Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em frangos conhecidas como colibacilose. A APEC MT78, ao contrário de outras linhagens APEC, foi capaz de invadir células não-fagocitárias no modelo de fibroblastos aviários (CEC-32). Considerando que as interações patógeno-hospedeiro envolvem modificações na abundância de proteínas e padrões de expressão, principalmente nas proteínas de superfície, nosso objetivo foi comparar o proteoma da MT78 crescida em meio de cultura celular com o proteoma de MT78 isolada de fibroblastos aviários infectados (condição de co-cultura). Desenvolvemos aqui a padronização das etapas de extração de proteínas totais, isolamento de células bacterianas do co-cultivo e análise proteômica de modo a obtermos uma análise proteômica global reprodutível e de qualidade. A análise da interação APEC MT78 e células CEC-32 por microscopia óptica e eletrônica de varredura revelou que essa cepa se associa à célula-alvo em um padrão de adesão localizada. A internalização de APEC MT78 pareceu ocorrer como resultado de uma interação entre bactéria-célula que dispara rearranjos do citoesqueleto de actina da célula-alvo, formando estruturas filo e lamelipodiais que são dependentes da viabilidade bacteriana. O reisolamento de células bacterianas intactas, observadas por microscopia eletrônica de transmissão, após o co-cultivo com CEC-32 foi obtido através da técnica de solubilização diferencial de membranas. As células bacterianas foram sonicadas e as proteínas digeridas em solução seguida de uma etapa de purificação. Nós identificamos 69 proteínas, distribuídas em 9 classes funcionais, incluindo as proteínas de membrana FimA, OmpA and OmpC. A proteína OmpA já foi associada a invasão do patógeno humano NMEC (neonatal meningitis-associated E. coli) à células HBMEC. Esses experimentos representam a primeira investigação proteômica global em E. coli patogênica aviária. As proteínas identificadas representaram diferentes rotas metabólicas, funções fisiológicas e diferentes localizações subcelulares. / In poultry, Avian Pathogenic Escherichia coli (APEC) cause localized extra- intestinal infections that often become systemic. APEC strain MT78 was able to invade non-phagocytic avian fibroblasts in vitro, raising the possibility that some APEC strains may invade epithelial cells and gain systemic access. Using light microscopy and scanning electron microscopy, we observed that viable MT78 strain associated with CEC-32 fibroblasts cells in clusters, and following association, MT78 internalization appeared to result from cytoskeleton rearrangements, such as filopodia and lamellipodia, in the eukaryotic membrane. Considering that host-pathogen interactions involve modifications of protein abundance and expression, mainly in surface proteins, we compared the proteome of MT78 harvested from culture medium with the proteome of MT78 isolated from infected avian fibroblasts (co-culture condition). For this purpose, we developed standard analytical procedures for global protein extraction and isolation of bacterial cells from infected CEC-32. Judged by transmission electron microscopy, we successfully reisolated intact APEC MT78 cells from CEC-32 fibroblasts using the differential membrane solubilization method. Bacterial cells were then sonicated and proteins digested in solution following a clean up procedure. We identified 69 proteins, distributed in 9 functional classes, including the membrane proteins FimA, OmpA and OmpC. The OmpA protein was already associated to invasion of the human pathogen called NMEC (neonatal meningitis-associated E. coli) to endothelial cell line HBMEC. Our results represent the first global proteomic investigation in APEC. The proteome of MT78 infecting avian fibroblasts may allow us to identify key proteins linked to the successful adhesion and/or invasion of host cells by APEC and thus throw light into the pathogenesis of avian colibacillosis.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23555号 / 生博第466号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 朝長 啓造, 教授 野田 岳志, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments. / Doctor of Philosophy / Fusobacterium, a member of the microbial community in our mouth, has been a captivating study target due to its association with human health and diseases. By nature, Fusobacterium lives in oxygen-free pockets between our teeth and gumline in which this organism has been correlated with a multitude of complications and diseases including periodontitis, inflammatory bowel disease, preterm birth, and most importantly colorectal cancer. Though the connection to human health is established, we still have to learn more about the mechanisms utilized by Fusobacterium to exacerbate diseases. This challenge is mainly hindered by the lack of efficient tools and resources to systematically investigate the relationship between the bacterium and its human host. Therefore, the work in this dissertation focuses on expanding the existing molecular toolkit to study clinically relevant Fusobacterium strain, which provides the power and convenience to discover novel mechanisms that Fusobacterium can take advantage of to be a successful pathogen. Accordingly, we first enhanced our ability to work with a wider range of Fusobacterium species. We successfully introduced exogenous genetic materials into a clinical strain of Fusobacterium, Fusobacterium nucleatum ATCC 25586. This breakthrough was built on the success of our current toolkit to make genetic modifications to a sister strain, Fusobacterium nucleatum ATCC 23726. With this newfound capacity to modify F. nucleatum ATCC 25586, we have described the importance of a novel protein aiding in the invasion of Fusobacterium to colorectal cancer. Furthermore, we have determined that certain proteins within the fusobacterial type 5a protein family can play a key role in governing binding and invasion of colorectal cancer cells in this study. Concurrently, for the first time, we provided the snapshot of a small protein and its role in fusobacterial long-term survival inside its targeted host cells. Altogether, the findings in this dissertation will bring forth an innovative framework to better the comprehension of current Fusobacterium-induced disease implications, while exploring alternative treatments for enhanced patient health.

The microbiome has long been an alluring target to study and recent advancements in microbial detection and omics-technologies has further revolutionized our view of how human diseases are impacted by the microbiome. A member of the human microbiome that has garnered such attention is Fusobacterium nucleatum, a Gram-negative, anaerobic bacterium, that normally inhabits the human oral cavity. Interestingly, F. nucleatum is highly invasive into surrounding cells and tissues of the periodontal pocket (below the gymline) and capable of disseminating throughout the entire body. Because of this, F. nucleatum is associated with a wide variety of diseases, most recently and strikingly, colorectal cancer. Despite the pathogenic potential of F. nucleatum, there is limited knowledge about the molecular mechanisms contributing to the invasive nature and virulence of this oral bacterium. This gap in knowledge can be attributed to the absence of genetic tools and resources to investigate and study host-pathogen interactions of Fusobacterium. Progress in dissecting the role of Fusobacterium in disease has been hindered by a lack of fully sequenced and annotated genomes, and the absence of genetic systems to generate target virulence gene deletions to validate mechanisms contributing to host-pathogen interactions. Breakthroughs discussed in this work focus on developing and expanding the genetic toolkits and resources available for studying F. nucleatum interactions in relation to human health and disease. As part of this work, herein, I introduce FusoPortal, an online database of fully sequenced and annotated Fusobacterium genomes, that enabled the bioinformatic annotation and correction of large protein encoding reading frames, that were previously misannotated. This database features a custom basic local alignment search tool (BLAST) server that establishes this resource as a powerful tool for identifying potential virulence factors that contribute to Fusobacterium pathogenesis. Most notably, FusoPortal facilitated my discovery of DNA uptake machinery involved in natural competence and transformation in F. nucleatum. This work is the first to characterize natural competence in a Fusobacterium species, and also enables the expansion of Fusobacterium genetics utilizing the newly found competence mechanism. The findings within this dissertation encompass a paradigm shift in efficient and robust tools to study F. nucleatum biology and pathogenesis. By creating tools for identifying key genes, proteins, and mechanisms involved in Fusobacterium induced or accelerated diseases, there is the potential to accelerate the development of novel therapeutics and vaccines against the emerging 'oncomicrobe' Fusobacterium nucleatum. / Doctor of Philosophy / The trillions of microbes living on or in the human body, collectively called the microbiome, has long been a captivating target to study and understand its role in human health and disease. Recent advances in technology have revolutionized our view of the individual components of the human microbiome, which has led to a renaissance in understanding how specific bacterial species could be used to modulate human health and fight a myriad of diseases. A member of this microbiome that has garnered such attention is Fusobacterium nucleatum, a bacterium that lives in oxygen free pockets along the gumline in the human mouth. A striking feature of F. nucleatum is its ability to invade surrounding tissue, driving bacterial spread throughout the entire body. This bacterium is associated with a wide variety of diseases, most importantly colon cancer. Although F. nucleatum is implicated in these diseases, we still know very little about the mechanisms used by Fusobacterium to promote disease. This roadblock in studying F. nucleatum can largely be attributed to the lack of molecular tools and resources to investigate and study the interactions between the bacteria and its human host. Therefore, research discussed in this work revolved around developing and resources available for studying F. nucleatum interactions in relation to human health and disease. One such resource developed was FusoPortal, an online website with fully sequenced and annotated genomes. This resource was critical in the bioinformatic annotation and correction of large proteins that were previously misannotated. This website features a tool that allows one to search this complete set of genes for a specific sequence establishing this resource as an important tool for identifying key genes and mechanisms that could influence F. nucleatum ability to infect and cause disease. Most notably, FusoPortal provided the means to discover a bacterial system that can import DNA and integrate it into the bacterial genome, a process called natural competence. This work is the first to characterize natural competence in a Fusobacterium species, and has allowed me to utilize the newly found natural competence mechanism to enhance Fusobacterium genetics. In summary, the findings within this dissertation brings about a new horizon for studying F. nucleatum biology, thereby, providing the framework for creating future therapeutic strategies to treat diseases including colorectal cancer.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates. Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status. Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates. The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates. The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death. We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic. / AFRIKAANSE OPSOMMING: Talle internasionale studies het verskeie endemiese metisillien vatbare Staphylococcus aureus (MSSA) klone geïdentifiseer en beskryf. Slegs 'n paar van hierdie klone word geassosieer met metisillien weerstandigheid (Klonale kompleks (KK) 5, KK8, KK22, KK30 en KK45). Studies oor die bevolking struktuur van S. aureus geïsoleer in Suid-Afrika is tot dusver beperk tot demografiese gebiede, fokus slegs op metisillien-weerstande S. aureus (MRSA) en het min klem op virulensie geplaas. Hierdie studie is onderneem om die bevolking struktuur van S. aureus, geïsoleer vanaf spesifieke kliniese bronne, in die pasiëntpopulasie van Tygerberg-hospitaal te ondersoek en om ondersoek in te stel na spesifieke gasheer-patogeen interaksies van verteenwoordigende isolate. Opeenvolgende, nie-herhalende en suiwer kliniese S. aureus isolate is versamel oor ´n periode van een jaar (September 2009/2010), tesame met pasiënt demografiese- en beperkte kliniese inligting. Stamme is deur PFGE en molekulêre merkers (spa, MLST, agr, SCCmec en PVL) beskryf. Verteenwoordigende isolate is gekies en ondersoek vir die teenwoordigheid van virulensie gene, aanhegting ( aan geïmmobiliseerde fibronektien [Fn], fibrinogeen [Fg], kollageen IV [CnIV] en kollageen VI [CnVI]), sellulêre indringing en die induksie van seldood. Statistiese assosiasies is bepaal tussen alle in vitro resultate en methicillin-weerstandigheid, klonaliteit, pasiënt MIV status en bakteriese PVL status. Fyftien persent van die isolate (n = 367) was MRSA. Vier-en-veertig van die isolate was PVL+. agrI-IV en SCCmec I-V is geïdentifiseer. Die MSSA bevolking was divers: ST22 (dominant), ST1865 en ST121 PVL +. ST45, ST1863 en ST15 was PVL+. PVL- MRSA was divers: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II en ST22-MRSA-IV. Die gene fnbA/B (fibronektien A/B), clfA/B (klontings faktor A/B), eap (ekstrasellulêre aanhegtings protein), nuc (nukease), coa (koagulase) en hld (delta toksien) was aangetref in alle verteenwoordigende isolate. Isolate van KK8 en KK6 het sterk aan alle ligande (100-700% van kontrole, ligand-afhanklike) aangeheg, terwyl isolate van KK45, KK22 en KK88 slegs sterk aand fibronektien en fibrinogeen aangeheg het. Isolate van KK30, KK15, en KK12 het baie sterk aan CnIV (> 300%) aangeheg en KK8, KK15, en KK6 and CnVI (> 200%). Isolate van KK30, KK8, KK15, KK6, KK12, KK97, KK88 en KK45 was hoogs indringend (> 100%). ST121 was nie-indringende (> 50%). Isolate van KK5, KK30 en KK121 was nie-sitotoksiese (<50%), terwyl isolate van KK22, KK8, KK15, KK45 en KK88 baie sitotoksies was (> 70%). Geen betekenisvolle verskil is waargeneem in die aanhegting of seldood induksie van MRSA teenoor MSSA klone of tussen isolate van MIV+ teenoor MIV- persone nie. PVL- isolate het hoër sellulêre indringing as PVL+ isolate vertoon. Die teenwoordigheid van ST612-MRSA-IV, ST22-MRSA-V en ST8-MRSA-V verwys na die plaaslike verwerwing van SCCmec, aangesien ons MSSA isolate beskryf het met dieselfde spa-tipes. Talle MSSA klone was algemeen, maar het nie 'n beduidende genetiese agtergrond met MRSA vertoon nie. PVL was baie algemeen onder MSSA isolate en die PVL gene is dalk onafhanklik van SCCmec verkry. Die vermoë om aan spesifieke geïmmobiliseer ligande in vitro aan te heg was divers en groepeer met die genetiese agtergrond, terwyl die meerderheid van die isolate indringend was en kon betekenisvolle sel dood veroorsaak. Ons kan aflei dat die bevolking van S. aureus by die Tygerberg hospitaal saamgestel is uit 'n groot aantal van MSSA en MRSA klone, wat verskillende patrone van aanhegting aan geselekteerde ligande vertoon en waarvan die meeste klone indringende en sitotoksies is. / DFG/NRF International Research and Training Group (IRTG) 1522 “HIV and associated infectious diseases in Southern Africa” / National Research Foundation / Medical Research Council, Medi-Clinic / Harry Crossley Fund (Stellenbosch University) / Stellenbosch University

Background: In order to optimize net transmission success, parasites are hypothesized to evolve towards causing minimal damage to their reservoir host while obtaining high shedding rates. For many parasite species however this paradigm has not been tested, and conflicting results have been found regarding the effect of arenaviruses on their rodent host species. The rodent Mastomys natalensis is the natural reservoir host of several arenaviruses, including Lassa virus that is known to cause Lassa haemorrhagic fever in humans. Here, we examined the effect of three arenaviruses (Gairo, Morogoro and Lassa virus) on four parameters of wild-caught Mastomys natalensis: body mass, head-body length, sexual maturity and fertility. After correcting for the effect of age, we compared these parameters between arenavirus-positive (arenavirus RNA or antibody) and negative animals using data from different field studies in Guinea (Lassa virus) and Tanzania (Morogoro and Gairo viruses). Results: Although the sample sizes of our studies (1297, 749 and 259 animals respectively) were large enough to statistically detect small differences in body conditions, we did not observe any adverse effects of these viruses on Mastomys natalensis. We did find that sexual maturity was significantly positively related with Lassa virus antibody presence until a certain age, and with Gairo virus antibody presence in general. Gairo virus antibody-positive animals were also significantly heavier and larger than antibody-free animals. Conclusion: Together, these results suggest that the pathogenicity of arenaviruses is not severe in M. natalensis, which is likely to be an adaptation of these viruses to optimize transmission success. They also suggest that sexual behaviour might increase the probability of M. natalensis to become infected with arenaviruses.

La multi-résistance aux drogues des bactéries est un problème majeur en clinique. L’un des mécanismes de résistance consiste à effluer les composés toxiques hors de la cellule grâce à des protéines de la membrane interne nommées pompes d’efflux. Ces protéines appartiennent à cinq familles (MFS, RND, MATE, SMR et ABC) et peuvent fonctionner en association avec deux autres types de protéines (protéine du périplasme et protéine de la membrane externe) pour former un canal. Dans le cadre d’une thématique de recherche basée sur l’étude des mécanismes de résistance auxdrogues de la bactérie pathogène Legionella pneumophila, une approche bioinformatique menée sur lesgénomes de trois souches séquencés (souches Lens, Paris et Philadelphia) a permis d’identifier des protéinespouvant participer à l’efflux. Notre but a été de vérifier l’implication de ces protéines dans la résistance auxdrogues et dans la virulence de Legionella en ciblant un ou plusieurs gènes codant pour des composants desystèmes d’efflux. Pour inactiver les gènes, nous avons choisi une stratégie de recombinaison homologue. Lesrecombinants ont été testés pour leur sensibilité à des composés toxiques afin de voir si les gènes ciblés jouentun rôle dans l’efflux d’E. coli. Un de ces mutants, le mutant MF201, altéré pour le gène codant pour une protéinehomologue à TolC chez E. coli s’est avéré être 2 à 16 fois plus sensible aux drogues testées comparé à lasouche sauvage. De plus, ce mutant présente un défaut important de virulence dans Acanthamoeba castellanii,Dictyostelium discoideum et les macrophages U937. Ce premier résultat implique que la protéine TolC-like deLegionella aurait un rôle clef dans la relation hôte pathogène et sous-tend un lien entre multi-résistance auxdrogues et virulence. Par ailleurs une étude de l’expression des gènes codant pour des pompes à efflux a étéinitiée afin de comprendre leur rôle au cours du cycle infectieux de Legionella. / Bacterial multi-drug resistance is of major concern in the case of clinic. One of the resistance mecanisms used by bacteria is the efflux of noxious compounds out of the cell thanks to inner membran proteins called efflux pumps. This proteins belong to five families (MFS, RND, MATE, SMR and ABC) and can function in close association with two partners (periplasmic protein and outer membrane protein) to form a canal. In our new research axis based on the study of the drug resistance of the bacterium Legionella pneumophila, we conducted a bioinformatical approach to identify efflux pumps proteins coded by the sequenced genome of three strains (strains Lens, Paris and Philadelphia). Our goal was to study the role of this proteins in Legionella drug resistance and in its virulence. The bioinformatic approach data allowed us to choose one or several genes coding for potential efflux pump components for genetic invalidation by an homologousrecombination strategy. The bacterial mutants were exposed to different noxious compounds in order to know ifthe target genes invalidated were implicated in the efflux of drugs. One of this mutants, strain MF201, which isdeleted for the gene encoding a protein homologous to E. coli TolC protein, revealed to be 2 to 16 times moresensitive to the drug tested compared to the wild-type strain. Furthermore, this mutant showed an importantvirulence defect in Acanthamoeba castellanii, Dictyostelium discoideum and U937 macrophages. This first resultsmeans that the TolC-like protein of Legionella could be a key factor in host-pathogen interaction and stronglysuggests a link between multi-drug resistance and virulence. We also initiated a transcriptomic approach to studyefflux pump genes expression in order to understand their role during the infectious cycle of Legionella.

La muqueuse des patients atteints de cancer colorectal (CCR) est anormalement colonisée par des souches d’Escherichia coli porteuses de l’îlot pathogène pks (E. coli/pks+), responsable de la synthèse de la génotoxine colibactine. Les E. coli/pks+ induisent des cassures double brin de l’ADN, l’accumulation d’aberrations chromosomiques ainsi que la sénescence, et favorisent le développement tumoral dans des modèles murins de CCR. L’autophagie est un processus des cellules eucaryotes qui permet la dégradation d’éléments cytoplasmiques par les lysosomes et est activé pour permettre l’adaptation des cellules en réponse à un stress. Un dysfonctionnement de l’autophagie est associé à plusieurs pathologies humaines, notamment les cancers. L’objectif de ce travail de thèse était d’étudier le rôle de l’autophagie dans la défense de l’hôte suite à l’infection par les E. coli/pks+. Nous avons montré une augmentation de l’expression des gènes de l’autophagie dans la muqueuse colique des patients atteints de CCR colonisée par des E. coli/pks+ comparativement à celle colonisée par des E. coli ne portant pas l’îlot pks. L’infection des cellules épithéliales intestinales humaines HCT-116 par des souches d’E. coli isolées de patients atteints de CCR entraîne l’activation de l’autophagie de façon dépendante de la présence de l’îlot pks. Les cellules déficientes pour l’autophagie présentent une augmentation des dommages à l’ADN induits par les E. coli/pks+, associée à un défaut de recrutement au niveau du noyau de la protéine de réparation des dommages à l’ADN, RAD51, ainsi qu’une augmentation de la sénescence et de la prolifération cellulaire induites par ces souches. Dans un modèle murin de CCR, le modèle de souris Apc Min/+ , déficient pour le gène de l’autophagie Atg16l1 spécifiquement dans les cellules épithéliales intestinales, nous avons montré un rôle complexe de l’autophagie dans la carcinogenèse colorectale. En effet, en condition non infectée, chez les souris Apc Min/+ , l’autophagie joue un rôle pro-tumoral. Cependant, suite à l’infection par la souche d’E. coli/pks+, 11G5, les souris déficientes pour l’autophagie présentent une augmentation de la tumorigénèse, accompagnée par une augmentation des dommages à l’ADN, de la prolifération cellulaire et de l’inflammation. Ces résultats suggèrent que l’autophagie est nécessaire pour inhiber les effets pro-tumoraux des souches d’E. coli/pks+ et ainsi limiter la carcinogenèse colorectale induite par ces dernières. De nombreuses perspectives d’études sur les mécanismes sous-jacents impliqués dans la progression tumorale induite par les souches d’E. coli/pks+ feront suite à ce projet. Notamment, l’impact du microenvironnement immunitaire et du microbiote sur la carcinogenèse colorectale seront analysés. Plus largement, cette étude permettra de mieux comprendre le rôle de l’autophagie dans la défense de l’hôte pour lutter contre les E. coli associés au CCR. À plus long terme, ce travail pourrait également contribuer au développement de nouvelles stratégies thérapeutiques basées sur la modulation de l’autophagie chez les patients présentant une colonisation par les souches d’E. coli/pks+. / Several studies have shown a role of intestinal microbiota in CRC etiology, which is the third cause of death by cancer in the world. Especially, colonic mucosa of CRC patient is abnormally colonized with E. coli strains which often carry the pathogenic pks island, leading to the synthesis of a genotoxin called by colibactin. Colibactin-producing E. coli strains induce DNA double strand breaks, chromosomic aberration and senescence in host cells enhancing the cellular proliferation and tumorigenesis in mouse models of CRC. The aim of this study is to investigate the role of autophagy, a key process in cellular homeostasis, in host defense against infection by pks-harboring E. coli (E. coli/pks+). We showed the increased expression of different autophagy-related genes in the mucosa of CRC patients colonized with E. coli/pks+ compared to that of patients colonized with E. coli without the pks island. In vitro and in vivo, we showed that autophagy is activated in intestinal epithelial cells upon infection in order to limit the pro-tumoral effects of E. coli/pks+. In a murine model of CRC, the ApcMin/+ mouse model, deficient for the Atg16l1 autophagy gene specifically in intestinal epithelial cells, we have shown a complex role of autophagy in colorectal carcinogenesis. Indeed, in uninfected conditions, autophagy plays a pro-tumoral role. However, following infection with the E. coli/pks+ 11G5 strain, mice deficient for autophagy exhibit increased tumorigenesis, accompanied by increased DNA damage, cell proliferation, and inflammation. These results suggest that autophagy is necessary to inhibit the pro-tumoral effects of E. coli/pks+ strains and thus limit the colorectal carcinogenesis induced by the latter. Future works using mouse models of CRC are required to study the role of autophagy in colonic tumorigenesis suppression following infection with E. coli/pks+. Different mechanism such as inhibition of cellular proliferation and immune response, modification of the composition of the gut microbiota will be analyzed. Together, those results will highlight the role of autophagy as a host defense mechanism against the pro-tumoral effects of pks-harboring E. coli strains. This work could also open the door to new therapeutic options in the treatment of CRC and therefore have a great impact on public health.

Philosophiae Doctor - PhD / In this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date