Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier

Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming

Protein modification with ISG15 (ISGylation) represents a major type I IFN–induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies

ATG12 Conjugation to ATG3 Modulates Mitochondrial Homeostasis and Cell Death

Autophagy is an intracellular response to starvation, which is conserved in eukaryotes from unicellular to metazoan organisms. While autophagy sustains an individual cell from death by a variety of cellular stresses, evidence suggests that it is also critically important on an organismal level for the prevention of cancer, neurodegenerative and autoimmune diseases. In light of the therapeutic implications of autophagy, much remains to be understood about the function of individual autophagy genes. Many of the autophagy genes participate in two ubiquitin-like conjugation systems. The first mediates conjugation of ATG12 to ATG5 and the second mediates lipidation of LC3/ATG8 by phosphatidylethanolamine. We hypothesized that ATG12 conjugates to other substrates in addition to ATG5, as is the case for most ubiquitin-like proteins (ubls). By increasing the cellular pool of ATG12, we were able to identify and characterize a novel covalent complex between ATG12 and ATG3. A small subset of the cellular pool of ATG3 is in complex with ATG12 at baseline conditions in mammalian cells. Formation of the ATG12-ATG3 complex requires activation by ATG7, the autophagy E1-like enzyme. ATG12 subsequently becomes auto-conjugated in cis by ATG3. Unlike ATG12-ATG5 complex formation, ATG12-ATG3 conjugation does not require the E2-like enzyme ATG10. The c-terminal glycine of ATG12 specifically binds to K243 of ATG3.The ATG12-ATG5 complex and ATG3 each modulate steps in early autophagosome expansion and closure. Therefore, we initially expected that the ATG12-ATG3 complex would affect autophagy. To our surprise, disruption of the ATG12-ATG3 complex had no effect on autophagy induced by starvation, rapamycin or chemical uncoupling of the mitochondria. Instead disruption of the ATG12-ATG3 complex leads to a highly fragmented mitochondrial network that is incapable of fusion, increased in mass and defective in mitochondrial autophagy. These phenotypes also correlate with resistance to mitochondrial cell death. In summary, we have demonstrated evidence for a previously unknown conjugate between ATG12 and ATG3. Conjugation between ATG12 and ATG3 endows each protein with an entirely separate function from either protein's known role in autophagosome biogenesis. Our work suggests that ATG12 may be a more broad-based protein modification than previously anticipated

Cytosolic innate immune sensing and signaling upon infection

International audienceCytosolic sensing of pathogens is essential to a productive immune response. Recent reports have emphasized the importance of signaling platforms emanating from organelles and cytosolic sensors, particularly during the response to intracellular pathogens. Here, we highlight recent discoveries identifying the key mediators of nucleic acid and cyclic nucleotide sensing and discuss their importance in host defense. This review will also cover strategies evolved by pathogens to manipulate these pathways

Lmo1656 is a secreted virulence factor of Listeria monocytogenes that interacts with the sorting nexin 6-BAR complex

Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm. Furthermore, deletion of the lmo1656 gene (Δlmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host

The new microbiology: a conference at the Institut de France

International audienceIn May 2012, three European Academies held a conference on the present and future of microbiology. The conference, entitled "The New Microbiology", was a joint effort of the French Academie des sciences, of the German National Academy of Sciences Leopoldina and of the British Royal Society. The organizers - Pascale Cossart and Philippe Sansonetti from the "Academie des sciences", David Holden and Richard Moxon from the "Royal Society", and Jorg Hacker and Jurgen Hesseman from the "Leopoldina Nationale Akademie der Wissenschaften" - wanted to highlight the current renaissance in the field of microbiology mostly due to the advent of technological developments and allowing for single-cell analysis, rapid and inexpensive genome-wide comparisons, sophisticated microscopy and quantitative large-scale studies of RNA regulation and proteomics. The conference took place in the historical Palais de l'Institut de France in Paris with the strong support of Jean-Francois Bach, Secretaire Perpetuel of the Academie des sciences

Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli.: SUMO sites mapping by quantitative proteomics

International audienceSUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease

Lmo1656 is a secreted virulence factor of Listeria monocytogenes that interacts with the sorting nexin 6–BAR complex

International audienceListeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood– brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm. Furthermore, deletion of the lmo1656 gene (lmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain– containing protein, as a host cell interactor of Lmol656. SNX6 colocal-izes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mam-malian host. The foodborne pathogen Listeria monocytogenes (Lm) 5 can cross several physiological barriers and infect multiple cell types. The pathogenic potential of Lm relies on the ability of this bacterium to cross multiple physiological barriers as well as its ability to enter and replicate within a wide variety of host cell types (for recent reviews, see Refs. 1 and 2). Upon binding to host cell surface receptors, Lm induces its internalization into both professional phagocytes and nonphagocytic cells (for a recent review, see Ref. 2). From there, Lm escapes into the cyto-sol by rupturing its vacuole. Lm is able to evade host cell immune responses (for a recent review, see Ref. 3) and subvert the host cell actin cytoskeleton to drive intra-and intercellular motility (for recent reviews, see Refs. 4 –6). Secreted and surface-exposed Lm proteins can encounter host components and serve as virulence factors. For example, the secreted pore-forming toxin listeriolysin O (LLO) is one of the most well-characterized and potent virulence factors of Lm (for a review, see Ref. 7). Secretion of LLO occurs prior to Lm entry into the host cell. It inserts into the host plasma membrane and makes large pores. The resulting ion flux drives a diverse array of responses within the cell from global deSUMOylation (8) to mitochondrial fragmentation (9). Upon entry, Lm can escape into the host cytosol by lysing the phagosomal membrane through the combined actions of secreted LLO and phospholipases A and B (PlcA and PlcB) (10 –12). Recent work has uncovered novel secreted Lm virulence factors and their binding partners in the host cell. The secreted protein Listeria nuclear targeted protein A (LntA) targets the host epigenetic regulator BAHD1, altering host cell transcription (13). The small secreted protein internalin C (InlC) sequesters Tuba, a Cdc42 guanine exchange factor, to induce relaxation of membrane cortical tension, thereby facilitating increased bacterial cell-to-cell spread (14, 15). InlC also directly binds to host IB kinase , interfering with host innate immunity (16). The recent plethora of genomics data and the rise of bioin-formatics pipelines have enabled the rapid comparison of mu