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<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems. One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy. MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

<p>Bacteria demonstrate an extraordinary capacity to survive and adapt to changing environments. In part, this ability to adapt can be attributed to horizontal gene transfer, a phenomenon which introduces novel genetic information that can be appropriated for use in particular niches. Nowhere is this more relevant than in pathogenic bacteria, whose acquisition of virulence genes have provided an arsenal that permits them to thrive within their selected host. Regulatory evolution is necessary for timely regulation of these acquired virulence genes in the host environment. <em>Salmonella enterica</em> serovar Typhimurium is an intracellular pathogen which possesses numerous horizontally-acquired genomic islands encoding pathogenic determinants that facilitate its host lifestyle. One island, <em>Salmonella</em> Pathogenicity Island (SPI)-2, encodes a type-III secretion system (T3SS) which is regulated by the two-component regulatory system SsrA-SsrB. This system coordinates expression of the SPI-2 T3SS as well as an array of virulence effectors encoded in horizontally-acquired regions throughout the <em>Salmonella</em> genome. The studies presented here investigated the mechanisms in which the transcription factor SsrB functions to integrate virulence processes through regulatory adaptation. This work identified the regulatory logic controlling SsrB and defined the associated SsrB regulon. Furthermore, SsrB was found to induce a regulatory cascade responsible for the expression of bacteriophage genes encoded within SPI-12, an island that also contributes to <em>Salmonella</em> virulence. These findings demonstrate the important contribution of regulatory evolution in pathogen adaptation to the host, and show that horizontally-acquired genes, once integrated into appropriate regulatory networks, can contribute to pathogen fitness in specific niche environments.</p> / Doctor of Philosophy (PhD)

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, contains five copies of the ESAT-6 gene cluster, each encoding a dedicated ESX protein secretion system which has been defined as a novel Type-VII secretion system. The ESX have been implicated in virulence and survival of M. tuberculosis, and as such present a promising target for novel treatment interventions. This study has investigated the evolution, regulation, functions and substrates of the ESX secretion systems. The evolutionary history of the ESX secretion systems was established using in silico and phylogenetic analyses of the sequenced mycobacteria, closely related actinomycetes and WXG-FtsK clusters from other bacteria. The ESX-4 gene cluster appears to have evolved with the start of the evolution of the mycomembrane, followed by the duplication of ESX-3, which marks the evolution of the genus Mycobacterium. The ESX-1 duplication occurred next, followed by ESX-2 and ESX-5 which occur only in the slow growing mycobacteria. Five additional ESX gene clusters were newly identified and named ESX-P1 to - P5. These additional ESX clusters occur, or are predicted to occur, on plasmid DNA, and appear to be progenitors of the genomic ESX-1 to -5 gene clusters, possibly indicating a plasmid-mediated mechanism of ESX duplication and evolution. The promoters expressing the M. tuberculosis ESX-1 to ESX-5 secretion systems were investigated using a promoter probe assay, and characterised using in silico analyses. Promoters were identified for ESX-1, -2, -3 and -5. The functions of the mycobacterial ESX secretion systems were investigated using whole proteomic, secretomic and metabolomic analyses of the fast growing, non-pathogenic M. smegmatis, which contains three of the ESX secretion systems, ESX-1, 3, and 4. ESX knockout strains of M. smegmatis were generated and used in comparative analyses with wild-type M. smegmatis. ESX-1 was highly expressed in wild-type M. smegmatis, however no specific pathways showed considerable variation when ESX-1 was deleted. Deletion of ESX-3 resulted in substantial variation to multiple cellular pathways, including amino acid, carbohydrate and fatty acid metabolism and oxidative stress. These and other differences indicate possible perturbed polyamine metabolism in the absence of ESX-3. Although no ESX-4 protein components were detected in wild type M. smegmatis, the ESX-4 knockout displayed substantial proteomic variation. Reduced levels of ESX-3 component proteins in the ESX-4 knockout suggest that ESX-4 influences ESX-3 expression. Other variation linked ESX-4 to cell division and molybdenum metabolism. Secretomic analyses of wild-type and ESX knockout M. smegmatis strains were used to search for novel substrates of the M. smegmatis ESX secretion systems. No prototype ESX substrates were identified in the culture filtrates, however 10 possible substrates of the ESX-1, -3 and -4 secretion systems, containing the general ESX secretion signal, YxxxD/E, were identified. The functions of some of these proteins correlate with the ESX functions identified in the proteomic and metabolomic analyses. This study sets the groundwork for future work in understanding the functional roles and expression patterns of these ESX secretion systems and in using global proteomic and metabolomic analyses to understand cellular changes in response to specific signals or genomic changes. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die veroorsakende agent van tuberkulose, bevat vyf kopieë van die ESAT-6 geengroep, wat elk 'n toegewyde ESX proteïen sekresiesisteem, omskryf as 'n nuwe Tipe-VII sekresiestelsel, kodeer. Die ESX sekresiesisteme is betrokke by virulensie en oorlewing van M. tuberculosis, en is dus belowende teikens vir nuwe behandelings. Hierdie studie het die evolusie, regulasie, funksies en substrate van die ESX sekresiesisteme ondersoek. Die evolusionêre geskiedenis van die ESX sekresiesisteme is bepaal met behulp van in silico en filogenetiese analises van die volgordebepaalde mikobakterieë, nouverwante actinomisete en WXG-FtsK groepe van ander bakterieë. Die ESX-4 geengroep het saam met die evolusie van die mikomembraan ontwikkel, gevolg deur die duplisering van ESX-3, wat die evolusie van die genus Mycobacterium merk. Die ESX-1 duplisering het volgende plaasgevind, gevolg deur ESX-2 en ESX-5, wat slegs in die stadiggroeiende mikobakterieë voorkom. Vyf addisionele ESX geengroepe is nuut geïdentifiseer in hierdie studie en is ESX-P1 tot -P5 genoem. Hierdie addisionale ESX groepe is op, of word voorspel om op, plasmied DNS voor te kom, en mag voorlopers van die genomiese ESX-1 tot -5 geengroepe wees, wat moontlik dui op 'n plasmied-gemedieërde meganisme van ESX duplisering en evolusie. Die promoters wat verantwoordelik is vir die uitdrukking van die M. tuberculosis ESX-1 tot ESX-5 sekresiesisteme is ondersoek deur middel van 'n promoter aktiwiteitstoets, en gekarakteriseer deur in silico analises. Promoters is geidentifiseer vir ESX-1, -2, -3 en -5. Die funksies van die mikobakteriële ESX sekresiesisteme is ondersoek deur proteomiese, sekretomiese en metabolomiese analises van die vinnig-groeiende, nie-patogeniese mikobakterium M. smegmatis, wat ESX- 1, -3 en -4 sekresiesisteme besit. ESX uitslaanmutante van M. smegmatis is gegenereer en gebruik in die vergelykende analises met die wilde-tipe M. smegmatis. ESX-1 is hoogs uitgedruk in wilde-tipe M. smegmatis, maar geen spesifieke metabolise weë het aansienlike variasie getoon wanneer ESX-1 verwyder is. Delesie van ESX-3 het gelei tot aansienlike variasie in verskeie sellulêre weë, insluitend aminosuur-, koolhidraat- en vetsuur-metabolisme en oksidatiewe stres. Hierdie en ander verskille dui op moontlike versteurde poli-amien metabolisme in die afwesigheid van ESX-3. Hoewel geen ESX-4 proteïenkomponente opgespoor is in wilde-tipe M. smegmatis nie, vertoon die ESX-4 uitslaanmutant aansienlike proteomiese variasie. Laer vlakke van ESX-3 proteïne dui daarop dat ESX-4 die uitdrukking van ESX-3 beinvloed. Baie van die proteomiese variasie kan geassosieer word met verlaagde ESX-3 uitdrukking, maar ander variasie mag ESX-4 koppel met seldeling en molibdeen metabolisme. Sekretomiese analises van wilde-tipe en ESX uitslaanmutant M. smegmatis stamme is gebruik om nuwe substrate van die M. smegmatis ESX sekresiesisteme te identifiseer. Geen prototipe ESX substrate is geïdentifiseer in die kultuurfiltraat, maar 10 moontlike substrate van die ESX-1, -3 en -4 sekresiesisteme, met die algemene ESX sekresiesein, YxxxD/E, is geïdentifiseer. Die funksies van sommige van hierdie proteïene korreleer met die funksies geïdentifiseer in die proteomiese en metabolomiese analises. Hierdie studie stel die grondslag vir toekomstige werk in die begrip van die funksionele rol en uitdrukkingspatrone van die ESX sekresiesisteme en in die gebruik van globale proteomiese en metabolomiese analises om sellulêre veranderinge in reaksie op spesifieke seine of genomiese veranderinge te verstaan. / The National Research Foundation / German Academic Exchange Service (DAAD), / The Harry Crossley Foundation / The Ernst and Ethel Erikson Trust / Stellenbosch University

Host-adapted bacteria are pathogens that, through evolutionary time and host-adaptive events, acquired the ability to manipulate hosts into assisting their own reproduction and spread. Through these host-adaptive events, free-living pathogens may be rendered unable to reproduce without their host, which is an irreversible step in evolution. Francisellaceae and Legionellales, two orders of Gammaproteobacteria, are cases where host-adaptation has lead to an intracellular lifestyle. Both orders use secretion systems, in combination with effector proteins, to invade and control their hosts. A current view is that Francisellaceae and Legionellales went through host-adaptive events at two separate time points. However, F. hongkongensis, a member of Francisellaceae shares the same secretion system as the order of Legionellales. Additionally, two host-adapted Gammaproteobacteria, Piscirickettsia spp. and Berkiella spp., swaps phylogenetic positions between Legionellales and Francisellaceae depending on methods applied - indicating shared features of Francisellaceae and Legionellales. In this study, we set up a workflow to screen public metagenomic data for candidate host-adaptive bacteria. Using this data, we attempted to assert the phylogenetic position and possibly resolve evolutionary events that occurred in Legionellales, F. hongkongensis, Francisellaceae, Piscirickettsia spp. and Berkiella spp. We successfully acquired 23 candidate host-adapted MAGs by (i) scanning for genes, among reads before assembly, using PhyloMagnet, and (ii) screening for complete secretion systems with MacSyFinder. The phylogenetic results turned out indecisive in the placement ofBerkiella spp. and Piscirickettsia. However, results found in this study indicate that, contrary to previous beliefs, it is possible that it was one intracellularization event of a common ancestor that gave rise to the intracellular lifestyle of Francisellaceae and Legionellales.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Les maladies parodontales sont causées par une infection bactérienne touchant les tissus entourant les dents, appelés parodonte. L’inflammation aggravée du parodonte peut conduire au déchaussement ou à la chute des dents. Porphyromonas gingivalis est une bactérie anaérobique qui sécrète des toxines appelées gingipaïnes, considérées comme le facteur de virulence majeur. Le système de sécrétion de type IX (T9SS) a été récemment mis en évidence exclusivement chez les membres de la famille des bacteroidetes. Chez P. gingivalis, ce système et directement relié à la sécrétion des gingipaïnes est donc sa pathogénicité. Des études ont montré que plus d’une quinzaine de protéines sont impliquées dans l’assemblage, la fonction et la régulation de ce système de sécrétion. Parmi ces protéines, PorK, PorL, PorM, PorN forment un complexe membranaire au cœur de la machinerie de sécrétion, enchâssé dans les deux membranes bactériennes. L’objectif de ce travail de thèse a été de mettre en place une méthodologie d’extraction et de solubilisation du complexe membranaire PorK-L-M-N afin d’étudier sa structure moléculaire par des méthodes de biochimie intégrative. Trois sous-complexes ont été étudiés successivement. Le complexe de membrane externe PorK-N, le complexe de membrane externe étendu PorK-N-M et le complexe de membrane interne PorL-M. Les résultats obtenus montrent que le complexe de membrane externe PorK-N présente une structure en forme d’anneau de 50nm de diamètre et le complexe de membrane interne PorL-M possède une structure étendue avec une base sphérique de 25nm. Ces résultats valident une méthodologie qui pourra par la suite être utilisée pour d'autres études du T9SS. / Periodontal diseases are caused by a bacterial infection affecting the tissues surrounding the teeth, called periodontal. The aggravated inflammation of the periodontium may lead to loosening or falling of the teeth. Porphyromonas gingivalis is an anaerobic bacterium able to secrete toxins called gingipains, considered as the major virulence factor. First called PorSS, the type IX secretion system (T9SS) was recently found exclusively in members of bacteroidetes. In P. gingivalis this system is directly related to the secretion of gingipains and is therefore its pathogenicity. Studies have shown that more than fifteen proteins are involved in the assembly, function and regulation of this secretory system. Among these proteins PorK, PorL, PorM, PorN form a membrane core complex, the central part of the secretory machinery embedded in the two bacterial membranes. The objective of this thesis was to set up a methodology of extraction and solubilization of the PorK-L-M-N membrane complex in order to study its molecular structure by integrative biochemistry methods. Three sub-complexes have been studied successively. PorK-N the outer membrane complex, PorK-N-M extended outer membrane complex and PorL-M the inner membrane complex. The results show that the PorK-N outer membrane complex has a ring-shaped structure of 50nm in diameter, confirming published results, and the PorL-M inner membrane complex has an extended structure of 25nm with a spherical base. These results validate the established methodology that can subsequently be used to continue the structural study of T9SS.