Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer. / Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer.

The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.

Chez les procaryotes, les protéines sont synthétisées dans le cytoplasme avant d'être transportés vers différentes destinations, intra- ou extra-cellulaires. Les bactéries Gram-négatives ont mis au point une grande collection de mécanismes et systèmes, appelés systèmes de sécrétion bactérienne, pour sécréter des protéines à travers leur paroi cellulaire vers l'extérieur. Le système de sécrétion de type VI, identifié dans les années 2006-2008, est une nano-machine polyvalente répandue chez les bactéries pathogènes. Il y a de nombreuses preuves que T6SS injecte des protéines toxiques (effecteurs) directement dans les cellules eucaryotes et procaryotes pour les tuer. Pour empêcher la destruction cellules provenant de la même espèce, les bactéries possédant un T6SS produisent également des protéines d'immunité qui neutralisent les effets toxiques des effecteurs de leurs congénères. Le T6SS est formé de 13 composants de coeur (nommés TssA-M) en une structure souvent comparée à un "bactériophage inversé". La queue, semblable à celle de phages, a une forme tubulaire (la gaine et le tube interne) et polymérise à partir d'une plaque basale ancrée sur un complexe membranaire trans-périplasmique. La contraction de la gaine fournit l'énergie nécessaire pour propulser le tube intérieur à travers la paroi vers les cellules proies. Dans le cadre de ma thèse, je me suis impliqué dans la détermination de la structure et la dynamique de certains composants du T6SS de la d’Escerichia coli enteroaggrégatif (EAEC). Plusieurs structures ont été déterminées et analysées. Quatre articles ont été publiés et deux autres sont en préparation. / In prokaryotes, proteins are synthesized in the cytoplasm before being transported to various destinations, intra- or extra-cellular. Gram-negative bacteria have developed a large collection of mechanisms and systems, termed bacterial secretion systems, to secrete proteins through their cell wall to the exterior. The type VI secretion system, identified in years 2006-2008, is a versatile nano-machine prevalent in pathogenic bacteria. There have been many evidences that T6SS delivers toxic proteins directly into both eukaryotic and prokaryotic cells to kill them. To prevent killing of sibling cells (cells from the same species), T6SS+ cells produce also immunity proteins that neutralize the toxic effects of their cognate effectors. T6SS contains 13 core-components (TssA-M), assembling a structure often quoted as an “inverted bacteriophage”. A phage-like tubular tail (the sheath and the internal tube) polymerizes from a baseplate-like complex, anchored to the cell internal and outer membranes via a membrane anchored complex spanning the periplasm. Contraction of the sheath provides the necessary energy to propel the internal tube through the wall towards the prey cells. In the framework of my PhD, I became involved in determining the structure and dynamics of some components of the EAEC sci1 T6SS, mostly on the membrane and baseplate subcomplexes. Several structures have been determined and analysed. Four articles have been published and two other are in preparation.

Toxoplasma gondii est un protiste apicomplexe responsable de la toxoplasmose. Ce parasite intracellulaire obligatoire possède des organites sécrétoires apicaux dont les rhoptries qui contiennent des facteurs de virulence essentiels à l'invasion et à la modulation de la cellule hôte qu'il infecte. Au cours de la division cellulaire de T. gondii, les protéines de rhoptries sont synthétisées selon la même cinétique. Dans le but d'identifier de nouvelles protéines dont la fonction est potentiellement liée aux rhoptries, nous avons recherché à partir des bases de données du génome de T. gondii, les protéines présentant ce profil particulier d'expression. La localisation subcellulaire de 12 candidats a été réalisée puis une caractérisation phénotypique de quatre d'entre eux a été entreprise. Nous avons identifié une nouvelle protéase de rhoptries, DegP, essentielle à la virulence du parasite in vivo. Nous montrons que DegP contrôle la phase aigüe de l'infection en modulant la réponse immune de l'hôte contribuant ainsi à la dissémination du parasite in vivo. Nous identifions également deux protéines homologues, Claw1 et Claw2, présentant une localisation atypique à l'extrémité apicale du parasite. Notre incapacité à déléter ces gènes pourrait indiquer un rôle essentiel de ces protéines au niveau du complexe apical de T. gondii. Enfin, bien que n'étant pas reliée aux rhoptries, ce crible a permis d'identifier la première protéine associée aux jonctions des vésicules constituant le complexe membranaire interne de Toxoplasma. La délétion de cette protéine, SIP, affecte la forme du parasite, entrainant un défaut de motilité, d'invasion et de virulence in vivo. / Toxoplasma gondii is an apicomplexan protist and the causative agent of toxoplasmosis. This obligate intracellular parasite harbors apical secretory organelles such as rhoptries that contain essential virulence factors responsible of the invasion and the modulation of the infected host cell. Along the cell cycle of T. gondii, rhoptry proteins share the same timing of expression. In order to identify new proteins involve in rhoptry content, biogenesis or secretion, we screened the genome database of T. gondii to isolate proteins that present this particular profile. We obtained the subcellular localization of 12 candidates and we investigated the biological functions for 4 of them. We showed that DegP, a rhoptry protease is essential for the in vivo virulence of T. gondii. DegP controls the acute phase during infection and modulate the host immune response leading to better parasite dissemination in vivo. Also, we identified Claw1 and its paralog Claw2 that present an atypical localization at the apical end of the parasite. To date, we were unable to disrupt the genes encoding these proteins suggesting that they may have an essential function related to the apical complex in T. gondii. Finally, we also examined a ‘hit' of this screening that was not related to rhoptries and we identified SIP, the first protein associated with the transversal junctions of the inner membrane complex in T. gondii. The disruption of SIP affects the shape of the parasite leading to an aberrant motility, defect in invasion and impaired parasite virulence in mice.

O habitat natural da Neisseria meningitis é a nasofaringe humana e a transmissão da bactéria é por contato direto ou por inalação de partículas durante a fase de transmissão N. meningitis é uma bactéria Gram-negativa responsável por uma significante mortalidade em todo o mundo. Embora existam vacinas polissacárides contras os sorogrupos A, C, W135 e Y , não há uma vacina adequada para crianças menores de 4 anos para o sorogrupo B. Estudos estão sendo direcionadas para pesquisa de antígenos vacinais que são derivados da proteínas de membrana externa(NOMV). Entretanto vacinas baseadas em NOMV são consideradas pouco imunogênicas , fazendo com que o uso de adjuvantes seja necessário. Este estudo investiga a imunogenicidade da NOMV de N. meningitidis administrada pela via intranasal/intramuscular em camundongos neonatos BALC/c, usando proteína de membrana externa (NOMC) obtido de uma cepa epidêmica de N. meningitidis B:4:P1:15. As cepas usadas para imunização dos camundongos foram selecionadas por colony-blot, usando anticorpo monoclonal anti L3,7,9 para maior expressão do LPS contra o imunotipo L3,7,9 presente na cepa (B:4:P1:15 3,7,9). Como adjuvantes de mucosa foram utilizados Bordetella pertussis (células íntegras) ou sobrenadante de cultura com 48 horas ou hidróxido de alumínio [Al(OH)3]. O soro dos camundongos imunizados foram analisados pelo método de ELISA à fim de se comparar os diferentes adjuvantes utilisados. O índice de avidez também foi determinado. IgG e IgM foram detectados nos soros dos camundongos após imunização, com índices de intermediária e alta avidez. Todos os adjuvantes foram capazes de aumentar a resposta imune contra NOMV de N.meningitidis. A via intranasal foi adequada para sensibilizar as células do sistema imune que foram rapidamente estimuladas pela via intramuscular usando os adjuvantes utilizados na presente investigação. Dados sugerem que o estudo da NOMV é importante na indução da imunidade de mucosa para N. meningitidis B, e que a qualidade e magnitude da resposta imune gerada pelas vacinas de mucosa são influenciadas tanto pelo adjuvante como pelo antígeno. Concluímos que NOMV juntamente com adjuvantes de mucosa tem considerável potencial no desenvolvimento de vacinas contra o meningococo do sorogrupo B. / The natural habitat of Neisseria meningitidis is the human nasopharynx, and the bacterium is transmitted by direct mouth-to-mouth contact or by the inhalation of released mucous particles during close contact. N meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharide-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Current studies are searching for vaccinal antigens that are derived from the native outer membrane (NOMV). However, vaccines based on NOMV are considered weak, making the use of adjuvants necessary. This study investigated the immunogenicity of NOMV of N. meningitidis administered intranasal/intramuscular in neonate BALB/c mice, using the native outer membrane complex (NOMC) obtained from an epidemic strain of N. meningitidis B:4:P1.15. The strains used for immunization of mice were selected by colony-blot, using anti L3,7,9 monoclonal antibodies, for the highest expression of LPS among the immunotypes (B:4:P1:15 L9á). As mucosal adjuvants, we used Bordetella pertussis (whole cells) or the supernatant of 48 h culture of this bacterium, followed by an intramuscular dose of the same protein adsorbed onto , B. pertussis (whole cells) or 48-h B. pertussis culture supernatant or aluminum hydroxide [Al(OH)3]. Sera of immunized mice were evaluated by ELISA in order to compare the different adjuvants used. We also determined their avidity index. IgG and IgM were detected in the serum of mice after immunization, with avidity indices that ranged from intermediate to high. All adjuvants were capable of increasing the immune response against NOMV of N. meningitidis in the homologous prime/boost schedule used. The intranasal route was suitable for sensitizing the cells of the immune system which were quickly stimulated by the intramuscular route using the adjuvants analysed in the present invertigation. Data suggest that the NOMV studied is important in the induction of mucosal immunity to N. meningitidis B, and that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the adjuvant as well as the antigen. In conclusion, nasal delivery of NoMV with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

O habitat natural da Neisseria meningitis é a nasofaringe humana e a transmissão da bactéria é por contato direto ou por inalação de partículas durante a fase de transmissão N. meningitis é uma bactéria Gram-negativa responsável por uma significante mortalidade em todo o mundo. Embora existam vacinas polissacárides contras os sorogrupos A, C, W135 e Y , não há uma vacina adequada para crianças menores de 4 anos para o sorogrupo B. Estudos estão sendo direcionadas para pesquisa de antígenos vacinais que são derivados da proteínas de membrana externa(NOMV). Entretanto vacinas baseadas em NOMV são consideradas pouco imunogênicas , fazendo com que o uso de adjuvantes seja necessário. Este estudo investiga a imunogenicidade da NOMV de N. meningitidis administrada pela via intranasal/intramuscular em camundongos neonatos BALC/c, usando proteína de membrana externa (NOMC) obtido de uma cepa epidêmica de N. meningitidis B:4:P1:15. As cepas usadas para imunização dos camundongos foram selecionadas por colony-blot, usando anticorpo monoclonal anti L3,7,9 para maior expressão do LPS contra o imunotipo L3,7,9 presente na cepa (B:4:P1:15 3,7,9). Como adjuvantes de mucosa foram utilizados Bordetella pertussis (células íntegras) ou sobrenadante de cultura com 48 horas ou hidróxido de alumínio [Al(OH)3]. O soro dos camundongos imunizados foram analisados pelo método de ELISA à fim de se comparar os diferentes adjuvantes utilisados. O índice de avidez também foi determinado. IgG e IgM foram detectados nos soros dos camundongos após imunização, com índices de intermediária e alta avidez. Todos os adjuvantes foram capazes de aumentar a resposta imune contra NOMV de N.meningitidis. A via intranasal foi adequada para sensibilizar as células do sistema imune que foram rapidamente estimuladas pela via intramuscular usando os adjuvantes utilizados na presente investigação. Dados sugerem que o estudo da NOMV é importante na indução da imunidade de mucosa para N. meningitidis B, e que a qualidade e magnitude da resposta imune gerada pelas vacinas de mucosa são influenciadas tanto pelo adjuvante como pelo antígeno. Concluímos que NOMV juntamente com adjuvantes de mucosa tem considerável potencial no desenvolvimento de vacinas contra o meningococo do sorogrupo B. / The natural habitat of Neisseria meningitidis is the human nasopharynx, and the bacterium is transmitted by direct mouth-to-mouth contact or by the inhalation of released mucous particles during close contact. N meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharide-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Current studies are searching for vaccinal antigens that are derived from the native outer membrane (NOMV). However, vaccines based on NOMV are considered weak, making the use of adjuvants necessary. This study investigated the immunogenicity of NOMV of N. meningitidis administered intranasal/intramuscular in neonate BALB/c mice, using the native outer membrane complex (NOMC) obtained from an epidemic strain of N. meningitidis B:4:P1.15. The strains used for immunization of mice were selected by colony-blot, using anti L3,7,9 monoclonal antibodies, for the highest expression of LPS among the immunotypes (B:4:P1:15 L9á). As mucosal adjuvants, we used Bordetella pertussis (whole cells) or the supernatant of 48 h culture of this bacterium, followed by an intramuscular dose of the same protein adsorbed onto , B. pertussis (whole cells) or 48-h B. pertussis culture supernatant or aluminum hydroxide [Al(OH)3]. Sera of immunized mice were evaluated by ELISA in order to compare the different adjuvants used. We also determined their avidity index. IgG and IgM were detected in the serum of mice after immunization, with avidity indices that ranged from intermediate to high. All adjuvants were capable of increasing the immune response against NOMV of N. meningitidis in the homologous prime/boost schedule used. The intranasal route was suitable for sensitizing the cells of the immune system which were quickly stimulated by the intramuscular route using the adjuvants analysed in the present invertigation. Data suggest that the NOMV studied is important in the induction of mucosal immunity to N. meningitidis B, and that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the adjuvant as well as the antigen. In conclusion, nasal delivery of NoMV with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.