Shigella flexneri est une entérobactérie Gram négatif responsable de la forme endémique de la shigellose, l’une des quatre causes majeures d’infection diarrhéique chez les jeunes enfants. La cible majeure de la réponse immunitaire lors d’une infection naturelle est le polysaccharide de surface (PS). Chez S. flexneri 1b, l’un des sérotypes prévalents dans les pays en voie de développement, le PS est défini par le pentasaccharide ramifié α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside [I] di-O-acétylé. Ces travaux s’intègrent dans un projet visant le développement d’un vaccin basé sur des sucres synthétiques à couverture large contre les infections par Shigella. Afin de concevoir des glycoconjugués efficaces et induisant une bonne réponse immunitaire chez les enfants, des synthèses multi-grammes des précurseurs mono- à pentasaccharidiques ont été optimisées permettant une stratégie par blocs en vue de l’obtention d’oligosaccharides de grande taille. Au cours de ces synthèses, l’obtention du trisaccharide ramifié C(E)D clé a nécessité de nombreuses optimisations, permettant la conception de synthons tri- à pentasaccharides. Un choix des groupements protecteurs orthogonaux nous a permis d’investiguer les différentes conditions de couplages nous donnant accès à 28 oligosaccharides déprotégés courts diversement acétylés. La validation de ces condensations avec des partenaires plus complexes a permis d’accéder à un large panel d’une cinquantaine d’oligosaccharides de di- à pentadécasaccharides sous leur forme libre, ou encore protégés avec divers degrés d’acétylation. / 700,000 children die each year due to diarrheal diseases, making it the second cause of death among this population. Shigella flexneri is a Gram negative enterobacterium responsible of the endemic form of shigellosis in developing countries. The O-antigen part of the bacterial lipopolysaccharide is the major target of the immune system during natural infection. The O-antigen of S. flexeni 1b, one of the prevalent serotypes, is defined by a ramified pentasaccharide made of three L-rhamnose, one D-glucosamine and one D-glucose with two non-stoichiometric sites for acetylation (I). This work is part of the project aimed at the development of a synthetic carbohydrate-based vaccine against Shigella infections. In order to obtain suitable glyconjugates inducing a high level of protection especially in children, the synthesis of mono- to pentasaccharide precursors was optimized, allowing a convergent synthesis of oligosaccharides with different acetylation patterns. Optimization of the glycosylation conditions, acetylations and protecting group manipulations enable the access to fragments from di to pentadecasaccharides representing S. flexneri type I O-antigen.

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Bakterien der Gattung Shigella sind die Erreger der bakteriellen Ruhr beim Menschen. Der wesent-liche Virulenzfaktor der Shigellen ist ihre Fähigkeit zur Invasion. Die Invasion in Epithel-zellen ist Ausdruck des erregerspezifischen Infektionsprozesses, der gekennzeichnet ist durch eine vom Bakterium induzierte zelluläre Aufnahme über einen Phagozytose-ähnlichen Mechanismus. In dessen Verlauf führt die charakteristische Reorganisation des Zytoskeletts der Zelle zur Ausbildung einer blütenartigen Membranstruktur an der bakteriellen Eintrittsstelle. Als essentielles Glied der Signalisationskaskade vom Bakterium zum zellulären Zytoskelett erwies sich das kleine G-Protein Rho. Es wurde gezeigt, daß Rho als Regulator der Veränderungen des Zytoskeletts selbst an den bakteriellen Invasionslocus rekrutiert wird. Diese Rekrutierung umfaßt drei Isoformen von Rho: RhoA, RhoB und RhoC. Trotz hoher Sequenzhomologie der Isoformen untereinander ( 85% ) existiert ein unterschiedliches Rekrutierungsmuster dieser Isoproteine an der Eintrittsstelle von Shigella flexneri. RhoA akkumuliert vorwiegend um die eindringenden Bakterien herum. Demgegenüber werden RhoB und RhoC in die bakterieninduzierten zellulären Protrusionen rekrutiert. Der Mechanismus dieser isoformspezifischen Rekrutierung ist nicht bekannt. Anhand unserer Experimente konnten wir zeigen, daß das allen kleinen G-Proteinen gemeinsame C-terminale Peptid-Motiv CAAX ( C = Cystein, A = aliphatische Aminosäure, X = Leucin ) eine wesentliche Voraussetzung für die Rekrutierung von Rho darstellt, sich die isoformspezifische Rekrutierung jedoch nicht anhand des CAAX-Motivs erklären läßt. Bedeutsam für die differentielle Rekrutierung der Isoproteine ist vielmehr die präterminale Region des Moleküls. Diese Beobachtung hat weitreichende Konsequenzen für die funktionelle Rolle von Rho bei der Epithelzellinvasion durch Shigella. / Shigella is the etiologic agent of human bacillary, an infectious large bowel disease. A major feature of Shigella's pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization requiring cytosceletal rearrangements; Shigella entry induces a blossom-like membrane structure at the bacterial entry site. This membrane folding process is dependent on the small GTPase rho. It has been shown that three rho isoforms rhoA, rhoB and rhoC are recruited into bacterial entry sites with different localization relative to the membrane structures. While rhoA preferentially accumulates in close vicinity to entering bacteria, rhoB and rhoC are recruited into the tips of Shigella-induced cellular protrusions. We could show that the C-terminal CAAX-region of rho is a prerequisite for recruitment, but not sufficient for different recruitment of rho isoforms. Additional information for differential recruitment patterns seems to come from the preterminal region of rho proteins. This result is of great importance for the functional role of rho during Shigella-invasion into epithelial cells.

Shigella flexneri verursacht im Verlauf der Infektion eine massive Enzündungsreaktion sowie Schädigung des humanen Darmepithels. Neutrophile sind die ersten Zellen des angeborenen Immunsystems, welche den Infektionsherd infiltrieren. Diese Zellen greifen Mikroorganismen mittels Phagozytose, Neutrophiler extrazellulärer Fallen (Neutrophil Extracellular Traps, NETs) oder Degranulierung an. In dieser Arbeit haben wir untersucht, wie die Degranulierung von Neutrophilen die Virulenz von Shigellen beeinflußt und konnten zeigen, dass die Exposition von Shigellen mit Proteinen aus den Granula von Neutrophilen die Invasion in Epithelzellen stark erhöht. Während dieser Exposition binden kationische Proteine der Granula an die Oberfläche von Shigella und bewirken eine verstärkte Adhesion, welche dann schließlich zu “Hyperinvasion” führt. Dieser Effekt wird durch Änderungen der Oberflächenladung bewirkt, da eine Lipopolysaccharid (LPS) Mutante mit negativer Oberflächenladung eine zusätzliche erhöhte Hyperinvasion im Vergleich zu Wildtyp Shigellen zeigt. Zusätzlich zur Hyperinvasion bewirkt die Infektion von Epithelzellen mit Shigellen, die mit Granula Proteinen in Kontakt gekommenen sind, eine Verminderung der IL-8 Sekretion. Dieses Zytokin bewirkt eine starke Rekrutierung von Neutrophilen. Daher stellen wir die Hypothese auf, dass Shigella in der Lage ist, antimikrobielle Proteine des Wirtes zur Erhöhung seiner Virulenz durch Hyperinvasion zu verwenden sowie eine weitere Rekrutierung von Neutrophilen durch Inhibition der IL-8 Sekretion zu verhindern. Somit unterwandert Shigella das angeborenen Immunsystem und nutzt dessen Angriff zu seinem Vorteil. / Shigella flexneri is an enteric pathogen that causes massive inflammation and destruction of the human intestinal epithelium. Neutrophils are the first cells of the innate immune system recruited to the site of infection. These cells can attack microbes by phagocytosis, Neutrophil Extracellular Trap (NET) formation and degranulation. Here, we investigated how neutrophil degranulation affects virulence and show that exposure of Shigella to granular proteins enhances infection of epithelial cells. During this process, cationic granular proteins bind to the Shigella surface causing increased adhesion which ultimately leads to hyperinvasion. This effect is mediated by changes in the surface charge, since a lipopolysaccharide (LPS) mutant with a negative surface shows enhanced hyperinvasion compared to wild-type Shigella. In addition, infection with Shigella exposed to granular proteins leads to the inhibition of secretion of the neutrophil attracting cytokine IL-8. We propose that Shigella uses host defense molecules to enhance its virulence by increased infection of its host cells and reduced recruitment of neutrophils after hyperinvasion through inhibition of IL-8 secretion. With this Shigella subverts the innate immune system and uses its attack for its own benefit.

Shigellen sind Erreger der bakteriellen Dysenterie beim Menschen. Ein notwendiger Schritt bei der Pathogenese der Shigellose ist die Invasion von Darmepithelzellen durch das Bakterium. Der Mikroorganismus löst dabei in der Wirtszelle Veränderungen des Aktinzytoskeletts aus, die zur Bildung einer blütenähnlichen Membranstruktur und schließlich zur Internalisierung des Pathogens führen. Diese Umbauvorgänge am Zytoskelett sind abhängig von einem Wirtszellprotein, der kleinen GTPase Rho. Drei Isoformen von Rho (A, B und C) sind beschrieben, deren Aminosäuresequenzen zu etwa 90% identisch sind. Während der Zellinvasion durch Shigella akkumulieren verschiedene Rho-Isoformen an unterschiedlichen Lokalisationen des Invasionskomplexes. Dabei werden RhoA vorwiegend um die eindringenden Bakterienherum, RhoB und RhoC hingegen hauptsächlich in die bakterieninduzierten zellulären Protrusionen rekrutiert. Durch Untersuchung von Rho-Hybridkonstrukten konnte gezeigt werden, daß ein prä-C-terminales, acht Aminosäuren umfassendes Modul die Rekrutierungsmuster von RhoA bzw. RhoC bestimmt. Der Austausch zweier Aminosäuren innerhalb des Moduls führte zu einer Konversion des Rekrutierungsmusters von RhoA. Wir konnten zeigen, daß die Rekrutierung von RhoA vom Funktionszustand der GTPase (Bindung von GTP/GDP) sowie von der Phosphorylierung durch die Proteinkinase A unabhängig ist. Schließlich wurde hier nachgewiesen, daß auch RhoD, das zu RhoA, B und C auf der Primärstrukturebene nur zu etwa 50% homolog ist, an die Bakterieneintrittsstelle rekrutiert werden kann. RhoD folgt dabei dem Rekrutierungsmuster von RhoB und RhoC. / Shigella causes bacillary dysentery in humans. Bacterial invasion of enterocytes is an essential step in the pathogenesis of shigellosis. Pathogen-triggered rearrangements of the host cell actin cytoskeleton induce a blossom-like membrane structure for internalisation of the microorganism. Actin remodeling requires activity of the host cell small GTPase rho. Three highly homologous rho isoforms (A, B and C) have been described with amino acid identities of about 90%. During Shigella invasion these rho isoforms accumulate at different sites of the invasion complex. While rhoA is chiefly recruited around entering bacteria, rhoB and rhoC are essentially translocated to the bacteria-induced cellular protrusions. Using a variety of rho hybrid constructs in a HeLa cell transfection-infection assay we were able to show that a pre-C-terminal stretch of eight amino acids determines the recruitment patterns of rhoA and rhoC. Exchange of two amino acids was sufficient for conversion of the rhoA recruitment pattern into a rhoC-like pattern. We could demonstrate that rhoA recruitment is independent of its functional state (GDP- or GTP-bound) or phosphorylation by the proteinkinase A (PKA). Finally, we have shown that rhoD, another member of the rho family which shares only 50% of its primary structure with rhoA, B or C, is also recruited to the bacterial entry site exhibiting a rhoB/C-like pattern of distribution.

Les bactéries du genre Shigella sont responsables de la shigellose, une maladie diarrhéique invasive du colon. L’entrée et la dissémination de Shigella à travers l’épithélium colique sont médiées par un système de sécrétion de type III (SST3) codé par un plasmide de virulence. Au sein de ce plasmide se trouve une région de 30-kb comportant les gènes impliqués dans l’entrée de la bactérie dans les cellules hôtes. Ces gènes sont regroupés en deux loci :le locus ipa-ipg qui code pour les protéines sécrétées et leurs chaperons ainsi que le locus mxi-spa codant pour les composants de l’appareil de sécrétion de type III (AST3), constitué d’un bulbe cytoplasmique, d’un corps basal transmembranaire et d’une aiguille se projetant au niveau extracellulaire. Ce système permet la sécrétion ordonnée et hiérarchique de différentes classes de protéines et la translocation de certaines d’entre elles (appelées effecteurs) dans le cytoplasme de la cellule hôte où elles interfèrent avec les voies de signalisation cellulaires. Avant le contact avec la cellule hôte, l’AST3 est inactif et verrouillé par les protéines IpaB et IpaD formant le complexe d’extrémité.<p>Chez Shigella, le gatekeeper MxiC séquestre les effecteurs au niveau du cytoplasme bactérien avant la transmission par l’aiguille du signal d’activation de la sécrétion mais les composants intermédiaires liant l’aiguille à MxiC restaient inconnus. Au cours de ce travail, nous avons montré que MxiC forme un complexe avec la sous-unité de la tige interne, MxiI, afin de bloquer l’entrée du canal de sécrétion et que cette interaction est conservée chez Yersinia et Salmonella. Nous démontrons que, suite au contact cellulaire, la dissociation de ce complexe facilite le switch de sécrétion des translocateurs aux effecteurs. Nos résultats révèlent également que MxiC est capable de s’associer au chaperon IpgC afin de réguler la sécrétion des translocateurs. De plus, nous avons identifié les domaines de MxiC engagés dans la régulation du SST3 et rapporté un nouveau rôle de MxiC dans l’échappement aux macrophage impliquant une possible inhibition de la voie apoptotique classique afin de promouvoir une pyroptose. Chez Shigella, IpaD gouverne la composition du complexe d’extrémité et est impliqué dans la régulation de la sécrétion. Nous avons développé une étude phénotypique de ses régions coiled-coil et centrale et montré que la composition du complexe d’extrémité permet de définir à la fois l’état d’inductibilité de l’AST3 et la sécrétion des effecteurs tardifs. Par ailleurs, notre étude fonctionnelle des domaines de MxiC et IpaD suggère que les capacités de Shigella à échapper au macrophage et à insérer un pore de translocation ne sont pas strictement couplées. <p>La dernière partie de ce travail s’est focalisée sur la caractérisation de la protéine Spa13 de Shigella. Nous avons découvert que le défaut de sécrétion du mutant spa13 est dû à l’instabilité de la sous-unité MxiH de l’aiguille et que Spa13 n’est pas sécrété par le SST3. Nos résultats indiquent également un rôle de Spa13 dans l’escorte de chaperons et l’activation de l’appareil d’exportation afin de promouvoir la sécrétion des substrats./Shigella is the causative agent of shigellosis, also known as bacillary dysentery, an invasive disease of the human colonic epithelium. During infection, Shigella uses a type III secretion system (T3SS) to penetrate enterocytes and to disseminate into the colonic epithelium, leading to destruction of the mucosal lining and shigellosis symptoms. Most of the virulence factors of Shigella are encoded by a large plasmid harboring a 30-kb region that is sufficient to promote bacterial entry into host cells. This entry region is organized in two loci, one corresponding to the the ipa-ipg genes encoding the secreted proteins and their cognate chaperones while the other encodes Mxi-Spa proteins that form the type III secretion apparatus (T3SA), consisting of a cytoplasmic bulb, a basal body spanning the bacterial envelope and a hollow needle. The T3SS allows the ordered and hierarchical secretion of effectors by inserting a translocation pore in the host cell membrane through which effector proteins are injected into the cytosol. Before host cell contact, the T3SA is inactive and plugged by the tip complex proteins IpaB and IpaD. <p>In Shigella, the gatekeeper MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle but the molecules involved in linking the needle and MxiC are unknown. We demonstrated that MxiC and the predicted inner-rod component MxiI form a complex plugging the T3SA entry gate and showed that this interaction is conserved in Yersinia and Salmonella. Dissociation of this complex seems to facilitate the switch in secretion from translocators to effectors upon host cell contact. Our results also revealed that MxiC binds to the chaperone IpgC to regulate translocators secretion. Moreover, we identified the domains of MxiC involved in the T3S regulation and reported a new role in macrophage escape by potential inhibition of the classical apoptosis to promote pro-inflammatory pyroptosis. <p>In Shigella, IpaD rules the composition of the tip complex and is involved in secretion control and translocon insertion. We therefore undertook a phenotypic analysis of its coiled-coil and central regions and showed that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Besides, our functional study on MxiC and IpaD domains suggests that Shigella abilities to escape macrophage vacuole and to insert the translocation pore are uncoupled.<p>The last part of this work is related to the characterization of the Spa13 protein of Shigella. We found that the secretion defect of the spa13 mutant is due to the instability of the needle component MxiH and that Spa13 is not a secreted substrat. Our results also support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch to promote substrates secretion. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les cellules épithéliales constituent la première ligne de défense face à l’infection et jouent un rôle actif dans l'immunité innée. Par la sécrétion locale de cytokines, ces cellules sont capables d'orchestrer la réponse immunitaire contre les pathogènes invasifs. L'activation des récepteurs de reconnaissance de pathogènes, qu’ils soient intracellulaires ou extracellulaires, conduit à une cascade de signalisation complexe. Cette dernière entraîne l'activation du facteur de transcription NF-kB ainsi que la production ultérieure de cytokines pro-inflammatoires. Cependant, les mécanismes moléculaires qui gouvernent ce processus n'ont pas été entièrement élucidés. La bactérie à Gram négatif Shigella flexneri est un pathogène humain majeur à l’origine de la dysenterie bacillaire. Cette maladie se caractérise par une inflammation aiguë du colon qui peut entraîner la destruction du tissu intestinal et même dans les cas les plus graves, la mort. En effet, S. flexneri peut envahir les cellules épithéliales du colon et se répliquer dans leur cytoplasme. Après la détection de bactéries intracellulaires, les cellules infectées et non infectées déclenchent des voies de signalisation inflammatoire, ce qui entraîne une production massive d'interleukine-8. En utilisant S. flexneri comme modèle d'infection, nous avons identifié une nouvelle voie de signalisation qui joue un rôle central dans l'activation de NF-kB et la production d'IL-8 qui en résulte lors des infections bactériennes à Gram négatif. Après la détection cytosolique des bactéries, les protéines TIFA forment des oligomères à travers un processus dépendant de leur thréonine en position 9, ainsi que de leur domaine « Forkhead-associated ». D’une part, ces oligomères interagissent avec TRAF6, ce qui conduit à l’oligomérisation de cette dernière et à l'activation subséquente de NF-kB. D'autre part, nous montrons que l'oligomérisation de TIFA dépend de la kinase ALPK1 et que cette voie est activée en réponse au métabolite bactérien heptose-1, 7-bisphosphate. Ces observations pourraient être étendues au pathogène entéro-invasif Salmonella typhimurium ainsi qu'à la bactérie extracellulaire Neisseria meningitidis. Nos résultats démontrent donc le rôle central de la voie de signalisation ALPK1-TIFA-TRAF6 en réponse aux pathogènes bactériens à Gram négatif intracellulaires et extracellulaires. Ainsi, ces travaux contribuent à une meilleure compréhension des mécanismes moléculaires régissant la réponse immunitaire des cellules épithéliales aux bactéries pathogènes. / Epithelial cells represent the first line of defense against pathogens and play an active role in innate immunity. Via local secretion of cytokines, they are able to orchestrate the immune response against invading pathogens. The activation of both intracellular and extracellular pathogen recognition receptors leads to a complex signaling cascade, resulting in the activation of the transcription factor nuclear factor kB(NF-kB)and the subsequent production of pro-inflammatory cytokines. However, the molecular mechanisms governing this process have not been fully elucidated. The Gram-negative bacterium Shigella flexneriis an important human pathogen and the causative agent of bacillary dysentery. This disease is characterized by acute inflammation of the colon resulting in the destruction of the intestinal tissue and, in severe cases, death. S. flexneri can invade and replicate within colonic epithelial cells. Following detection of the bacteria, both infected and uninfected bystander cells initiate inflammatory signaling pathways, which result in massive interleukin-8 (IL-8) production by the latter. Using S. flexneri as a model of infection, we have identified a novel signaling pathway, which is central to the activation of NF-kB and the subsequent production of IL-8 during Gram-negative bacterial infections. Following the cytosolic detection of bacteria, the protein TRAF-interacting factor with forkhead-associated domain (TIFA) forms oligomers, a process dependent on its threonine at position 9 and theforkhead-associated domain. These oligomers interact withTNF receptor associated factor (TRAF)6, leading to its oligomerization and the subsequent activation of NF-kB. In addition, we show that oligomerization of TIFA is dependent on the kinase alpha-kinase(ALPK)1 and that this pathway is activated in response to the detection of the bacterial metabolite heptose-1, 7-bisphosphate (HBP). These observations could be extended to the enteroinvasive pathogen Salmonella typhimurium as well as the extracellular bacteria Neisseria meningitidis. Our results therefore demonstrate the central role of the ALPK1-TIFA-TRAF6 signaling pathway in response to HBP of both intracellular and extracellular Gram-negative bacterial pathogens, and offer a better understanding of the molecular mechanisms governing the epithelial cell immune response to pathogenic bacteria.

Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC. / Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.