The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to ER-Chlamydia Inclusion Membrane Contact Sites

Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development

Making Contact: VAP Targeting by Intracellular Pathogens

In naïve cells, the endoplasmic reticulum (ER) and the ER-resident V esicle-associated membrane protein- A ssociated P roteins (VAP) are common components of sites of membrane contacts that mediate the nonvesicular transfer of lipids between organelles. There is increasing recognition that the hijacking of VAP by intracellular pathogens is a novel mechanism of host–pathogen interaction. Here, we summarize our recent findings showing that the Chlamydia inclusion membrane protein IncV tethers the ER to the inclusion membrane by binding to VAP via the molecular mimicry of two eukaryotic FFAT motifs. We extend the discussion to other microorganisms that have evolved similar mechanisms

Pathogen vacuole membrane contact sites – close encounters of the fifth kind

Vesicular trafficking and membrane fusion are well-characterized, versatile, and sophisticated means of ‘long range’ intracellular protein and lipid delivery. Membrane contact sites (MCS) have been studied in far less detail, but are crucial for ‘short range’ (10–30 nm) communication between organelles, as well as between pathogen vacuoles and organelles. MCS are specialized in the non-vesicular trafficking of small molecules such as calcium and lipids. Pivotal MCS components important for lipid transfer are the VAP receptor/tether protein, oxysterol binding proteins (OSBPs), the ceramide transport protein CERT, the phosphoinositide phosphatase Sac1, and the lipid phosphatidylinositol 4-phosphate (PtdIns(4)P). In this review, we discuss how these MCS components are subverted by bacterial pathogens and their secreted effector proteins to promote intracellular survival and replication

Macrophages from Mice with the Restrictive Lgn1 Allele Exhibit Multifactorial Resistance to Legionella pneumophila

Although Legionella pneumophila can multiply in diverse cell types from a variety of species, macrophages from most inbred mouse strains are nonpermissive for intracellular replication and allow little or no growth of the bacteria. This phenomenon is likely genetically controlled by the mouse naip5 (birc1e) gene located within the Lgn1 locus. In this study, we have investigated the resistance of C57BL/6J macrophages to L. pneumophila infection by examining the fate of both the bacterium and the infected cells compared to that in macrophages from the permissive A/J strain. Our results indicate that although the trafficking of the L. pneumophila-containing vacuole is partially disrupted in C57BL/6J macrophages, this cannot account for the severity of the defect in intracellular growth observed in this strain. Infected macrophages are lost shortly after infection, and at later times a larger fraction of the C57BL/6J macrophages in which L. pneumophila undergoes replication are apoptotic compared to those derived from A/J mice. Finally, a loss of bacterial counts occurs after the first round of growth. Therefore, the resistance mechanism of C57BL/6J macrophages to L. pneumophila infection appears to be multifactorial, and we discuss how early and late responses result in clearing the infection

Legionella pneumophila Replication Vacuole Formation Involves Rapid Recruitment of Proteins of the Early Secretory System

Legionella pneumophila vacuole biogenesis was analyzed by using a cell-free system. We show that calnexin, Sec22b, and Rab1 are recruited to the vacuole very shortly after bacterial uptake, and we have identified Rab1 as a potential host factor involved in the endoplasmic reticulum recruitment process

CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram-positive bacteria

International audienceclpP and clpC of Bacillus subtillis encode subunits of the Clp ATP-dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR, the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC, as well as clpE, which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNasel footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram-positive bacteria, including Listeria monocytogenes, Streptococcus salivarius, S. pneumoniae, S. pyogenes, S. thermophilus, Enterococcus faecalis, Staphylococcus aureus, Leuconostoc oenos, Lactobacillus sake, Lactococcus lactis and Clostridium acetobutylicum. CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram-positive bacteria

The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37 degrees C

International audienceCtsR (class three stress gene repressor) negatively regulates the expression of class III heat shock genes (clpP, clpE and the clpC operon) by binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAA NAN A/GGTCAAA). CtsR-dependent genes are expressed at a low level at 37 degrees C and are strongly induced under heat shock conditions. We performed a structure/function analysis of the CtsR protein, which is highly conserved among low G+C Gram-positive bacteria. Random chemical mutagenesis, in vitro cross-linking, in vivo co-expression of wild-type and mutant forms of CtsR and the construction of chimeric proteins with the DNA-binding domain of the lambda CI repressor allowed us to identify three different functional domains within CtsR: a helix-turn-helix DNA-binding domain, a dimerization domain and a putative heat-sensing domain. We provide evidence suggesting that CtsR is active as a dimer. Transcriptional analysis of a clpP'-bgaB fusion and/or Western blotting experiments using antibodies directed against the CtsR protein indicate that ClpP and ClpX are involved in CtsR degradation at 37 degrees C. This in turn leads to a low steady-state level of CtsR within the cell, as CtsR negatively autoregulates its own synthesis. This is the first example of degradation of a repressor of stress response genes by the Clp ATP-dependent protease

A <em>C. trachomatis</em> Cloning Vector and the Generation of <em>C. trachomatis</em> Strains Expressing Fluorescent Proteins under the Control of a <em>C. trachomatis</em> Promoter

<div><p>Here we describe a versatile cloning vector for conducting genetic experiments in <i>C. trachomatis</i>. We successfully expressed various fluorescent proteins (i.e. GFP, mCherry and CFP) from <i>C. trachomatis</i> regulatory elements (i.e. the promoter and terminator of the <i>incDEFG</i> operon) and showed that the transformed strains produced wild type amounts of infectious particles and recapitulated major features of the <i>C. trachomatis</i> developmental cycle. <i>C. trachomatis</i> strains expressing fluorescent proteins are valuable tools for studying the <i>C. trachomatis</i> developmental cycle. For instance, we show the feasibility of investigating the dynamics of inclusion fusion and interaction with host proteins and organelles by time-lapse video microscopy.</p> </div

The CAD domain of STIM1 is required for STIM1 localization to ER-Inclusion MCSs.

<p>A. Schematic representation of the major domains of the STIM1 protein, their respective amino acid residue position and their respective cellular localization (ER lumen or Cytosol). Signal: signal peptide; EF: EF-hand; SAM: sterile alpha motif; TM: transmembrane domain; CC1: coiled-coil 1; CC2: coiled-coil 2; CAD: CRAC activation domain; S/P: Serine-proline-rich region; K: lysine-rich region. B. Confocal micrographs of HeLa cells expressing the indicated mCherry-STIM1 construct (red) and infected with <i>C</i>. <i>trachomatis</i> for 24h. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion and N indicates the nucleus. Scale bar: 10μm. C. Quantification of inclusion association of the indicated mCh-STIM1 constructs compared to full-length mCh-STIM1. *** p value <0.001. D. Confocal micrographs of HeLa cells expressing YFP-CAD (CAD(342–448), yellow), and infected for 24h with a strain of <i>C</i>. <i>trachomatis</i> expressing CFP (cyan). The merge is shown on the right. Two representative examples of YFP-CAD pattern on the inclusion are shown. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. Scale bar: 10μm.</p