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Type VI Secretion System Effectors

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Type VI Secretion System Effectors THESE DE DOCTORAT DE L’UNIVERSITE D’AIX-MARSEILLE Soutenue par Mme Thi Thu Hang LE Pour obtenir le grade de Docteur de l’Université d’Aix-Marseille Spécialité: Biochimie structurale Type VI secretion system effectors Soutenue le 22 Février 2017 devant le jury : Dr. Valerie CAMPANACC (I2BC, Gif-sur-Yvette) Rapporteur Prof. Gérard LAMBEAU (IPMC, Nice-Sophia Antipolis) Rapporteur Prof. Sophie BLEVES (IMM, AMU, Marseille) Examinateur Dr. Coralie BOMPARD (USTH, Lille) Examinateur Dr. Tâm MIGNOT (LCB, Marseille) Examinateur Dr. Alain ROUSSEL (AFMB, Marseille) Directeur de thèse Dr. Christian CAMBILLAU (AFMB, Marseille) Co-directeur Dr. Stéphane CANAAN (IMM, Marseille) Invité TABLE OF CONTENTS SUMMARY…………………..…………………..……………....…………………….p 3 INTRODUCTION……………………………..…………………..…………………..p 4 I. Bacterial secretion systems: diversity and functions………..…………………….p 4 1.1. Two-step secretion mechanism …………………………………………….……p 7 1.1.1. Type V secretion system (T5SS) …………………………………………….……p 8 1.1.2. Chaperone– usher (CU) pathway T7SS……………………………………..……p 9 1.1.3. Curli biogenesis system T8SS……………………………………………….……p 10 1.1.4. Type II secretion system (T2SS) ……………………………………………….…p 11 1.1.5. Por Secretion System (PorSS or T9SS) ………………………………….….……p 12 1.2. One step secretion systems…………………………….…………………….……p 13 1.2.1. Type I secretion system (T1SS) ………………………………………….….……p 13 1.2.2. Type III secretion system (T3SS) ……………..…………………………….……p 14 1.2.3. Type IV secretion system (T4SS) ……………..…………………………….……p 15 1.2.4. Type VI Secretion System (T6SS) ……………..…………………………….……p 16 Structural assembly of T6SS……………..…………………………..…………….……p 16 Contruction/delivery effector proteins……………..………………...…………….……p 21 Disassembly……………..………………………………………………………….……p 24 II. T6SS effectors……………..……………………………..…………………….……p 24 2.1. Cell wall targeting……………..………………………….………………….……p 24 2.2. Membrane-targeting effectors……………..……….……………………….……p 31 2.3. Nucleic acid-targeting effectors ……………..…………………………..….……p 35 AIMS OF THE THESIS……………..…………………………………….…….……p 37 RESULTS CHAPTER 1 ……….……..…………………………………………………………...p 38 Foreword ……….……..………….……………………………………………………p 38 Publication: A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery ……..…….……….p 41 Chapter 1b supplementary experiments (not published) ………………..….………….p 83 1b.1. Crystallization and data collection of the Tle1-Tli1 complex. ….…..….………….p 83 1b.2 Nanobodies generation against Tle1 (SciW). ………………..….………………….p 84 1 1b.3 Structure of the nanobody nbTle1-15 against Tle1. ………………..….……….….p 85 Conclusion and perspectives of Chapter 1b. ………………..………………………….p 87 CHAPTER 2 Foreword ………………………………………………………………..….………….p 88 Publication (in preparation) Purification, characterization, crystallization and preliminary X-ray studies of the type VI secretion effector/immunity protein Tle3AIEC/ Tli3AIEC from Adherent-Invasive Escherichia coli ..….…………………………………...….……….p 91 1 - Introduction …………………………………………..……………..….………….p 91 2 – Results …………………………………………..……………..….……………….p 92 2.1 Cloning and protein expression ……………………………….….………….…….p 93 2.2. Protein purification ……………………………….….………….…….……….….p 94 2.3. Phospholipase A1 fluorescent assays and inhibition studies ..….……………...….p 97 2.4. Analytical Gel Filtration Analysis and MALS/QELS/UV/RI coupled size exclusion chromatography ….….…………………………..…….……….p 99 2.5. Crystallization of the Tle3AIEC - Tli3AIEC complex 101..….……………….……….p 101 2.6. Data collection and processing ..….……………………………………….……….p 101 2.7. Analysis of preliminary X-ray diffraction results ..….…………………….……….p 101 2.8. Generation of llama nanobodies against Tle3 ..….………………………..……….p 103 3 Conclusion ..….……………….………………………………………………….…..p 104 GENERAL CONCLUSIONS..….……………….….…………………………….…..p 106 PERSPECTIVES..….……………….….………………………………………….…..p 108 REFERENCES..….……………………………….….…………………………….…..p 109 ANNEXES - Inhibition of Type VI Secretion by an Anti- TssM Llama Nanobody………..….p120 - Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody ………..………....p134 2 SUMMARY The Type VI secretion system (T6SS) is a multi-protein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels an inner tube capped by a spike toward target cells, allowing the delivery of protein effectors. In 2006, for the first time the Type VI Secretion System (T6SS) has been described in two bacterial genus, Vibrio cholerae and Pseudomonas aeruginosa. Later, this system has been found in various bacteria including Burkholderia mallei, Burkholderia cenocepacia, Edwardsiella tarda, Serratia marcescens, Escherichia coli, Agrobacterium tumefaciens, Aeromonas hydrophila, Helicobacter, Campylobacter as well as other organisms. The genomic analysis suggested that, T6SSs are found in approximately 25% of all sequenced Gram-negative bacteria, making T6SS the most widespread specialized secretion system. Here, we analyzed the Entero-aggregative Escherichia coli Sci-1 T6SS toxin effectors. We identified Tle1, a toxin effector encoded by this cluster and show that Tle1 possesses phospholipase A1 and A2 activities required for the inter-bacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located in the C-terminal regions of VgrG proteins. Meanwhile, a pathogenic group of E. coli, called adherent-invasive E. coli (AIEC), has been extensively implicated in human Crohn’s disease (CD) and is currently one of the most exciting players in the pathogen story. There are at least two gene clusters in the AIEC genome that encode T6SS components named AIEC LF82 T6SS1 and AIEC LF82 T6SS2. The protein 435 from the pathogen AIEC LF82 has been predicted to be a phospholipase of the Tle3 effector family with PLA1 activity from a T6SS1 gene cluster. Its toxicity can be neutralized by the cognate immunity protein 434 that is a putative Tli3, by forming Tle3 - Tli3 protein complex. The two separated proteins and their complex were then called Tle3AIEC, Tli3AIEC and Tle3AIEC - Tli3AIEC complex proteins, respectively. In order to further investigate 3 the related mechanism of Tle3AIEC and Tli3AIEC, we performed expression, purification, characterization, crystallization of the two proteins and preliminary X-ray crystallographic studies of the Tle3AIEC - Tli3AIEC complex in order to understand how Tle3AIEC protein recognizes and binds to its cognate Tli3AIEC effector and inhibits its activity. X-ray diffraction data were collected from selenomethionine-derivatize Tle3AIEC SeMet - Tli3AIEC crystals to a resolution of 3.8 Å. INTRODUCTION I. Bacterial secretion systems: diversity and functions Bacterial infections have caused several serious diseases continuing threat to human health. Thus, to fight against this threat, it is very important to understand how such bacterial survive and prosper [3]. Meanwhile, bacterial colonize almost everywhere in different environments on the earth. They rarely live in isolation even when it diversity species is low. Therefore, in bacterial life cycle, their growth is influenced by interbacterial interaction [4]. Bacterial cells develop collaborative or antogonistic mechanisms to communicate, exchange information or compete for space and nutrition [5]. By using protein secretion systems, bacteria deliver specific proteins to the extracellular environment or directly into target cells. Bacterial secretion systems, are molecular nano-machines, play essential roles in pathogenesis and also in maintaining lines of communication between bacterial cells and their host [6]. These systems are responsible for the secretion of various substrates including small molecules, virulence factors, proteins and DNA outside the cell [7, 8]. These substrates, in term of the response of a bacterium to its environment, were showed to be the important factors in several physiological processes like adhesion, adaptation, pathogenicity, and survival. In addition, these substrates were secreted in a variety mean of transport. Depending on the targeting purpose, they are injected into a target cell, released into the extracellular space or even only remain associated with the bacterial outer membrane (OM) [8, 9]. In recent years, advances in structural biology have revealed the diversity of structures and molecular mechanisms of several bacterial secretion systems. These studies have strongly improved our knowledge on the complex structures and how secretion machineries deliver their substrates into the extracellular milieu (EM) or into target cells [8, 9]. Most secretion systems are relatively complex and each secretion type is identified by the characteristics of the constituents involved but they share a number of comment features and rules that are 4 hallmarks of
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