Research / Scientific departments / Cell Biology and Infection / Units and groups / Biology of Cell Interactions / Selected Published Work
Selected Published Work
Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?
Ball SG, Subtil A, Bhattacharya D, Moustafa A, Weber AP, Gehre L, Colleoni C, Arias MC, Cenci U, Dauvillée D. (2013) Plant Cell.;25(1):7-21.
Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.
The signalling factor PI 3-kinase is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors.
Basquin C, Malardé V, Mellor P, Anderson DH, Meas-Yedid V, Olivo-Marin JC, Dautry-Varsat A, Sauvonnet N. (2013) J Cell Sci.
Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent endocytic routes exist but the molecular mechanism of each pathway remains to be uncovered. This study focuses on a clathrin-independent, dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Rac1 and its targets the p21-activated kinases (Pak) are specific regulators of this pathway, acting on cortactin and actin polymerization. Here, our study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2R endocytosis. Firstly, the inhibition of the catalytic activity of PI 3-kinase strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI 3-kinase activation, is specifically involved in IL-2R entry. The second action of PI 3-kinase is via its regulatory subunit, p85α, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85α mutant missing the Rac1 binding motif, leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85α mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85α acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85α, Rac1 and IL-2R that is enhanced by IL-2. In summary our results point out a key role of class I PI 3-kinase in IL-2R endocytosis that creates a link with IL-2 signalling.
Shigella effector IpaB-induced cholesterol relocation disrupts the Golgi complex and recycling network to inhibit host cell secretion.
Mounier J, Boncompain G, Senerovic L, Lagache T, Chrétien F, Perez F, Kolbe M, Olivo-Marin JC, Sansonetti PJ, Sauvonnet N. (2012) Cell Host Microbe.;12(3):381-9.
Shigella induces bacillary dysentery by invading and causing the inflammatory destruction of the human colonic epithelial barrier. Here we show that Shigella invasion induces major damage to receptor recycling and to the Golgi complex. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting transferrin recycling. Noteworthy, Shigella induces the fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport. We demonstrate that the molecular process underlying this effect responds to a massive redistribution of cholesterol at the plasma membrane to the sites of Shigella entry. IpaB, a virulent factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. As the Golgi disorganization was also observed in vivo, this new mechanism affecting the sorting of cell surface molecules is likely to contribute to epithelial barrier disruption.
"Identification of a family of effectors secreted by the type III secretion system that are conserved in pathogenic Chlamydiae."
Muschiol, S., Boncompain G., Vromman, F., Dehoux, P., Normak, S., Henriques-Normark, B., Subtil, A. (2011). Infect Immun 79(2): 571-80.
Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.
"Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins."
Dehoux, P., R. Flores, Dauga, C., Zhong, G., Subtil, A. (2011). BMC Genomics, 12(1): 109.
BACKGROUND: Chlamydiae are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species.
RESULTS: Inc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the C. trachomatis and C. pneumoniae sequences tested, validating the data obtained in silico. We identified a macro domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions.
CONCLUSIONS: The Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.
Rac1 inactivation by lethal toxin from Clostridium sordellii modifies Focal Adhesions upstream of actin depolymerization.
Geny, B, Grassart, A, Manich, M Chicanne, G Payrastre, B Sauvonnet, N MR. Popoff. (2010). Cell Microbiol, 12(2):217-32.
Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1(V12). Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, beta-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization.
Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis.
Pennini, M.E., Perrinet, S., Dautry-Varsat, A., Subtil, A. (2010). PLOS Pathog, 6(7): e1000995.
Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.
Pak1 Phosphorylation Enhances Cortactin –N-WASP Interaction in Clathrin-Caveolin-Independent Endocytosis.
Grassart, A., Meas-Yedid, V., Dufour, A., Olivo-Marin, J-C., Dautry-Varsat, A., Sauvonnet, N. (2010). Traffic, 11(8) :1079-91.
Growing evidence indicates that kinases are central to the regulation of endocytic pathways. Previously, we identified p21-activated kinase 1 (Pak1) as the first specific regulator of clathrin- and caveolae-independent endocytosis used by the interleukin 2 receptor subunit (IL-2R). Here, we address the mechanism by which Pak1 regulates IL-2Rbeta endocytosis. First, we show that Pak1 phosphorylates an activator of actin polymerization, cortactin, on its serine residues 405 and 418. Consistently, we observe a specific inhibition of IL-2Rbeta endocytosis when cells overexpress a cortactin, wherein these serine residues have been mutated. In addition, we show that the actin polymerization enhancer, neuronal Wiskott-Aldrich syndrome protein (N-WASP), is involved in IL-2Rbeta endocytosis. Strikingly, we find that Pak1 phosphorylation of cortactin on serine residues 405 and 418 increases its association with N-WASP. Thus, Pak1, by controlling the interaction between cortactin and N-WASP, could regulate the polymerization of actin during clathrin-independent endocytosis.
Production of recative oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis.
Boncompain, G., Schneider, B., Delevoye, C., Kellermann, O., Dautry-Varsat, A., Subtil, A. (2010). Infect. Immun, 78(1) :80-7.
Reactive oxygen species (ROS) are many-faceted compounds involved in cell defense against pathogens, as well as in cell signaling. Their involvement in the response to infection in epithelial cells remains poorly documented. Here, we investigated the production of ROS during infection with Chlamydia trachomatis, a strict intracellular pathogen, in HeLa cells. C. trachomatis induced a transient increase in the ROS level within a few hours, followed by a return to basal level 9 hours after infection. At this time point, the host enzyme dedicated to ROS production, NADPH oxidase, could no longer be activated by external stimuli, such as interleukin-1beta. In addition, Rac, a regulatory subunit of the NADPH oxidase complex, was relocated to the membrane of the compartment in which the bacteria develop, the inclusion, while other subunits were not. Altogether, these results indicate that C. trachomatis infection elicits the production of ROS and that the bacteria rapidly target the activity of NADPH oxidase to shut it down. Prevention of ROS production at the onset of the bacterial developmental cycle might delay the host response to infection.
Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells.
Muschiol, S., Normark, S., Henriques-Normark B., Subtil A. (2009). BioMed Cent., 9 :75.
BACKGROUND: Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.
RESULTS: We demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry.
CONCLUSION: INPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.
SNARE Protein Mimicry by an Intercellular Bacterium.
Delevoye C., Nilges M., Dehoux P., Paumet F., Perrinet S., Dautry-Varsat A., Subtil A. (2008). PLoS Path., 4(3), e1000022.
Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.
Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2.
Grassart, A., Dujeancourt A., Lazarow P., Dautry-Varsat A., Sauvonnet N. (2008). EMBO Reports, 9(4), 356-362.
There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway.