IV secretion system in the human pathogen Legionella pneumophila. Identifying, detecting, and monitoring the translocation of each of the effector proteins represents an active field of research and is key to understanding the bacterial molecular weaponry. Translational fusion of an effector with a reporter protein of known activity remains the best method to monitor effector translocation. The development of a fluorescent substrate for the TEM-1 beta-lactamase has turned this antibiotic-resistant protein into a highly versatile reporter system for investigating protein transfer events associated with microbial infection of host cells. Here we describe a simple protocol to assay the translocation of an effector protein by the Icm/Dot system of the human pathogen Legionella pneumophila.