The type II secretion system (T2SS) is widespread in Gram-negative bacteria that cause disease in animals and plants. In human and animal pathogens toxins are secreted (e.g. cholera toxin) and in plant pathogens lytic enzymes that breakdown the plant cell wall are exported in to the extracellular milieu (e.g. pectate lyase). Structurally the T2SS comprises at least 11 core proteins that form three major subassemblies spanning the inner-membrane, periplasmic space and outer-membrane: (i) the inner-membrane platform and associated cytoplasmic ATPase (E); (ii) the pseudopilus, which consists of five pseudopilins, G to K; and (iii) a large, pore-forming outer-membrane complex secretin D. The inner-membrane platform comprises three single transmembrane helix proteins, and one three transmembrane helix protein, OutF. The evidence from cryo-electron microscopy on the related type IVa pilus machine (T4PS) places the protein corresponding to OutF at the centre of this platform. This platform is responsible for assembling the pilus and for communicating between the periplasm and the cytoplasmic ATPase. To date, no high-resolution structure of a full-length OutF/PilC family protein is available. A low-resolution electron microscopy reconstruction of isolated PilG (PilC ortholog from Neisseria meningitides T4PS) showed a tetrameric two lobed structure. Here I report the results of studying the structure of the inner-membrane protein OutF from Dickeya dadantii and the complete inner-membrane platform comprising 9 proteins: OutEFGHIJKLM. This work involved cloning the corresponding operon, purifying the proteins, and using crystallography and electron microscopy. Key results reported here are the crystal structure of the first cytoplasmic domain of Dickeya dadantii, OutF65-172 and a preliminary three-dimensional model of the Dickeya dadantii inner-membrane platform. This model, and higher-resolution models to come, will provide valuable information about the oligomeric state, and arrangement of the inner-membrane proteins. These studies will help us to understand how the type II secretion system works.

<i>Aeromonas hydrophila</i> uses the type II secretion system to transport protein toxins across the outer membrane. The trans-envelope system is comprised of more than ten proteins, including ExeA and ExeB, which form a complex in the inner membrane and are required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan binding domain (Pfam protein families database number PF01471) is present in the periplasmic region of ExeA (pExeA), leading to the hypothesis that ExeA generates gaps in peptidoglycan, a barrier for trans-envelope transport and apparatus assembly, to allow ExeD to assemble into the outer membrane.<p> In this study, interactions between ExeA and peptidoglycan were examined both <i>in vivo</i> and <i>in vitro</i>. Wild type ExeA, but not the mutants containing substitution mutations of three highly conserved amino acid residues in the putative peptidoglycan binding domain, was cross-linked to peptidoglycan in vivo with DTSSP. Furthermore, the presence of wild type ExeA was also required for co-crosslinking of ExeB and ExeC to peptidoglycan. <i>In vitro</i> cosedimentation revealed that purified pExeA was able to bind to highly purified peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in cross-linking and co-gel filtration experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at temperatures above 25 °C. Two pExeA constructs, which disrupted the putative peptidoglycan binding domain, greatly reduced the cosedimentation, accompanied by decreased multimerization in the presence of peptidoglycan fragments. These results provide evidence that the putative peptidoglycan binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions cause ExeA to multimerize, possibly forming a ring-like structure on the peptidoglycan, to generate a gap large enough to accommodate the secretion apparatus and/or to form an assembly scaffold.<p> The putative peptidoglycan binding domain of ExeA was also analyzed by comparing its amino acid sequence with that of other homologues. The highly conserved amino acid residues were found to cluster at one pocket on the surface in the crystal structure of hydrolase metallo (Zn) DD-peptidase that also contains this domain. We propose that this pocket is the binding site for the peptidoglycan ligand.

<i>Aeromonas hydrophila</i> uses the type II secretion system to transport protein toxins across the outer membrane. The trans-envelope system is comprised of more than ten proteins, including ExeA and ExeB, which form a complex in the inner membrane and are required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan binding domain (Pfam protein families database number PF01471) is present in the periplasmic region of ExeA (pExeA), leading to the hypothesis that ExeA generates gaps in peptidoglycan, a barrier for trans-envelope transport and apparatus assembly, to allow ExeD to assemble into the outer membrane.<p> In this study, interactions between ExeA and peptidoglycan were examined both <i>in vivo</i> and <i>in vitro</i>. Wild type ExeA, but not the mutants containing substitution mutations of three highly conserved amino acid residues in the putative peptidoglycan binding domain, was cross-linked to peptidoglycan in vivo with DTSSP. Furthermore, the presence of wild type ExeA was also required for co-crosslinking of ExeB and ExeC to peptidoglycan. <i>In vitro</i> cosedimentation revealed that purified pExeA was able to bind to highly purified peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in cross-linking and co-gel filtration experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at temperatures above 25 °C. Two pExeA constructs, which disrupted the putative peptidoglycan binding domain, greatly reduced the cosedimentation, accompanied by decreased multimerization in the presence of peptidoglycan fragments. These results provide evidence that the putative peptidoglycan binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions cause ExeA to multimerize, possibly forming a ring-like structure on the peptidoglycan, to generate a gap large enough to accommodate the secretion apparatus and/or to form an assembly scaffold.<p> The putative peptidoglycan binding domain of ExeA was also analyzed by comparing its amino acid sequence with that of other homologues. The highly conserved amino acid residues were found to cluster at one pocket on the surface in the crystal structure of hydrolase metallo (Zn) DD-peptidase that also contains this domain. We propose that this pocket is the binding site for the peptidoglycan ligand.

The exeC gene, found in the gram-negative bacteria Aeromonas hydrophila codes for a 31 kDa, three domain, bitopic inner membrane protein. The components of the ExeC protein include an amino-terminal cytoplasmic domain, a trans-membrane helix and two periplasmic domains. The two periplasmic domains are involved in recognition and selection of protein substrates which are subsequently transported across the outer membrane and free of the cell. This study focuses exclusively on the two periplasmic domains referred to hereafter as the HR and the PDZ domains. Three constructs were used throughout the course of this study. Two of them were designed, cloned and expressed for this study. The third is a result of previous work. Two constructs contained both the HR and PDZ domains while the other consists of the amino-terminal periplasmic HR domain. Only one construct was used to grow single crystals for analysis by X-ray crystallography. Crystals comprised of the PDZ domain from a degraded construct grew in a hexagonal space group with a hexagonal bi-pyramidal morphology. Crystals diffracted anisotropically to a maximum resolutions of 2 Å along the c axis and 3 Å in the a/b plane. Anisotropy in combination with twinning drastically complicated structure solution. Efforts toward elucidating the crystal structure will be discussed.

Researchers of <i>Clostridium perfringens</i>, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when <i>C. perfringens</i> was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins. / Master of Science

<p><i>Francisella tularensis</i> is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. <i>F. tularensis</i> can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of <i>F. tularensis</i>: the highly virulent <i>tularensis</i> (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent <i>holarctica</i> (Type B) and <i>mediasiatica</i>, and the essentially avirulent subspecies <i>F. novicida</i>. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between <i>F. tularensis</i> subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of <i>F. tularensis</i>, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the <i>Francisella</i> pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in <i>Neisseria gonorrhoeae. pilA</i> was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to <i>F. tularensis</i> and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (<i>pilA</i>) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them <i>in cis</i> could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.</p>

Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Le système de sécrétion de type II (T2SS) est largement exploité par les bactéries à Gram négatif pour sécréter divers facteurs de virulence depuis le périplasme vers le milieu extra-cellulaire. La bactérie phytopathogène Dickeya dadanti (ex. Erwinia chrysanthemi) utilise ce système, appelé Out, pour la sécrétion de pectinases responsable de la maladie de la pourriture molle chez de nombreuses plantes. Les deux composants essentiels du système Out, la protéine de membrane interne OutC et la sécrétine OutD, formant un pore dans la membrane externe, sont impliqués dans la spécificité de sécrétion. L'interaction entre OutC et OutD pourrait assurer l’intégrité structurelle et fonctionnelle du système de sécrétion en reliant les deux membranes. Nous avons entrepris une étude structure-fonction de ces deux composants afin d’identifier et caractériser leurs sites d’interaction et de mieux comprendre leurs rôles. Nous avons appliqué une approche intégrative impliquant une analyse in vivo par cystéine-scanning et pontage disulfure, une analyse in vitro par GST pull down et une analyse structurale d’OutC et OutD et de leurs interactions par RMN. Nos résultats indiquent la présence d'au moins trois sites d'interaction entre les régions périplasmiques d’OutC et d’OutD et suggèrent que ces interactions s’établissent par un mécanisme d’addition des brins β. Nous avons démontré qu’un site situé sur le domaine HR d’OutC pouvait interagir avec deux sites distincts d’OutD suggérant un mode d’interaction alternatif. La présence d’exoprotéines et/ou des composants de membrane interne du système OutE-L-M, modifie différemment l’affinité de ces trois sites d'interaction entre OutC et OutD. Nous proposons que ces interactions alternatives entre divers sites d’OutC et OutD pourraient refléter une succession d’étapes fonctionnelles lors du processus de sécrétion. Pour étudier le mécanisme d’adressage et d’assemblage de la sécrétine OutD dans la membrane externe, nous avons exploité les interactions entre OutD et deux composants auxiliaires du T2SS, la protéine de la membrane interne OutB et la lipoprotéine de la membrane externe OutS. Nous avons montré une interaction directe entre le domaine périplasmique d’OutB et le domaine N0 d’OutD. Une analyse structure-fonction du complexe OutS-OutD a révélé que la pilotine OutS interagit fortement avec 18 résidus à l’extrémité C-terminale de la sécrétine, entraînant la structuration sous forme hélicoïdale de cette région initialement non structurée. Ce travail nous permet de mieux comprendre le mécanisme d’assemblage et de fonctionnement du système de sécrétion de type II. / The type II secretion system (T2SS) is widely exploited by Gram-negative bacteria to secrete diverse virulence factors from the periplasm into the extra-cellular milieu. The phytopathogenic bacterium Dickeya dadanti (ex. Erwinia chrysanthemi) uses this system, named Out, to secrete several cell-wall degrading enzymes that cause soft-rot disease of many plants. The two core components of the Out system, the inner membrane protein OutC and the secretin OutD, which forms a secretion pore in the outer membrane, are involved in secretion specificity. The interaction between OutC and OutD could assure the structural and functional integrity of the secretion system by connecting the two membranes. To understand structure-function relationships between these two components and characterize their interaction sites, we applied an integrative approach involving in vivo cysteine scanning and disulfide cross-linking analysis, truncation analysis of OutC and OutD combined with in vitro GST pull-down, and structural analysis of these proteins and of their interactions by NMR. Our results indicate the presence of at least three interacting sites between the periplasmic regions of OutC and OutD and suggest a β-strand addition mechanism for these interactions. We demonstrated that one site of the HR domain of OutC can interact with two distinct sites of OutD suggesting an alternative mode of their interactions. The presence of exoproteins or/and the inner membrane components of the system OutE-L-M differently alters the affinity of the three OutC-OutD interacting sites. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machinery between different functional states. To study the mechanism of the targeting and assembly of the secretin OutD into the outer membrane, we exploited the interactions between OutD and two auxiliary proteins, i.e., the inner membrane protein OutB and the outer membrane lipoprotein OutS. We showed a direct interaction between the periplasmic domain of OutB and the N0 domain of OutD. Structure-function analysis of OutS-OutD complex shows that the pilotin OutS binds tightly to 18 residues close to the C-terminus of the secretin subunit causing this unstructured region to become helical on forming the complex. This work allows us to better understand the assembly and function mechanism of the type II secretion system.

Les bactéries à Gram négatif sont entourées par une enveloppe cellulaire qui, contrairement aux bactéries à Gram positif, possèdent une organisation membranaire complexe composée d’une membrane interne appelée généralement membrane cytoplasmique, un espace périplasmique contenant une matrice de peptidoglycane et une membrane externe asymétrique constituée d’une monocouche de phospholipides surmontée d’une assise de lipopolysaccharide (LPS). Afin de franchir cette barrière, les bactéries à Gram négatif ont développé différentes voies de sécrétions spécifiques dédiées à l’export des protéines (effecteurs) du milieu intracellulaire vers le milieu extracellulaire. Jusqu'à présent, six systèmes de sécrétion ont été identifiés chez ces bactéries. Chez Pseudomonas aeruginosa, une bactérie pathogène opportuniste, le système de sécrétion de type II appelé aussi sécréton Xcp constitue l’un des facteurs principales de sa virulence. Le sécréton Xcp est un complexe macromoléculaire formé par 12 protéines, nommées XcpAO et XcpPC-XcpZM. Ce complexe macromoléculaire est organisé en trois sous-complexes : i) une plateforme d’assemblage ancrée dans la membrane interne formé par les protéines XcpRESFYLZM ii) un pore de sécrétion localisé dans la membrane externe formé par l’oligomérisation d’une protéine appelé la sécrétine XcpQD. Le pore de sécrétion est connecté à la plateforme de la membrane interne par une protéine appelée XcpPC iii) un pseudopilus périplasmique sous forme de fibre hélicoïdale qui est formé par la multimérisation d’une protéine appelée la pseudopiline majeure XcpTG. D’autres protéines appelées les pseudopilines mineures XcpUH-VI-WJ-XK intègrent le pseudopilus. La première partie du travail effectué au cours de cette thèse a eu pour but d’étudier et de comprendre par des approches structurales, biochimiques et biophysiques le mécanisme d’assemblage des pseudopilines en pseudopilus. La deuxième partie de ce travail a porté sur l’étude des réseaux d’interactions entre les substrats sécrétés et les composants de la machinerie Xcp. Durant cette thèse, nous avons ainsi i) identifier grâce à l’étude des interactions protéine-protéine l’existence d’un complexe quaternaire entre les pseudopilines mineures XcpUH-VI-WJ-XK localisées au sommet du pseudopilus ii) déterminer les structures de la pseudopiline majeure XcpTG par RMN et de la pseudopiline mineure XcpWJ par cristallographie aux rayons X iii) déterminer les différents éléments du sécréton qui interagissent avec les exoprotéines du sécréton. Ce réseau d’interaction nous a permis de proposer un modèle de fonctionnement du sécréton qui élucide le cheminement des exoprotéines dans le sécréton afin qu’elles soient exportées vers le milieu extracellulaire. / Gram-negative bacteria are characterized by a complex organization of their cell envelope composed by the inner membrane (IM) called cytoplasmic membrane, the periplasmic space containing a peptidoglycan layer and the outer membrane (OM) covered by the lipopolysaccharide matrix. Gram-negative bacteria have evolved several specialized machines called secretion systems to export their effectors from the intracellular medium to the extracellular milieu or to the host cells. Up to now, at least six secretion systems have been identified. In the opportunistic pathogen Pseudomonas aeruginosa, the type II secretion system called the Xcp secreton is the major pathway for the release of virulence factors. The Xcp secreton is a macromolecular complex composed by 12 proteins called XcpAO, XcpPC-XcpZM. This machinery is organized in 3 sub-complexes: i) the assembly platform localized in the IM implicating XcpRESFYLZM proteins ii) the OM pore composed by the oligomerization of the secretin XcpQD. The connection between the assembly platform and the secretin is performed by XcpPC anchored in the IM iii) a periplasmic pseudopilus consisting of the multimerization of the so-called major pseudopilin XcpTG. The pseudopilus is a helicoidally filament spanning the periplasmic area and pushing the substrate into the secretin pore. Four other proteins, the minor pseudopilins XcpUH-VI-WJ-XK, were found in the pseudopilus. In the present work we first focused on the study of the pseudopilus components by biochemical, biophysical and structural strategies to understand their assembly. Secondly, we investigate the protein interactome between periplasmic secreton component and secreted substrates. Thus, we revealed the presence of a quaternary complex composed by XcpUH-VI-WJ-XK located at the tip of the pseudopilus. To understand at atomic scale the regulation of the pseudopilus, we determined the structure of two components of the pseudopilus XcpTG by NMR and XcpWJ by X-ray crystallography. Using systematic protein-protein interaction studies between secreton components and purified exoproteins of Pseudomonas aeruginosa, we identified 5 proteins of the secreton able to interact with exoproteins. This interaction network allowed us to propose a model for the secretion process including the sequential steps followed by exoproteins inside the secreton to leave the cell envelop.