Dissertations / Theses on the topic 'Type II secretion system ; T2SS'

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.

Les protéines synthétisées et sécrétées par les bactéries jouent des rôles importants pour leur survie. Les bactéries à Gram négatif ont développé des voies de sécrétion en tant qu'armes principales pour transporter des facteurs de virulence dans l'environnement extracellulaire ou dans des cellules hôte. L'un de ces systèmes, le T9SS a été principalement étudié chez l'agent pathogène oral Porphyromonas gingivalis et chez la bactérie mobile Flavobacterium johnsoniae. Un autre complexe, le T2SS est le principal déterminant de la virulence de la bactérie Pseudomonas aeruginosa, un agent pathogène de la fibrose kystique. Dans le cadre de ma thèse, j'ai résolu la structure atomique de plusieurs composants centraux du T9SS et du T2SS. Concernant le projet T9SS, j'ai essayé de cristalliser le domaine cytoplasmique de GldL de F. johnsoniae. La co-cristallisation de GldL avec des Nbs a été réalisée sans succès. Néanmoins, les structures cristallines de deux nanobody contre GldL ont été résolues par remplacement moléculaire. De plus, j'ai également travaillé sur la protéine PG1058 de P. gingivalis. J'ai résolu sa structure par diffraction anomale à la longueur d’onde du selenium. Concernant le projet T2SS, je me suis concentré sur la partie N-terminale de XcpQ, une sous-unité de la sécrétine. J'ai résolu la structure cristalline de XcpQN012 seul et en complexe avec le nanobody vhh04 à une résolution de 2,98 Å et de 2,9 Å, respectivement. Enfin, j'ai participé à la détermination structurale de TssK, un composant de plaque de base du système de T6SS et déterminer la structure cristalline d'un nanobody contre le domaine périplasmique de PorM
Proteins synthesized and secreted by bacteria serve many important roles in their survival. In particular, Gram-negative bacteria have evolved secretion pathways as the main weapons for transporting virulence factors into target cells or into the extracellular environment. One of these systems, the type IX secretion system (T9SS) or the Por secretion system, has been studied mainly in the oral pathogen Porphyromonas gingivalis and the gliding bacterium Flavobacterium johnsoniae. Another complex, the type II secretion system (T2SS) is the main determinant of the virulence of Pseudomonas aeruginosa, a cystic fibrosis pathogen. In my PhD thesis, I solved the atomic structure of several core components of both T9SS and T2SS.For the T9SS project, I tried to crystallize the cytoplasmic domain of GldL from F. johnsoniae. The co-crystallization of GldL with Nbs was unsuccessfull. The crystal structures of two nanobodies against GldL were solved by molecular replacement. I also worked on the PG1058 protein of P. gingivalis. I obtained crystals of the selenomethionine-derivatized PG1058 OmpA_C-like domain that diffracted up to 1.55 Å, and solved its structure by single-wavelength anomalous diffraction. For the T2SS project, I focused on the N-terminal part of XcpQ, a subunit of the secretin. I solved the crystal structure of XcpQN012 alone and in complex with nanobody vhh04 at a resolution of 2.98 Å and 2.9 Å, respectively. In addition, I also took part in the structural determination of the base plate component TssK of the T6SS and determined the crystal structure of one nanobody (vhh19) against the periplasmic domain of PorM

The type II secretion system (T2SS) is the major terminal branch of the general secretory pathway. It is composed of 12-15 proteins, most in multiple copies, and spans the inner and outer membranes of Gram-negative bacteria. The T2SS secretin subunits form a large dodecameric torus-like structure in the outer membrane. The secretin is the only essential component in the outer membrane and secreted proteins and virulence factors pass through the pore in the toroidal secretin dodecamer and out into the environment. The interaction between the secretin and its partners plays a key role in regulation of the T2SS. The interaction between the so-called homology region of the innermembrane protein GspC (GspC-HR) and secretin provides the structural and functional integrity of the secretion machinery across the two cell membranes. The interaction between secretin and its pilotin translocates the secretin subunits to the outer membrane. In this Thesis, the interactions between secretin and its partners are studied at molecular level. The GspC-HR structure is solved using NMR spectroscopy. Its interaction with secretin (GspD) is elucidated using several biochemical and biophysical approaches and a model of the complex is proposed. Also, the interaction between secretin (GspD) and pilotin (GspS) is further charicterisied. An 18 residues secretin sequence is identified as responsible for interacting with pilotin. Upon binding to the pilotin, the unstructured secretin forms a helical structure.

Le système de sécrétion de type II (SST2) permet la sécrétion de protéines repliées à travers la membrane externe chez les bactéries à Gram-négatif. Le SST2 est une nano-machine enchâssée dans l’enveloppe bactérienne, proche par sa composition et structure aux systèmes d’assemblage des pili de type IV (PT4) impliqués, entre autres, dans d’adhésion et motilité. Chez Klebsiella oxytoca, la surexpression des gènes pul codant le SST2 permet l’assemblage de pili composées des sous-unités PulG. Ceci suggère qu’en conditions physiologiques l’assemblage d’un pseudopilus périplasmique permet la sécrétion du substrat spécifique du SST2, la pullulanase. Dans ce projet nous avons exploré le mécanisme moléculaire de l’assemblage du pseudopilus en se focalisant sur les interactions de PulG avec les composants du SST2 dans la membrane interne. En utilisant l’approche de double-hybride bactérien, nous avons établi le réseau d’interactions de PulG avec les pseudopilins mineures PulH, I, J et K et avec la plateforme d’assemblage (PA). Pour valider ces interactions, nous avons combiné des techniques de biochimie (co-purification par affinité, pontage cystéine et chimique) avec des analyses fonctionnelles de sécrétion et de formation du pseudopilus. Nous avons mis en évidence des interactions entre PulG et les protéines de la PA, PulF et PulM, et nous avons analysé en détail l’interface PulG-PulM. Les résultats suggèrent la formation d’un complexe PulK-I-J-H-G dans la membrane interne impliqué dans des étapes précoces de la formation du pseudopilus, à travers les interactions de PulG et PulH avec PulM et PulF. Nos données expérimentales suggèrent un rôle majeur de PulM dans la sécrétion, vraisemblablement durant l’assemblage du SST2 et l’élongation du pseudopilus. Nos travaux collaboratifs mettant en jeu l'analyse par spectroscopie de masse et en dynamique moléculaire in silico révèlent le rôle essentiel des résidus conservés Glu5 et Thr2 de PulG, requis pour l’interaction avec PulM. Ces données suggèrent que Glu5 participe à l'extraction de PulG de la membrane, en neutralisant la charge positive de son peptide N-terminal par des interactions intramoleculaires. Ces résultats permettent d'établir un modèle détaillant les étapes initiales de l’assemblage des pseudopili dans la membrane interne, relevant pour de futures études sur le SST2 et nanomachines homologues. sécrétion de protéinespili de type 4 assemblage de fibres complexes protéiques membranairesinteractions protéine-protéinemicroscopie à immuno-fluorescence simulations en dynamique moléculairedouble-hybride bactérien spectrométrie de masse nanomachines bacteriennes
The type II secretion system (T2SS) drives the translocation of folded, periplasmic proteins across the outer membrane in Gram-negative bacteria. Secretion is carried out by an envelope-spanning nanomachine that is similar to the apparatus that builds type IV pili (T4P), bacterial surface filaments involved in adhesion, motility and other functions. In the Pul T2SS of Klebsiella oxytoca, overexpression of pul genes in plate-grown bacteria allows the assembly of T4P-like surface fibres made of PulG subunits, suggesting that a periplasmic pseudopilus fibre plays a role in the secretion of the type II substrate pullulanase under physiological conditions. In this project, we explored the molecular mechanism of pseudopilus assembly by focusing on the interaction between PulG and the T2SS inner membrane and pseudopili components. The network of interactions of PulG with the minor pseudopilins PulH, I, J and K and the assembly platform (AP) components was established using bacterial two-hybrid analysis. To validate these interactions, we combined biochemical approaches (affinity co-purification, chemical or cysteine cross-linking) with functional assays of secretion and pseudopilus formation. We provide evidence of the interaction between PulG and the AP proteins PulF and PulM, and delve into the PulG-PulM interface. Our results point to the formation of a PulK-I-J-H-G complex in the plasma membrane involved in early steps of fibre assembly, with a determinant role for PulG and PulH interaction with PulM and PulF. We obtained experimental evidence supporting a major role for PulM in pseudopilus assembly and protein secretion, probably by intervening in the assembly of the T2SS apparatus and in pseudopilus elongation. The results of experimental and in silico studies in collaboration with experts in mass spectrometry and molecular dynamics support the essential role of the highly conserved PulG residues Glu5 and Thr2, which participate in PulM binding. In addition, Glu5 probably favours PulG membrane extraction by neutralising its N-terminal positive charge through intra-molecular interaction. These findings shed new light on early membrane events during fibre assembly, and open new and exciting avenues in research on T2SSs and related nanomachines.protein secretiontype 4 pilifibre assemblymembrane protein complexprotein-protein interactionsimmunofluorescence microscopymolecular dynamics simulationsbacterial two-hybrid assaymass spectrometrybacterial nanomachines

The type three-secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called “translocators”. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats (TPRs). However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultra-centrifugation and equilibrium chemical denaturations showed that LcrH is a weak and thermodynamically unstable dimer (KD ≈ 15 μM, ΔGH2O = 7.4 kcalmol-1). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (ΔGH2O = 1.7 kcalmol-1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O = 5.7 kcalmol-1). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far larger translocator cargo.

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

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.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22495号
農博第2399号
新制||農||1076(附属図書館)
学位論文||R2||N5275(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 木岡 紀幸
学位規則第4条第1項該当

Le système de sécrétion de type II (T2SS) assure le transport de protéines sous une forme repliée du périplasme dans le milieu extracellulaire. Ce système est largement exploité par les bactéries à Gram négatif pathogènes des plantes, des animaux et de l'homme où il permet la sécrétion de facteurs de virulence (des toxines et des enzymes lytiques). La bactérie phytopathogène Dickeya dadantii utilise le T2SS appelé Out, pour sécréter une douzaine de pectinases qui dégradent les parois des cellules végétales. Les protéines sécrétées par le T2SS n'ont pas de motif de sécrétion apparent et leur sécrétion implique plusieurs interactions transitoires avec les composants du système. La nature moléculaire de ces interactions n'est pas connue. Afin de capter ces interactions transitoires lors du processus de sécrétion, j'ai utilisé le pontage dirigé in vivo. Cette technique repose sur l'incorporation d'un analogue photoréactif d'un acide aminé (le para-benzoyl Lphénylalanine, pBpa) à la place des résidus soupçonnés de faire partie d'un site d'interaction. Le pontage est ensuite activé par une courte exposition des cellules aux UV ce qui permet la formation des complexes protéiques. Tout d'abord, cette technique a été utilisée pour introduire le pBpa dans plusieurs régions exposées à la surface d'une exoprotéine, PelI. Cette stratégie a permis de mettre en évidence qu'un élément structural, la boucle 3 du domaine Fn3 de PelI, est impliquée dans l'interaction avec la sécrétine OutD, le composant du T2SS situé dans la membrane externe, et avec le domaine PDZ d'OutC, un composant de la membrane interne. Ces résultats suggèrent que la boucle 3 fait partie d'un motif de sécrétion. Deux autres régions ont été identifiées au sein de PelI : le linker entre les deux domaines de PelI qui est impliqué dans l'interaction avec OutD et une région exposée du domaine catalytique qui interagit avec la protéine OutC. La même approche a été utilisée pour introduire le pBpa dans les deux composants du T2SS, OutC et OutD. Ces expériences ont suggéré que le domaine PDZ d'OutC interagit avec une autre exoprotéine, PelB. Cette étude, de façon complémentaire à d'autres approches, nous a permis de démontrer certains détails moléculaires essentiels de la sécrétion par le T2SS
The type II secretion system (T2SS) transports folded proteins from the periplasm through the outer membrane into the milieu. In many pathogenic Gram-negative bacteria, the T2SS secretes various virulence factors in host tissue and is directly involved in pathogenesis. The phytopathogen Dickeya dadantii secretes a dozen of pectinases through a T2SS named Out. The secreted proteins are lacking an obvious common signal and secretion is thought to involve multiple transient interactions of folded exoproteins with several T2SS components. Molecular nature of these interactions remains unknown. To address this question we used an in vivo sitespecific photo-crosslinking approach to capture such transient interactions within the functional T2SS of D. dadantii. In this technique, the photo-crosslinker para-benzoyl-L-phenylalanine, pBpa, is introduced in vivo in place of a residue of interest and UV-irradiation of living cells provokes the formation of complexes between the protein of interest and its partners. First, in a systematic approach, pBpa was introduced at several surface-exposed sites of the secreted protein PelI. This strategy permitted us to identify that one structural element, loop 3 of Fn3 domain in PelI, interacts both with the secretin, the outer membrane T2SS component, and with the PDZ domain of OutC, an inner membrane T2SS component. These results suggest that this loop 3 is a part of the secretion motif. The same approach permitted us to identify two other regions of PelI interacting with the T2SS: a linker situated between the two domains of PelI, which interacts with OutD, and an exposed region of the catalytic domain of PelI interacting with OutC. In another approach, pBpa was introduced into the T2SS components, OutC and OutD. These experiments suggested that the PDZ domain of OutC interacts with the secreted protein PelB. This study, in complement with other approaches, allowed us to uncover some important molecular features of the protein secretion by the T2SS

Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui est caractérisée par son ubiquité et sa grande capacité adaptative. Cette faculté lui est notamment permise par de nombreux systèmes de perception et de régulation, la sécrétion d'un large arsenal d'exoprotéines, une capacité à alterner entre deux modes de vie, une haute résistance naturelle aux antibiotiques ainsi qu'un génome riche soumis à une importante plasticité génomique. Cette dernière, associée aux pressions de sélection exercées par la grande diversité d'environnements rencontrés par P. aeruginosa, a permis l'émergence de nombreuses souches aux caractéristiques génotypiques et phénotypiques qui leur sont propres. Durant ma thèse, nous avons réalisé une analyse transcriptomique globale comparative entre les souches connues PA14, PAO1 et un nouvel isolat clinique atypique multirésistant aux antibiotiques, la souche PA7. Cette étude nous a permis de suggérer que cette souche, dépourvue des armes principales de la cytotoxicité, tendait naturellement vers un mode de développement associé à la formation de biofilm. Nous avons également caractérisé l'îlot génomique RGP69, unique à la souche PA7 qui code un troisième système de sécrétion de type II, Txc, qui sécrète dans le milieu extracellulaire une protéine d'affinité à la chitine, CbpE, sous le contrôle régulationnel d'un nouveau système de régulation à deux composants, Tts. Cet îlot génomique serait directement impliqué dans la physiologie particulière de la souche PA7
Pseudomonas aeruginosa is an opportunistic bacterial pathogen, characterized by its ubiquity and its high adaptative property. This faculty is particularly due to many systems of perception and regulation, the secretion of a wide arsenal of exoproteins, an ability to switch between two life styles, a high natural resistance to antibiotics and a rich genome submitted to an important genomic plasticity. The latter, combined with the selection pressure exerted by the wide variety of environments encountered by P. aeruginosa, has allowed the emergence of many strains with their own genotypic and phenotypic characteristics.During my thesis, we performed an overall comparative transcriptomic analysis between the known strains PA14 and PAO1, and a new atypical clinical isolate multiresistant to antibiotics, the PA7 strain. This study allowed us to determine that this strain, lacking the main weapons of cytotoxicity, naturally tended to a life-style associated with biofilm formation. We also characterized the RGP69 genomic island, unique in the PA7 strain, which encodes a third type II secretion system, Txc, that secretes in the extracellular medium a chitin-binding protein, CbpE, under the regulatory control of a component system, Tts. This genomic island could be directly involved in the particular physiology of the PA7 strain

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

Le système de sécrétion de type II (T2SS) est largement répandu chez les bactéries à Gram négatif. Il permet la sécrétion d'enzymes lytiques et de toxines. Chez la bactérie phytopathogène Erwinia chrysanthemi, les pectinases, sécrétées par ce système appelé Out, dégradent la pectine, provoquant les symptômes de pourriture molle. La sécrétion par le T2SS se passe en 2 étapes : les protéines traversent la membrane interne par le système Sec ou le système Tat. Une fois dans le périplasme, elles sont repliées et transloquées par le T2SS à travers la membrane externe. Le système Out est composé de 14 protéines intégrées ou associées à l'une des deux membranes. Son assemblage et son fonctionnement restent obscurs. Une plateforme serait formée dans la membrane interne par OutE, -F, -L, -M et -C. Ces trois derniers composants sont des protéines bitopiques dont la stœchiométrie et le rôle sont inconnus. Pour identifier des interactions entre ses composants, nous avons utilisé le double-hybride bactérien, basé sur la reconstitution de l'activité d'adénylate cyclase. Nous avons démontré que le domaine de type ferrédoxine, situé en C-terminus d'OutL et d'OutM, est directement impliqué dans l'homo- et l'hétérodimérisation de ces protéines. Une interaction entre les régions périplasmiques d'OutC et d'OutD a été aussi détectée (Login et al., 2010). Pour mieux analyser les multiples interactions au sein du T2SS, des expériences de triple-hybride ont été réalisées en co-exprimant différentes combinaisons des régions solubles de trois composants. Nos résultats suggèrent qu'OutL empêche l'interaction entre OutC et OutD. Par ailleurs, OutL est impliquée dans l'activation de l'ATPase OutE, le moteur du système (Camberg et al., 2007). OutL serait donc impliquée dans la transmission du signal entre le périplasme et le cytoplasme et pourrait intervenir dans la dissociation du complexe OutD/OutC. Afin d'analyser le rôle des segments transmembranaires (TMS) de composants du T2SS, nous avons adapté la technique du double-hybride. Le domaine de la protéine rapporteur Cya a été fusionné au N-terminus du TMS et BlaM au C-terminus. BlaM sert à contrôler la topologie correcte des fusions dans la membrane. Plusieurs interactions bi-partenaires entre les TMS d'OutC, OutL et OutM ont été ainsi détectées. Ce travail a été complété par une étude in vitro (pull-down) et par mutagenèse dirigée. Ces interactions TMS-TMS pourraient intervenir dans la transmission du signal du périplasme vers le cytoplasme à travers la membrane interne.

Le système de sécrétion de type II (T2SS) est largement répandu chez les bactéries à Gram négatif et est, entre autre, exploité par de nombreux pathogènes pour sécréter des facteurs de virulence dans le milieu extérieur. Le T2SS est constitué de 12 à 15 protéines différentes s’associant en une machinerie complexe qui traverse la totalité de l’enveloppe bactérienne. Ce système assure la sécrétion de protéines repliées du périplasme au milieu extracellulaire. Le mode de fonctionnement de cette machinerie n’est toujours pas connu. Pour comprendre les mécanismes moléculaires régissant la sécrétion des protéines par le T2SS, nous avons utilisé comme modèle le T2SS de la bactérie phytopathogène Dickeya dadantii, nommé Out, qui assure la sécrétion de pectinases entrainant la pourriture molle chez de nombreux végétaux. Nous avons employé des approches de pontage disulfure, double hybride bactérien et GST-pull down afin d’étudier l’arrangement et l’organisation des composants au sein du système de sécrétion. Nous avons ainsi montré que les composants de la membrane interne et la sécrétine de la membrane externe se coordonnent entre eux grâce à un réseau d’interactions complexe et dynamique qui peut être modifié par la présence d’une protéine à sécréter. En combinant des approches génétiques, biochimiques, structurales et bioinformatiques, nous avons étudié le mécanisme de reconnaissance de la pectinase PelI, par deux composants majeurs du système, la protéine de membrane interne OutC et la sécrétine OutD qui forme le pore du T2SS dans la membrane externe. Nous avons montré que PelI interagit avec les domaines périplasmiques HR et PDZ d’OutC et N0 et N1 d’OutD. La présence de N1 renforce l’interaction PDZ/PelI suggérant que le processus de sécrétion pourrait être régi par une succession de contacts synergiques. PDZOutC reconnait une boucle de 9 résidus au sein de l’exoprotéine PelI. Cette boucle constitue un motif d’adressage spécifique contrôlant le recrutement de PelI par la machinerie de sécrétion Out. Des études in silico et in vivo ont montré l’existence de régions similaires à cette boucle au sein d’autres pectinases sécrétées par D. dadantii. Par ailleurs, l’interaction N1OutD/PelI impliquerait un contact de brins β ainsi que la région non structurée située en amont de N1. Ces travaux constituent la première démonstration expérimentale du rôle de signal de sécrétion d’un élément structural précis d’une exoprotéine sécrétée par un T2SS. Ils ont également permis pour la première fois de caractériser des sites précis d’interactions entre une protéine sécrétée et des composants du T2SS. Cette étude constitue une avancée majeure dans la compréhension des mécanismes moléculaires qui gouvernent le recrutement et la sécrétion des protéines par le système de type II
The type II secretion system (T2SS) is widespread in Gram-negative bacteria. It is notably exploited by various pathogenic bacteria to secrete virulence factors into the extracellular milieu and host tissues. The T2SS is composed of 12 to 15 proteins that assemble together into a complex machine that spans the bacterial envelope. It allows the translocation of fully folded proteins from the periplasm across the outer membrane. The exact mode of action of this sophisticated machine is still unknown. The phytopathogenic bacterium Dickeya dadantii uses a T2SS, named Out, to secrete several plant cell-wall degrading enzymes that cause the soft rot disease of many plants. We used the Out system of this bacterium as a model to study the molecular mechanism of protein secretion by T2SS. In order to study the mutual arrangement of the different components of this machinery, we used disulfide bonding, bacterial two hybrid and GST-pull down. We showed that the components of the inner membrane platform interact together and we characterized several interfaces between the inner membrane component OutC and the outer membrane secretin OutD. These various contacts create a complex and dynamic network within the secretion machine that can be modulated by the presence of a protein to be secreted. Subsequently, we combined genetic, biochemical, structural and bioinformatics approaches to study how the pectinase PelI is recognized by the inner membrane component OutC and the pore-forming secretin OutD. We showed that PelI interacts with the periplasmic domains HR and PDZ of OutC and N0 and N1 of OutD. The presence of N1OutD positively modulates the PDZ/PelI interaction, suggesting that protein progression through the T2SS could involve a succession of synergistic contacts. The OutC PDZ domain recognizes a short loop of PelI. This loop acts as a specific secretion signal that controls exoprotein recruitment by the T2SS. Concerted in silico and in vivo approaches suggest the occurrence of equivalent secretion motifs in other exoproteins. The interaction between PelI and OutD could involve a β-strand contact and an intrinsically disordered region located upstream of N1. This work provides the first experimental evidence of molecular mechanisms that govern exoprotein recruitment by the T2SS. Notably, we identified a short structural element acting as a secretion signal and characterized for the first time the interfaces between the T2SS components and a protein to be secreted. This study provides important new mechanistic insights to understand the functioning of this secretion machine

Peu de protéines nécessaires à l’infection des plantes communes aux bactéries, aux champignons et aux oomycètes ont été identifiées à part les enzymes dégradant les parois des cellules végétales. Nous avons caractérisé la structure et les propriétés d’une protéine, IbpS, sécrétée par la bactérie phytopathogène nécrotrophe Dickeya dadantii. Des homologues de cette protéine sont présents non seulement chez des bactéries à Gram+ mais aussi chez des champignons, des oomycètes et quelques animaux. Ce gène d’origine bactérienne a été transféré une fois chez les oomycètes et probablement plusieurs fois chez les champignons. IbpS peut fixer le fer et le cuivre, des métaux à activité redox. Elle a une structure en Venus Fly Trap classique mais avec des caractéristiques originales : elle forme des dimères en solution et possède un nouveau mode de fixation du métal. IbpS est impliquée dans le processus infectieux de D. dadantii et de Botrytis cinerea, un champignon phytopathogène nécrotrophe. Nous proposons qu’IbpS, une fois sécrétée, fixe le fer et le cuivre exogène, réduisant ainsi leur concentration intracellulaire et la formation de ROS dans le microorganisme. La sécrétion de cette protéine fixant les métaux semble être un mécanisme de protection contre l’oxydation requis pendant l’infection partagé par des phytopathogènes nécrotrophes
Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Herein, we have characterized the structure and properties of a protein (IbpS) secreted by the plant pathogenic bacterial necrotroph Dickeya dadantii. Homologs of this protein are present in not only Gram+ bacteria but also in fungi, oomycetes, most phytopathogens, and some animals. The gene originating from bacteria was transferred once in oomycetes and most likely several times in fungi. IbpS is capable of binding the redox-active metals iron and copper and has a classical Venus Fly trap fold with some original characteristics: it forms dimers in solution and has a novel metal binding site. IbpS is involved in D. dadantii and of the Botrytis cinerea, a fungal necrotroph, infection process. We propose that secreted IbpS binds exogenous iron and copper, reducing their intracellular concentrations of these metals and ROS formation in the microorganisms. Secretion of this metal scavenging protein appears to be a common antioxidant protection mechanism shared by necrotrophic phytopathogens and required during infection

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.

碩士
國立中興大學
生物化學研究所
100
The type II secretion system (T2SS) of Xanthomonas campestris pv. campestris is constituted of twelve components spanning the inner and outer membrane. This study is intended to determine significance of the major pseudopilin XpsG in foci formation of the T2SS ATPase XpsE-ECFP during secretion. By taking homologous recombination approach, I replaced the chromosomal xpsE gene with the xpsE-ecfp gene in the genetic background with or without the xpsG gene. Fluorescence microscopic observations were then made for comparing cellular distribution of the XpsE-ECFP. Foci formation of XpsE-ECFP at cell boundary was observed in strains with or without the xpsG gene, suggesting the XpsG protein is not essential for XpsE-ECFP foci formation. Introduction of a plasmid-encoded xpsG gene into the xpsG-null strain resulted in the XpsG protein expressed at elevated level. Meanwhile, the XpsE-ECFP foci appeared more diffused than those in the xpsG-null strain, implicating that presence of abundant XpsG may have a dispersing effect on the XpsE-ECFP foci. As observed before, in absence of the secretin gene encoding component of the secretion channel, the XpsE-ECFP foci were detected at cell boundary. Simultaneous disruption of the xpsG gene enhanced XpsE-ECFP foci formation, as suggested by forming XpsE-ECFP foci with increased fluorescence intensity and abundance, relative to those observed in the xpsG-null or the secretin-null strain. Further enhancement of the XpsE-ECFP foci formation was observed when the inner membrane protein XpsF was overproduced in the xpsG-, secretin-doubly deleted strain. The fluorescence appearing diffused in cytoplasm diminished while the fluorescent foci became even sharper. Introduction of the plasmid-encoded xpsG gene reversed the effect, revealed as increased fluorescence intensity in cytoplasm and less sharp focal-appearing XpsE-ECFP. This observation agrees with the hypothesis that assembly of pseudopilus from its major constituent XpsG may cause XpsE-ECFP foci disperse. Presumably, the foci-appearing XpsE-ECFP is recruited to provide energy for pseudopilus assembly, which in turn generates mechanical force pushing the secreted protein through the secretion channel. As suggested previously, the XpsE-ECFP becomes dispersed when secretion completes. It is plausible that in type II secretion process, successful passage of secreted protein through secretion channel may be signaled to the assembled ATPase to disperse through pseudopilus formation from its building block the major pseudopilin.

碩士
國立中興大學
生物化學研究所
101
Type II secretion system is utilized by Xanthomonas campestris pv. campestris for translocating proteins across the outer membrane to attack the host plant cells. Previous studies showed that the only cytoplasmic protein XpsE forms oligomer when it binds to ATP and associates with inner membrane through its interaction with the inner membrane protein XpsL. It has been postulated that ATP hydrolysis by XpsE provides the energy for the assembly of pseudopilus from pseudopilin. The growing pseudopilus may push the exoprotein through the secretion channel constituted of secretin XpsD in the outer membrane. Pseudopilus comprises of one major component XpsG named major pseudopilin and 4 minor components XpsHIJK named minor pseudopilins. As suggested by studies in the literature, minor pseudopilins may self-assemble to form so-called tip complex and prime the pseudopilus elongation approaching the outer membrane by incorporating XpsG one by one following the right handed helix rule. Previous studies in this laboratory showed that XpsE-ECFP in the xpsD- strain gathered as focused spots at the cell boundary. It was observed by the former student Ting-Ru Liu that the XpsE-ECFP foci in the xpsD- strain became diffused in cytoplasm when XpsG was overproduced. It is hypothesized that perhaps pseudopilus assembly from the major pseudopilin may cause diffusion of the gathered ATPase. To examine this hypothesis and the requirement of minor pseudopilins in XpsE-ECFP foci formation, I performed experiments to analyze the cellular distribution of XpsE-ECFP in xpsHIJK- strain. The number of foci formed by the chromosome-encoded XpsE-ECFP appeared to increase in xpsHIJK-, as well as in the xpsG- strain. The results implicate that depletion of the major or all minor pseudopilins favors XpsE-ECFP foci formation. I further examined if additionally expressed major, minor or both pseudopilins influences the cellular distribution of plasmid-encoded XpsE-sfGFP in the xpsD- strain. In presence of additionally expressed minor psedopilins in xpsD- strain, there was no significant difference in the cellular distribution of XpsE-sfGFP. However, when the major pseudopilin alone was additionally expressed in the xpsD- strain, the brightness of the XpsE-sfGFP fluorescence in the cytoplasm decreased and the intensity of the XpsE-sfGFP foci gathered at the cell boundary increased. Similar results were observed in the xpsD+ strain. These observations implicate that increasing protein abundance of the major pseudopilin alone in the cell enhances gathering of the ATPase.

碩士
國立中興大學
生物化學研究所
100
Type II secretion system is utilized by Xanthomonas campestris pv. campestris for translocating proteins from periplasm across the outer membrane. Previous studies showed that the only cytoplasmic protein XpsE forms oligomer when it binds to ATP and associates with inner membrane through its interaction with the inner membrane protein XpsL, providing energy for the secretion through the secretion pore constituted of the outer membrane protein XpsD. As suggested from previous studies, enhanced cyan fluorescent protein (ECFP)-tagged XpsE produced in the xpsD- strain formed fluorescent foci at cell boundary in most cells. It was proposed that foci appeared as a consequence of accumulation of secreted protein in the periplasm. In the xpsD– xpsL- strain, XpsE-ECFP distributed evenly in cytoplasm, suggesting that XpsL is absolutely essential for the focal assembly of XpsE-ECFP in the xpsD- strain. XpsF is an inner membrane protein with three transmembrane regions. It may interact with XpsL and XpsE through its two cytoplasmic domains. To better understand the significance of XpsF in the interaction between XpsL and XpsE, I monitored the XpsE-ECFP foci formation in the presence or absence of XpsD by varying the protein abundance of XpsF. In the xpsF- strain, secretion was defective and XpsE-ECFP appeared diffused in the cytoplasm. When XpsD was absent, secretion was defective as well and XpsE-ECFP appeared as foci at the cell boundary. However, no matter XpsF was present or not, foci could still be observed, indicating that XpsF is not required for foci formation in the absence of XpsD. When XpsF was overproduced in the xpsD– strain, the fluorescent intensity and the number of cells exhibiting foci increased. Furthermore, the fluorescent brightness and the abundance of XpsE-ECFP foci were greatly enhanced by simultaneous overproduction of the secreted protein a-amylase in the xpsD- strain that overproduced XpsF. This observation suggested that enhancement of XpsE-ECFP foci formation in the xpsD- strain by overproducing XpsF may be related to the accumulation of secreted protein in the cell. In agreement, overproduction of XpsF in the wild-type genetic background caused secretion partially inhibited and secreted protein accumulated in the cell. Meanwhile, the XpsE-ECFP foci were detected, suggesting that overproduction of XpsF by itself is sufficient to enhance the XpsE-ECFP foci formation.

博士
國立中興大學
生物化學研究所
98
(Part I: Structural studies of bacterial type II secretion system component EpsL) The type II secretion system (T2SS) is used for the translocation of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria. At least 12 distinct protein components are required for the functioning of T2SS by forming a secretion nano-machine that spans both the outer and inner membranes, providing a direct connection between the cytoplasm and outer membrane. Such a trans-envelop assembly couples ATP hydrolysis, taking place exclusively in the cytoplasm, to protein translocation. In T2SS, the cytosolic secretion ATPase GspE is recruited to the membrane-associated secretion complex by interacting with the cytoplasmic membrane proteins GspL. Therefore, GspL appears to serve as a critical link between ATP utilization and exoprotein secretion. Full-length GspL is known to form dimers in vivo, yeast two-hybrid analyses further suggested that both the cytoplasmic and periplasmic domain of GspL can form homomeric interactions. From crystallographic data, cyto-EpsL and N1-EpsE/cyto-EpsL structures of Vibrio cholerae also exist as dimers in the crystals. The existence of homodimer may be important for GspL function. To obtain additional insights on GspL function, we have determined the crystal structure of cytoplasmic domain of GspL from Vibrio parahaemolyticus (cyto-EpsL) at 2.67 Å resolution by the multiwavelength anomalous diffraction method. The cyto-EpsL adopts a dimeric architecture that was first observed in the V. cholerae protein as well as its complex with the N1 domain of EpsE protein. Results from cross-linking assay showed that dimer interface affected cyto-EpsL dimerization efficiency, indicating the crystallographic dimer may indeed exist in solution. It remains to be determined whether this interface is important for protein secretion. Together with the results from gel filtration analysis, a possible mechanism by which the cyto-EpsL dimer interacts with the secretion ATPase was proposed. (Part II: Structural studies of bacterial transcription regulator MerR1 of mercury resistance operon TnMERI1) The mercurial compounds are best known for their extreme toxicity to living organisms due to their high affinity towards all thio-containing proteins and a tendency to substitute and block the functions of essential metals. For some bacteria, carrying a suite of cotranscribed genes, termed the mercury resistance mer operon, allows them to survive in environments contain mercurial compounds. The mer operon is consisted of MerP, MerT, MerC, MerE, MerF, MerA, and MerR genes, which encode proteins capable of converting inorganic (Hg(II)) and organiomercurial compounds (such as methylmercury, MeHg) to less toxic form (Hg(0)). The mer operon transcription is regulated by mercury resistance operon repressor (MerR) protein. Although its name suggests a repressive role during transcription regulation, MerR may also function as a transcription activator. In the absence of Hg(II), MerR binds and represses the transcription of mer operon. However, MerR is converted into a transcription activator upon Hg(II) binding. To understand how MerR regulates the transcription of mer operon, we have determined the structure of MerR protein from Gram-positive bacteria Bacillus megaterium MB1 by the multiwavelength anomalous diffraction method. The MerR1 monomer contains a DNA-binding domain, a dimerization helix and a metal-binding motif. Like most other transcription factors, dimerization of MerR1 is required for function. A total of four MerR1 dimers are present in the asymmetric unit, all exhibiting similar quaternary structure. Compared with the structures of other MerR family members, the metal binding domain of one MerR1 monomer winds around the dimerization helix of the other monomer, suggesting that Hg(II) binding may alter the quaternary structure of MerR1. Such a structural transition may reposition the two DNA-binding domains, thus allows the promoter DNA to interact productively with the RNA polymerase to turn on transcription.

碩士
國立清華大學
分子醫學研究所
101
Pseudomonas aeruginosa is a common nosocomial pathogen causing acute infections in immunocompromised patients and chronic infections in cystic fibrosis patients. Gram-negative bacteria, especially pathogens, have developed diversified secretion systems to deliver effector molecules through their complex double membrane structure. P. aeruginosa contains three gene clusters named T6SS-I, -II, and -III that can encode a type VI secretion system. The valine-glycine repeat protein G (VgrG), structurally similar with the complex puncturing device of T4 bacteriophage, is one of the proteins secreted by type VI secretion systems P. aeruginosa PAO1 contains 10 vgrG-like genes. While PA0091, PA0095, and PA2685 have been shown to be the VgrGs of T6SS-I, the VgrG associated with T6SS-II is less clear. Based on comparative genomic analysis, either PA1511, PA0262, or PA5266 could be the VgrG associated with T6SS-II. Protein domain analysis revealed that PA1511 and PA0262 contain a functionally unknown extended segment in the C-terminal region. Further cytotoxicity assay of PA1511 and PA0262 by transfecting the genes into cultured HCT-8 cells failed to detect any significant effects. This study then constructed the vgrG deletion strains and examined their biological properties. Among commonly assayed phenotypes, no significant effect could be observed resulting from the vgrG deletions. In contrast, cytotoxicity and virulence to Chinese cabbage leaves are reduced in the ΔPA1511 and ΔPA5266 mutants. Finally, this study demonstrated that PA5266 and Hcp2 are secreted when PAO1 was grown in M8 minimal medium. Although the exact VgrGs secreted by T6SS-II and their functions remain to be determined, this study has established a strong basis for future analysis of the three VgrGs.

Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...