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.

Emerging infectious diseases represent a threat to biodiversity, posing significant challenges to wildlife conservation globally. Infectious diseases can cause population declines, local extirpations and, in rare cases, complete species extinction. Among emerging pathogens, pathogenic fungi have been responsible for drastic declines in several high-profile vertebrate taxa, such as Batrachochytrium dendrobatidis causing chytridiomycosis in many species of amphibians worldwide. Recently, an emerging infectious disease, 'snake fungal disease' (SFD), caused by the fungal pathogen Ophidiomyces ophidiicola, is affecting the health of snake populations in North America by causing skin infections which can be fatal. Given the potential impact of this disease on snake biodiversity worldwide, compounded by the pressure of anthropogenic stressors that already jeopardize the viability of many snake populations, there is a clear need for ecological research in this understudied system. This dissertation is comprised of 4 data chapters focusing on the disease dynamics of snake fungal disease in Europe, and the factors resulting in differential infection. In chapter 2, I develop a large field-based data collection in 10 countries in Europe to investigate the presence of disease hotspots and the variation of disease prevalence across host species, and to examine the pathogen genotypes that are present on the landscape. I found isolated areas of disease hotspots, and models including an interactive effect of host species and which pathogen clade are present on the landscape were best at explaining disease prevalence. In chapter 3, I perform a virulence challenge assay using 120 corn snakes (Pantherophis guttatus) and 7 strains of O. ophidiicola (3 collected from Europe, 4 from the USA). This experiment reveals that pathogen genotypes associated with higher disease prevalence in Europe also have higher pathogen virulence, and that different strains from the USA show variation in virulence. These results also match both physiological host responses measured in the lab and landscape patterns of disease. In chapter 4, I explore two mitigation-driven snake translocation projects in Europe that were complicated due to O. ophidiicola outbreaks. One snake species, N. tessellata, appears highly susceptible to SFD, indicating that under stressful conditions, O. ophidiicola can cause mortality regardless of pathogen genotype, and that this snake species may be important in pathogen maintenance. Finally in chapter 5, I report the presence of a different fungal pathogen in Spain, Parannannizziopsis sp., never reported in wild snakes in Europe before. Broadly, my dissertation demonstrates coevolutionary relationships between hosts and pathogens and has important implications to snake conservation over large scales. / Doctor of Philosophy / Biodiversity conservation is under significant threat globally due to the ever-growing human population. Threats such as habitat loss, climate change, pollution and infectious diseases are all important factors that are affecting wildlife populations. Snakes, and specifically infectious diseases of snakes, have been understudied compared to that of other types of wildlife in Europe. My dissertation focuses on understanding the disease ecology and conservation implications of snake fungal disease, an infectious disease that can be life-threatening to snakes. I first developed a study of this disease in multiple countries over three years to understand how common the disease is across the landscape, which species of snakes are most infected, and how severe skin infections are. I found specific areas in Europe where disease prevalence is high and attributed these higher disease areas to specific snake species and fungal strains that interact together to cause higher infection rates (chapter 2). Secondly, I designed a controlled experimental study in the laboratory to test the capacity of different pathogenic strains, collected in Europe and the USA, to harm its host. I chose a single host species, the corn snake (Pantherophis guttatus) to test this question. I found that different pathogen strains vary in their ability to cause severe disease, and these results were confirmed with field observations. I also found that one strain collected from coastal Virginia is able to cause more severe disease when compared to all other strains (chapter 3). Finally, I found that under captive stress, strains that might not be able to cause severe disease in the wild can lead to snake mortality, specifically in one snake species that might be more susceptible than others (chapter 4). Broadly, this dissertation describes complex interactions between snakes and fungal pathogens and has relevance to reptile conservation.

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

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) disease results in approximately 2 million deaths annually and is the leading cause of death due to a single infectious agent. Previous studies have indicated that host genetics play an important role in the development of TB. This together with pathogen and environmental factors intensifies the complexity of this disease. The Major Histocompatibility Complex (MHC) and Leukocyte Receptor Complex (LRC) comprise several genes which are known to be important modulators of the host immune response. The human leukocyte antigen (HLA) class-I genes of the MHC are involved in the presentation of pathogenic antigens on the surfaces of infected cells, while the killer cell immunoglobulin-like receptors (KIRs) of the LRC are involved in the recognition of self and non-self cells. Natural Killer (NK) cells through their KIRs are thus able to kill non-self cells through recognition of the class-I molecules expressed. Additionally, HLAs and KIRs are extremely polymorphic and differ markedly across populations of different ethnicities. Here we studied these genes and their polymorphisms in the South African Coloured (SAC) population to determine their involvement in susceptibility to TB, susceptibility to disease caused by specific Mycobacterium tuberculosis subtypes, and understanding their ancestral contribution to the SAC with regards to the development of TB. We showed that the KIR3DS1 gene and KIR genotypes with five or more activating KIRs, and the presence of 3DS1, protected against the development of active TB in the SAC population. Several HLA class-I alleles were identified as susceptibility factors for TB disease. With regards to genes of the MHC and LRC, several loci were found to alter susceptibility to TB in the SAC population, including MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 and the intergenic regions between HLA-C/WASF5P and LAIR1/TTYH1. We showed that the Beijing strain occurred more frequently in individuals with multiple disease episodes, with the HLA-B27 allele lowering the odds of having an additional episode. Associations were identified for specific HLA types and disease caused by the Beijing, Latin America-Mediterranean (LAM), Low-Copy Clade (LCC), and Quebec strains. HLA types were associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential co-evolutionary events between host and pathogen. Finally, we showed that the SAC population is the most diverse SA population with regards to HLA alleles and KIR genotypes, as would be expected given the admixture of the SAC. Based on the HLA allele class-I profiles across SA populations, we noted that the Ag85BESAT- 6, Ag85B-TB10.4 and Mtb72f vaccines currently undergoing clinical trials would have low efficacy across most SA populations. We showed that the MHC and LRC regions in SAC healthy controls are predominantly of European ancestry, and that SAC TB cases are more closely related to Khoisan and black SA population groups. Our work highlights the importance of investigating both host and pathogen genetics when studying TB disease development and that understanding the genetic ancestral contributions to the SAC population can contribute to the identification of true and novel TB causing variants. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is jaarliks verantwoordelik vir ongeveer 2 miljoen sterftes en is die hoofoorsaak van dood as gevolg van „n aansteeklike siekte. Vorige navorsingstudies het aangedui dat die genetiese samestelling van die gasheer „n beduidende rol speel in die ontwikkeling van TB. Die kompleksiteit van hierdie siekte word vererger deur die betrokkenheid van die gasheer genoom sowel as bakteriële en omgewings faktore. Die Major Histocompatibility Complex (MHC) en Leukocyte Receptor Complex (LRC) bestaan uit verskeie gene wat die gasheer immuunrespons verstel. Die human leukocyte antigen (HLA) klas I gene van die MHC is betrokke by die aanbieding van patogeniese antigene op die oppervlak van geïnfekteerde selle, terwyl die killer cell immunoglobulin-like receptors (KIRs), geleë in die LRC, betrokke is by die herkenning van eie en vreemde selle. NK selle, deur middel van hul KIRs, kan dus vreemde selle uitwis aangesien hulle die uitgedrukte klas I molekules kan herken. Beide HLA en KIRs is hoogs polimorfies en verskil beduidend tussen etniese groepe. In hierdie studie is die bogenoemde gene en hul polimorfismes in die Suid Afrikaanse Kleurling bevolking (SAC) ondersoek om vas te stel tot watter mate dit genetiese vatbaarheid vir TB, asook vatbaarheid vir TB wat deur spesifieke Mycobacterium tuberculosis subtipes veroorsaak word, beïnvloed. Daar is ook gepoog om te verstaan hoe die voorouerlike bydrae van hierdie gene die SAC met betrekking tot TB vatbaarheid affekteer. Die resultate van die studie het aangedui dat die KIR3DS1 geen en KIR genotipes met vyf of meer aktiewe KIRs en die teenwoordigheid van 3DS1, die SAC bevolking beskerm teen die ontwikkeling van aktiewe TB. Verskeie HLA klas I allele is geïdentifiseer as vatbaarheidsfaktore vir TB. Talle lokusse van die MHC en LRC gene is ook as vatbaarheidsfaktore vir TB in die SAC bevolking geïdentifiseer, insluitende MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 en die intergeniese areas tussen HLA-C/WASF5P en LAIR1/TTYH1. Die studie het aangedui dat die Beijing stam meer voorkom in individue wat verskeie kere TB gehad het en dat die HLA-B27 alleel die kanse om „n verdere episode te hê, verlaag het. Assosiasies is geïdentifiseer tussen spesifieke HLA tipes en siekte veroorsaak deur die Beijing, LAM, LCC, en Quebec TB stamme. HLA tipes was geassosieer met siekte veroorsaak deur TB stamme van Euro-Amerikaanse en Oos-Asiëse afkoms. Die frekwensies van hierdie allele, in hul ooreenstemmende mensbevolkings, dui op „n potensïele koevolusionêre gebeurtenis tussen die gasheer en patogeen. Die studie het ook vasgestel dat die SAC populasie die mees diverse SA bevolking is met betrekking tot die HLA allele en KIR genotipes, soos verwag sou word gegewe die gemengde genetiese herkoms van die SAC. Gebaseer op die HLA allele klas I profiel van verskillende SA bevolkings merk ons op dat die Ag85B-ESAT-6, Ag85B-TB10.4 en Mtb72f vaksiene, wat huidiglik kliniese toetsing ondergaan, nie so effektief in die meeste SA bevolkings sal wees nie. Die studie het ook bewys dat die MHC en LRC streke in gesonde SAC kontroles, grootliks afkomstig was van „n Europese nalatenskap en dat die SAC TB gevalle meer verwant is aan die Khoisan en swart SA bevolkings. Hierdie studie beklemtoon die noodsaaklikheid om beide gasheer en patogeen genetika te bestudeer wanneer die ontwikkeling van TB ondersoek word en dat die verstaan van die genetiese voorouerlike bydrae van die SAC bevolking kan bydra tot die identifisering van ware en nuwe TB-veroorsakende variante.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.

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.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family. The specificities and functions of the majority of DUBs are unknown, although their impact on many biological and pathological processes is widely appreciated. This dissertation entails a detailed characterization of otubain 1 (OTUB1), an ovarian tumor domain-containing DUB. The presented work describes OTUB1’s specificity, localization, protein interactions, importance in infection with Yersinia, and proposes a novel model of regulation of its enzymatic activity. I first discuss the structural and biochemical properties of OTUB1, demonstrating its selectivity towards ubiquitin and NEDD8. Moreover, I show that OTUB1 cleaves lys48- but not lys63-linked polyubiquitin, emphasizing its role in ubiquitin-mediated proteasomal degradation. Mass spectrometric identification of interaction partners and localization studies suggest possible involvement of OTUB1 in RNA processing and cell morphology. Furthermore, I demonstrate that invasion of the host cells by the enterobacteria Yersinia can be altered by changing OTUB1 expression. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize RhoA-GTP prior to infection. YpkA and OTUB1 modulate RhoA-GTP stability in opposing ways, leading to cytoskeletal rearrangements that may be involved in bacterial invasion. Moreover, OTUB1 is post-translationally modified by phosphorylation that modulates its ability to stabilize RhoA-GTP and counteracts its effect on bacterial invasion. These findings provide a novel entry point for the manipulation of the host—pathogen interactions. Lastly, a kinase screen revealed that FER, an oncogenic kinase with a role in cell morphology, phosphorylates OTUB1, as demonstrated by overexpression, siRNA and in vitro studies. The phosphorylated site was mapped to tyr26 and the activity-based labeling revealed that this modification interferes with the deubiquitinating activity of OTUB1. In summary, the results presented in this thesis confirm that OTUB1 exerts properties of a “classical DUB” and uncover some of its physiological functions.

Legionella pneumophila est la bactérie responsable de la légionellose, une pneumonie atypique dans les pays industrialisés. Les souches pathogènes sont issues de notre environnement après multiplication à l’intérieur d’amibes, sont disséminées par la technologie humaine, puis peuvent infecter les macrophages alvéolaires humains. Ce travail vise à caractériser une famille d’effecteurs du système de sécrétion de type IV Dot/Icm transloqués dans le cytoplasme de la cellule hôte, des protéine kinases, et en particulier à établir le rôle de la protéine kinase LegK2 dans la virulence. L’analyse in silico et des tests de phosphorylation in vitro ont permis d’identifier 5 protéine kinases fonctionnelles, LegK1-LegK5, codées par la souche épidémique L. pneumophila Lens. Des tests de translocation ont montré qu’à l’exception de LegK5, les protéine kinases de Legionella sont transloquées dans la cellule hôte de façon Icm/Dot dépendante. LegK2 joue un rôle clé dans la virulence, comme démontré par inactivation de gène. Les vacuoles contenant le mutant legK2 présentent un recrutement moins efficace de reticulum endoplasmique, ce qui entraine une réplication intracellulaire retardée. Un mutant de substitution déficient pour l’activité kinase présente les mêmes défauts de virulence, ce qui démontre le rôle central de la phosphorylation dans le contrôle de ce processus. Les mécanismes moléculaires contrôlés par LegK2 sont actuellement recherchés par identification de partenaires et/ou substrats protéiques / Legionella pneumophila is the most common causative agent of the severe pneumony legionellosis. Legionella pathogenic strains are emerging from the environment after intracellular multiplication in amoeba, are dissiminated by water aerosols technologies, and are able to infect alveolar macrophages of human lungs. This work aims to characterize one family of effectors translocated into the host cytoplasm, namely the protein kinase family, and particularly the role of LegK2 protein kinase in virulence. In silico analysis and in vitro phosphorylation assays allowed the identification of 5 functional protein kinases LegK1-LegK5 encoded by the epidemic L. pneumophila Lens strain. Translocation assays showed that except LegK5, the Legionella protein kinases are translocated. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles display less efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. A kinase-dead substitution mutant of legK2 exhibits the same virulence defects. Molecular mechanisms controled by LegK2 have been investigated by searching LegK2 partner and substrate proteins

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.

Au sein du clade Splendidus, Vibrio tasmaniensis et Vibrio crassostreae sont deux populations de vibrios virulentes pour l’huître qui ont été associées au syndrome de mortalité des huîtres juvéniles. Nous nous sommes intéressés à la diversité des mécanismes d'interaction entre ces vibrios et leur hôte, l'huître creuse Crassostrea gigas. Dans un premier temps, nous avons précisé le processus de pathogénèse de la souche V. tasmaniensis LGP32 et montré qu’elle possédait une activité cytotoxique sur les cellules immunitaires de l’huître, les hémocytes, qui dépendait de sa phagocytose. Le transcriptome intracellulaire de LGP32 a révélé le rôle important des systèmes antioxydants et de l’efflux du cuivre dans la survie du vibrio à l’intérieur du phagosome. D’un point de vue fonctionnel, nous avons montré que ces mécanismes jouaient un rôle important au cours de la pathogénèse in vivo. Dans un second temps, nous avons réalisé une étude comparative des interactions entre des souches représentatives des deux populations V. tasmaniensis et V. crassostreae et l’huître. Nous avons montré que les souches virulentes des deux populations étaient cytotoxiques pour les hémocytes mais que cette cytotoxicité était indépendante de la phagocytose chez les V. crassostreae, à l’inverse des V. tasmaniensis. Le transcriptome des huîtres a montré que les souches virulentes des deux populations réprimaient l’expression de gènes impliqués dans la réponse antibactérienne. Des réponses spécifiques à chaque souche virulente ont également été identifiées, révélant la diversité des interactions. In vivo, les souches virulentes se sont montrées capables de coloniser les tissus de l'huitre, contrairement aux souches non virulentes contrôlées par les hémocytes. L’ensemble de nos travaux montre que, bien qu’il existe un degré de diversité et de spécificité des interactions entre différents vibrios du clade Splendidus et l’huître, les deux populations virulentes sont cytotoxiques pour les cellules immunitaires, et ce processus est essentiel pour leur succès infectieux. Ainsi, la capacité de dépasser les défenses hémocytaires est un phénotype conservé entre les populations virulentes du clade Splendidus. Les processus évolutifs ayant conduits à l’émergence de ces traits de virulence communs entre populations de vibrios pathogènes différentes méritent d’être explorer. / In the Splendidus clade, Vibrio tasmaniensis and Vibrio crassostreae are two populations of virulent vibrio for oysters that are associated to "juvenile oyster mortality syndrome". Here we were interested in the diversity of interaction mechanisms between the vibrios and their host, the oyster Crassostrea gigas. First, we investigated the pathogenesis process of the strain V. tasmaniensis LGP32 and showed that it exerts a cytotoxic activity against oyster immune cells, the hemocytes, which depend on its entry into the cells through phagocytosis. Transcriptomic analysis of LGP32 response during intracellular stage revealed a crucial role for antioxydant systems and copper efflux in intraphagosomal survival of the bacteria. From a functional point of view, we showed that this virulence mechanisms of LPG32 play a major role in pathogenesis in vivo. Second, we realized a comparative study of the interaction mechanisms between representative strains of the two populations V. tasmaniensis and V. crassostreae with the oyster. Virulent strains from both populations were cytotoxic for hemocytes but this cytotoxicity was independant of phagocytosis in the case of V. crassostreae, in contrary to V. tasmaniensis. Transcriptomic analysis of the oyster responses during infection showed that virulent strains of both populations repressed the expression of genes involved in antibacterial responses. However, some pecific responses were also identified for each virulent strain, highlighting some diversity of interactions. In vivo, virulent strains were able to colonize oyster tissues, in contrary to non-virulent strains, which were controlled by hemocytes. Our work show, although a certain degree of diversity and specificity exist in the interactions between different vibrios of the Splendidus clade and oysters, both virulent populations are cytotoxic for immune cells, and this process is essential for their infectious success. Thus, the capacity to overcome the hemocyte defenses is a conserved phenotype between distinct virulent populations of vibrios from the Splendidus clade. Hence, it would be of particular interest to determine the evolutionary processes that drove the emergence of common virulence traits in distinct populations of pathogens.