The type VII secretion system is a protein export pathway linked to diverse phenotypes in both Actinobacteria and Firmicutes. The Actinobacterial subtype of the T7SS, referred to as T7SSa, has been shown to play a critical role in various aspects of Mycobacterial life including virulence, conjugation, and metal homeostasis. The T7SSb of Firmicutes bacteria on the other hand has similarly been shown to influence virulence but by the direct growth inhibition of competitor bacteria. Structure-function analyses of the T7SSa apparatus as well as various effectors and chaperones have begun to build a more mechanistic understanding of how T7SSa functions. In contrast, we know little of how the T7SSb functions despite its noted importance to both pathogens and environmental bacteria such as Bacillus, Staphylococcus, Enterococcus, and Streptococcus. During my thesis work, I have addressed several gaps in our understanding of T7SSb function. The three major questions that I have studied are: (1) how do T7SSb immunity proteins inhibit the toxicity of their cognate toxins, (2) how does the T7SSb export effectors through the thick Gram-positive cell wall, and (3) what is the role of chaperone proteins in facilitating T7SSb effector export? / Thesis / Doctor of Philosophy (PhD) / Bacteria require space and various nutrients to survive and grow and must therefore compete against other bacteria for access to these resources. To gain advantage over their competitors, many bacteria have developed molecular weapons that target and kill other closely related bacteria. Some of these weapons take the form of protein secretion machines that export antibacterial toxins. Gram-positive bacteria use the type VIIb secretion system (T7SSb) to inhibit the growth of other Gram-positive bacteria. In this work, I explore several aspects of T7SSb including: (1) how toxins are inhibited by immunity proteins, (2) how toxins are secreted through the cell envelope, and (3) how toxins are recognized by the secretion apparatus. The goal of this work is to better understand how T7SSb functions at the molecular level.

Mycobacterium abscessus est une mycobactérie à croissance rapide, et un pathogène opportuniste responsable d’infections pulmonaires notamment chez les patients atteints de la mucoviscidose, et d’infections cutanéomuqueuses. La source de contamination pourrait être environnementale mais des contaminations interhumaines ne sont pas exclues. Les amibes environnementales pourraient jouer un rôle de réservoir. M. abscessus est capable de résister aux mécanismes de défense bactéricides des phagocytes environnementaux et humains. Le génome complet de M. abscessus a été séquencé mettant en évidence de nombreux facteurs de virulence non mycobactériens. Certains sont des facteurs de virulence connus dans le monde bactérien, comme la phospholipase C ou le facteur de captation du magnésium MgtC. Ces facteurs ont été montré induits en présence d’amibes, mais ne peuvent à eux seuls expliquer la survie intracellulaire et la virulence de M. abscessus. Nous avons donc, au cours de ce projet, criblé une banque de mutants générée par transposition chez M. abscessus, à la recherche de mutants dénués de croissance intracellulaire en amibes et macrophages. Cette approche a permis d’identifier, de façon majeure, 5 gènes du locus ESX-4 de M. abscessus codant un système de sécrétion de type VII avec tous ces composants cœur conservés. Pour mieux comprendre la contribution d’ESX-4 dans la survie intracellulaire de M. abscessus, un mutant obtenu par double recombinaison au sein du gène eccB4 dans la souche type de M. abscessus (ΔeccB4) a été construit. EccB4 est un élément structurel central du système de sécrétion codé par ESX-4. ΔeccB4 présente un défaut de survie au sein des cellules, lié à déficit de blocage de l’acidification phagosomale ainsi qu’un défaut de dégradation de la membrane phagosomale, empêchant un contact phagosome-cytosol. Ce travail a permis de révéler pour la première fois dans le monde mycobactérien le rôle d’un locus ancestral de sécrétion ESX-4 au sein d’une mycobactérie. L’étude des protéines secrétées par ce locus est actuellement en cours au laboratoire, afin d’envisager des approches thérapeutiques et vaccinales pour contrer cette mycobactérie multirésistante aux antibiotiques. / Mycobacterium abscessus is a fast growing mycobacterium, and an opportunistic pathogen responsible for lung infections particularly in patients with cystic fibrosis, and for mucocutaneous infections. The source of contamination could be environmental but human-to-human contaminations are not excluded. Environmental amoeba could play a role as a reservoir. M. abscessus is able to resist to the bactericidal defense mechanisms of environmental and human phagocytes. The complete genome of M. abscessus has been sequenced and presents numerous non-mycobacterial virulence factors. Some are known virulence factors in the bacterial world, such as phospholipase C or the magnesium uptake factor MgtC. These factors have been shown to be induced in the presence of amoeba, but cannot alone explain the intracellular survival and virulence of M. abscessus. We thus, in the course of this project, screened a library of mutants generated by transposition in M. abscessus, in search of mutants lacking intracellular growth in amoeba and macrophages. This approach made it possible to identify, in a major way, 5 genes of the ESX-4 locus of M. abscessus encoding a type VII secretion system with all these conserved core components. In order to better understand the contribution of ESX-4 to the intracellular survival of M. abscessus, a mutant obtained by double recombination within the eccB4 gene in the M. abscessus type strain (ΔeccB4) was constructed. EccB4 is a central structural element of the secretion system encoded by ESX-4. ΔeccB4 has a defect of survival within the cells, linked to deficiency of blockage of the phagosomal acidification as well as a defect of degradation of the phagosomal membrane, preventing phagosome-cytosol contact. This work made it possible to reveal for the first time in the mycobacterial world the role of an ancestral locus ESX-4 secretion within a mycobacterium. The study of the proteins secreted by this locus is currently underway in the laboratory, in order to consider therapeutic and vaccine approaches to counter this multiresistant antibiotic mycobacterium.

Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The genome of the pathogen Mycobacterium tuberculosis contains five copies of the ESAT-6 (ESX) gene cluster region, which encodes for a novel type VII secretion system. These gene cluster regions, which are directly involved in pathogenicity and phagosomal escape, contain genes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action of the ESX secretion system however, remains largely unknown. This study focused on ESX gene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and is also present in other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor. This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosis in the model organism Mycobacterium smegmatis by means of Mycobacterial Protein Fragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs two cloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferring resistance to kanamycin). Genes of interest are cloned into these vectors and co-transformed into the model organism M. smegmatis after which it is expressed as fusion proteins. Interaction of the proteins allows selective growth on a medium containing the antibiotic trimethoprim. Various interactions were identified throughout this region, including selfinteractions as well as the expected interaction between the ESAT-6 and CFP-10 protein family members esxT and esxU. Since this region is ancestral, ESX-4 provides the basic model of the mechanism of secretion of the type VII secretion system. Many similarities were apparent when the interactions identified for ESX-4 were compared to the interactions previously identified in ESX-3. Interactions identified by means of M-PFC provide a basis for the further study of the structure of this secretion system, and should be confirmed by means of other techniques, such as co-immunoprecipitation. Despite the ability of M-PFC to identify protein-protein interactions in a mycobacterial system, and thus overcoming some of the limitations of the classical yeast two-hybrid model, it must still be regarded as a fishing experiment for potential interactions. A further aim of the project was to construct a knock-out of ESX-4 in the model organism M. smegmatis, which contains three ESX regions, namely ESX-1, -3 and -4. Homologous recombination proved to be an effective technique for the construction of the knock-out, also indicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-out strain showed no morphological differences to the wild type strain of M. smegmatis. The knock-out strain will in future be compared to the wild type strain in various functional studies in order to determine the function of the ancestral ESX region. / AFRIKAANSE OPSOMMING: Die genoom van die patogeen Mycobacterium tuberculosis bavat vyf kopieë van die ESAT-6 geen groep gebiede wat kodeer vir ‘n unieke tipe VII sekresie sisteem. Die geen groep gebiede, wat direk betrokke is by patogenisiteit en fagosomale ontsnapping, bevat gene wat kodeer vir die gesekreteerde T-sel antigene ESAT-6 en CFP-10. Die meganisme van die ESX sekresie sisteem is egter steeds tot ‘n groot mate onbekend. Hierdie studie het gefokus op die ESX geen groep gebied 4 (ESX-4), wat voorheen bepaal is om die vroegste kopie van die gebied te wees en wat ook in ander species van Mikobakterieë en hoë G+C Gram-positiewe bakterieë, soos Corynebacterium diptheriae en Streptomyces coelicolor, voorkom. Hierdie projek was daarop gemik om die proteïen-proteïen interaksies van ESX-4 van M. tuberculosis in die model organisme Mycobacterium smegmatis te ondersoek deur middel van Mikobakteriële Proteïen Fragment Komplementasie (M-PFK). M-PFK is ‘n twee-hibried tegniek wat van twee kloningsvektore, naamlik pUAB300 (wat weerstand teen hygromycin B bied) en pUAB400 (wat weerstand teen kanamycin bied) gebruik maak. Gene van belang word in die vektore ingekloneer en in die model organisme, M. smegmatis geko-transformeer, waarna dit as fusieproteïene uitgedruk word. Indien ‘n interaksie tussen die proteïene plaasvind, sal selektiewe groei op ‘n medium wat die antibiotikum trimethoprim bevat, waargeneem word. Verskeie interaksies is in hierdie gebied geïdentifiseer, insluitende self-interaksies, sowel as die verwagte interaksie tussen die ESAT-6 en CFP-10 proteïen familielede esxT en esxU. Aangesien hierdie gebied die vroegste kopie is, bied ESX-4 die basiese model vir die meganisme van sekresie van die tipe VII sekresie sisteem. Wanneer interaksies wat vir ESX-4 geïdentifiseer is met die wat voorheen vir ESX-3 geïdentifiseer is vergelyk word is daar heelwat ooreenkomste. Interaksies wat deur middel van M-PFK geïdentifiseer is, verskaf ‘n basis vir die vêrdere studie van interaksies van hierdie gebied, en sal bevestig moet word deur gebruik te maak van aanvullende tegnieke, soos ko-immunopresipitasie. Ten spyte van die vermoë van M-PFK om proteïen-proteïen interaksies in ‘n mikobakteriële sisteem, wat dus sommige van die beperkings van die klassieke gis twee-hibriedmodel oorkom, te bestudeer, behoort dit steeds as ‘n voorlopige metode van identifikasie beskou te word. ‘n Vêrdere doel van die projek was om ‘n uitslaanmutant van ESX-4 in die model organisme M. smegmatis, wat drie van die ESX gebiede, naamlik ESX-1, -3 en -4 bevat, te skep. Homoloë rekombinasie is bewys om ‘n effektiewe tegniek te wees vir die skep van ‘n uitslaanmuntant en het daarop gedui dat ESX-4 nie essensieel is vir die in vitro groei van M. smegmatis nie. Die uitslaanstam het ook geen morfologiese verskille getoon teenoor die oorspronklike stam nie. Die uitslaanmutant sal in die toekoms gebruik word in ‘n verskeidenheid funksionele studies waar dit vergelyk sal word met die oorspronklike stam, ten einde die funksie van die vroegste ESX-gebied te bepaal. / Medical Research Council of South Africa / National Research Foundation of South Africa / Ernst and Ethel Eriksen Trust

La première partie de nos travaux a consisté au séquençage de génomes complets de trois souches pathogènes et de trois souches de portage de Staphylococcus lugdunensis pour les comparer aux 15 génomes complets disponibles sur NCBI. Aucun déterminant génétique associé au contexte de virulence ou de portage de S. lugdunensis n’a été identifié. Cependant, nous avons mis en évidence la présence d’éléments génétiques mobiles et des variations dépendantes des complexes clonaux,définis par MultiLocus Sequence Typing, au sein de loci potentiellement associés à la virulence. Des variations ont été observées dans un locus homologue à celui de Staphylococcus aureus codant le système de sécrétion Ess/type VII (SST7). Nous avons mis en évidence huit organisations génétiques chez cette espèce présentant pourtant une structure de population clonale. La seconde partie de nos travaux a consisté à la caractérisation phénotypique et moléculaire du SST7 chez S. lugdunensis par la formation d’un mutant de délétion du gène essC codant une protéine essentielle à la sécrétion. Nos résultats suggèrent que le SST7 serait impliqué dans la translocation de protéines prédites in silico comme impliquées dans la virulence. Néanmoins, dans des modèles de cytotoxicité cellulaire et d’infection du nématode Caenorhabditis elegans, aucune atténuation de la virulence n’a été observée chez la souche mutante malgré une perte de sa capacité à lyser les erythrocytes, comparativement à la souche sauvage. Nos travaux ont également permis de développer et d’évaluer le pouvoir discriminant de trois nouvelles méthodes de typage constituant des outils très prometteurs pour l’épidémiologie moléculaire des infections à S. lugdunensis. / The first part of this study consisted in whole genome sequencing of three pathogenic and three carriage strains of S. lugdunensis and comparison with the 15 genomes available in the NCBI. No genetic determinant was associated to the pathogenic or carriage context. However, we have highlighted the presence of mobile genetic elements and MultiLocus Sequence Typing clonal complex dependent variations within loci potentially associated with virulence. Variations wereobserved in the ess locus homologous to that of Staphylococcus aureus encoding the type VII secretion system (T7SS). We showed eight genetic organizations in this species with a clonal population structure. The second part of our work consisted in a phenotypic and molecular characterization of T7SS in S. lugdunensis by construction of a deletion essC gene mutant. This gene encodes a protein requiredfor protein secretion. Our results suggest that T7SS could be involved in translocation of proteins predicted as implicated in virulence in silico. Nevertheless, no virulence attenuation was observed in cells cytotoxicity assay and Caenorhabditis elegans virulence assays between wild-type and mutant strains which yet has lost the ability to lyse erythrocytes. We also developed and evaluated discriminating power of three new typing methods, which are very promising tools for the molecular epidemiology of S. lugdunensis infections.