Vibrio splendidus LGP32 est une bactérie pathogène associée aux épisodes de mortalités estivales qui affectent la production d'huître Crassostrea gigas depuis des décennies. Nous avons montré ici que la porine OmpU était un effecteur majeur de l'interaction V. splendidus / C. gigas. Nous avons pour cela construit un mutant ΔompU de V. splendidus. Celui-ci nous a permis de mo ntrer l'implication de OmpU (i) dans la résistance de V. splendidus aux antimicrobiens, incluant ceux de l'huître, (ii) dans la « fitness » chez l'huître, et (iii) dans la virulence en infections expérimentales (mortalités de 56 % pour le sauvage versus pour le 11% mutant). En accord avec ces résultats, nous avons montré que la délétion de ompU modifiait la sécrétion de protéines dont l'expression est contrôlée par les voies de régulation de la virulence (ToxR) et de l'intégrité membranaire (SigmaE). Par ailleurs, nous avons montré que OmpU jouait un rôle essentiel dans la reconnaissance par les hémocytes. En effet, (i) in vivo, les gènes hémocytaires répondent différemment à l'infection par le Vibrio sauvage ou ΔompU, et (ii) in vitro, OmpU est nécessaire à l'invasion hémocytaire par V. splendidus. Cette invasion utilise la phagocytose dépendante de l'intégrine b et la SOD extracellulaire du plasma d'huître comme opsonine qui lie OmpU. Ainsi, OmpU est un facteur de virulence majeur qui permet l'infection des hémocytes dans lesquels il est capable de survivre en inhibant la formation de radicaux oxygénés et de vacuoles acides. La résistance du Vibrio aux antimicrobiens hémocytaires de l'huître, elle-même dépendante de OmpU, est probablement un élément supplémentaire favorable à la survie intra-cellulaire. / Vibrio splendidus LGP32 is a bacterial pathogen associated to the summer mortality outbreaks that have affected the production of Crassostrea gigas oysters over the past decades. We showed here that the OmpU porin is a major effector of the V. splendidus / C. gigas interaction. For that, we have constructed a ΔompU mutant of V. splendidus, and shown that the OmpU porin is implicated (i) in the resistance of V. splendidus to antimicrobials, including those of oyster, (ii) in its in vivo fitness, and (iii) in its virulence in oyster experimental infections (mortalities have been reduced from 56 % to 11 % upon mutation). In agreement, we have shown that the ompU deletion modified the expression of secreted proteins controlled by the virulence (ToxR) and the membrane integrity (SigmaE) regulation pathways. Furthermore, we have shown that OmpU has a major role in the recognition of V. splendidus by oyster hemocytes. Indeed, (i) in vivo, hemocyt e genes displayed differential responses to an infection with the wild-type or the ΔompU mutant, and (ii) in vitro, OmpU was necessary for hemocyte invasion by V. splendidus. This invasion process required the hemocyte b-integrin and the oyster plasma extracellular SOD, which was found to act as an opsonin recognizing OmpU. Thus, OmpU is a major virulence factor that allows infection of hemocytes in which V. splendidus is able to survive by inhibiting the production of reactive oxygen species and the formation of acidic vacuoles. Resistance of V. splendidus to hemocyte antimicrobials, which is also OmpU-dependant, is probably an additional determinant of V. splendidus intracellular survival.

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.

Shigella flexneri is a highly adapted pathogen that invades the host cytosol and causes bacillary dysentery. Shigella has evolved powerful countermeasures to disarm host defense mechanisms; amongst them a family of twelve bacterial E3 ubiquitin ligases (IpaH) that are structurally unrelated to eukaryotic enzymes. IpaH ligases are injected into the host cytosol via the bacterial type III secretion system (T3SS) to manipulate the host cell and counteract anti-bacterial defense pathways. My work demonstrated that IFN-induced guanylate-binding proteins (GBPs) are novel targets for IpaH9.8. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria unless they are ubiquitylated by IpaH9.8 and consequently degraded by the proteasome. IpaH9.8 targets GBP1, GBP2, and GBP4, thereby causing a transient poly-ubiquitin coat comprising K48 and K27-linked chains around S. flexneri, which leads to the proteasome-dependent destruction of existing GBP coats and the re-establishment of bacterial motility and cell-to-cell spread. So far, ubiquitylation of bacteria has mostly been associated with anti-bacterial autophagy or immune signaling. However, the ubiquitin coat assembled around intracellular Shigella by IpaH effectors, in particular IpaH9.8, serves a pro-bacterial function, the first observed so far. In addition, I characterized IpaH1.4 and IpaH2.5 for their ability to prevent NF-κB activation by targeting LUBAC. I found that IpaH1.4 specifically binds the LUBAC component HOIP and mediates its proteasomal degradation, thus abolishing linear ubiquitylation of bacteria and consecutive NF-κB activation via NEMO and autophagy induction via optineurin. Lastly, I identified novel potential ubiquitylation targets for IpaH effectors in human cells using a mass spectrometry-based approach. The resulting IpaH interactome presents the groundwork for further investigations and will help to identify potentially unknown cellular defense mechanisms that are antagonized by Shigella flexneri.

Salmon Pancreas Disease Virus (SPDV) is the only viral species of the genus Alphavirus, family Togaviridae, affecting fish. SPDV induces two conditions historically recognised independently as Pancreas disease (PD) and Sleeping disease (SD), affecting Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss), respectively. Infection by SPDV can lead to clinical disease with characteristic acinar pancreatic necrosis and a range of myopathies of the skeletal and heart muscle. Mortality is not a necessary outcome of the disease and usually is not significant. However, affected fish stop eating and therefore present a reduced growth rate and the disease can also leave visible lesions at the fillet level that lead to downgrading at slaughter. SPDV can affect in the fresh and sea water environments, but a higher and most relevant impact reported in the latter. Historically, PD has posed a significant challenge to the Atlantic salmon farming industry in the UK, as well as in other salmon producing countries. This thesis was developed and conducted at Marine Scotland Science (MSS), the Scottish National Reference Laboratory, with the aim to contribute to knowledge gaps identified by the industry and research communities. The focus was on development and improvement of in vivo and in vitro infection models to assist with host pathogen interaction studies. In vivo work was to establish an experimental challenge model to induce SPDV infection in a more natural way than by intra-peritoneal (IP) injection. The first step involved selection of an infective SPDV isolate through a comparative IP challenge study. An infective isolate was then used to establish a co-habitation challenge model in "post smolts", the sea-water stage predominantly affected by PD. Additionally, during this experiment assessment of viral tissue tropism along time and potential intra-subtype differences in infectivity was undertaken. In vitro work accounted for the more innovative part of this thesis with the development, optimization and application of an ex vivo cardiac primary culture originated from Atlantic salmon embryos. While fish origin aggregates of self-contracting cardiomyocytes had been previously isolated and suggested as a robust tool on human biomedical research and pharmacological and toxicology testing, paradoxically very little has been done to explore the approach of ex vivo primary cultures as a disease model with the specific goal for health issues affecting fish. The work involved an adaptation and refinement to produce salmon cardiac primary cultures (SCPCs). Once this was achieved, SCPCs could be kept under laboratory conditions with minimal maintenance for periods up to 6 months. Following this work, SCPCs were successfully challenged with different SPDV isolates as well as another cardiotropic viral agent (Infectious Salmon Anaemia, ISA). The kinetics of SPDV and ISA viral infection and one element of the immune response (i.e. expression of mx gene) were studied. As part of this study, the comparative response of SCPCs of diverse genetic backgrounds (i.e. IPN resistant vs. IPN sensitive) was also assessed. Differences were observed, which highlights potential usefulness of SCPCs to examine genotype-based differences in response to viral disease. Finally, SCPCs were used to examine the SPDV infection cycle ultrastructure by transmission electron microscopy (TEM). This work resulted in novel insights on the replication cycle of SPDV, drawing from the extensive literature in mammalian alphavirus work. With SPDV and other virus associated myocarditis severely affecting Atlantic salmon aquaculture at present, I believe that the SCPCs model represents the most relevant contribution of this PhD.

Innate immunity relies on a repertoire of germline-encoded non-rearranging pattern recognition receptors that bind to invariant microbial surface molecules. This event initiates a number of signal transduction cascades that lead to humoral and cellular defense responses like synthesis of antimicrobial peptides, phagocytosis and coagulation – mechanisms that efficiently fight infectious microorganisms and have been evolutionary conserved to exist in parallel with the antibody-based adaptive immunity found in vertebrates. The fruit fly, Drosophila melanogaster represents a widely used animal model for studies of a pristine innate immune system. Its immune responsive intracellular signalling pathways display a high degree of similarity with the NF-κB /Rel-signalling pathways that mediate the inflammatory response in mammals. Insects are also vectors for medically important parasitic diseases which can trigger immune responses in the vector so basal knowledge about the regulation and function of insect immune systems can contribute to our understanding of inflammation and microbial disease in higher animals and open new strategies for biological vector control. Drosophila hemocytes play a key role in executing and coordinating local and systemic defenses in response to infection. This thesis describes in vitro studies of Drosophila gene expression in response to bacterial infection using the larval hemocyte-like cell line – mbn-2. Our results show that immune challenge with bacterial cell wall components and intact live bacteria induces differential gene expression that gives clues to how cellular immune responses could be activated and regulated.

Post-translational modification is an intracellular process that modifies the properties of proteins to extend the range of protein function without spending energy in de novo peptide synthesis. There are many post-translational modifications, for example, phosphorylation, ubiquitination, and S-nitrosylation. S-Nitrosylation is a post-translational modification which adds nitric oxide (NO) to sulfhydryl groups at cysteine residues to form S-nitrosothiol (SNO), and is required for plant immunity and fertility. Cellular NO changes between a pool of free NO and bound SNO. During pathogen infection, nitrosative stress in plants is mainly controlled by Snitrosothiolglutathione reductase (GSNOR) via the decomposition of GSNO. GSNOR is an alcohol dehydrogenase type 3 (ADH3) which has both GSNOR and formaldehyde dehydrogenase (FDH) activities. The roles of S-nitrosylation in mammals overlap with those in plants. This conservation led us to explore the relationship between S-nitrosylation, immune response, and fertility in Drosophila melanogaster as it might prove to be a good genetic model for further analysis of the role of S-nitrosylation in animals. I have identified fdh as the likely gsnor in D. melanogaster and have knocked this out using an overlapping deficiency technique in order to observe the effect on immunity and fertility. There are two main pathways in the Drosophila innate immune response, the Toll pathway for protecting against gram-positive bacteria and fungi, and the Imd pathway against gram-negative bacteria. I have investigated the effect of removing GSNOR on sensitivity to gramnegative bacteria (Escherichia coli and Erwinia carotovora) by septic and oral infection, and to fungi (Beauveria bassiana). Susceptibility to infection by the gram negative bacteria was similar to wild-type but susceptibility to B. bassiana was increased. This increase in susceptibility correlated with reduced anti-fungal antimicrobial peptide (AMP) production after B. bassiana infection. This suggests that GSNOR might be required for the normal activity of the Toll pathway or novel Toll-independent processes. We also observed that gsnor knockout impairs fertility and development of embryos.

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Much of our current mechanistic understanding of the innate immune response in animals has grown out of empirical work in insect models, especially the fruit fly Drosophila melanogaster. The mainstream understanding of the fly immune response to bacteria has been that it exists in two parts; a cellular and a humoral response. Drosophila also harbor substantial genetic variation for antibacterial defense and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life-span and reproduction. My first study (chapter 2) aimed to understand the way in which insects invest in fighting bacterial infection. We selected for survival following systemic infection with the opportunistic pathogen, Pseudomonas aeruginosa in wild-caught D. melanogaster over 10 generations. We then examined genome wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen to specifically identify the genetic basis of the evolved immune response. In response to selection, population level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. Females usually produce a more robust immune response and are often less susceptible to infection. This female bias has been documented in humans, mice and some birds and reptiles. The most common explanation is that males increase their mating success at the cost of immune investment whilst females invest in immunity to maximize life-time egg production. In insects, however, there is growing evidence of male-biased immune performance. Using fly survival data from my first study, I found that males exhibited higher post-infection survival than females. In my second study (chapter 3), I related these differences in survival rate to changes in gene transcription. Firstly, we examined the expression of a set of immunity genes in both sexes in the presence and absence of infection. We found that male-biased survival may be partially attributable to a higher baseline expression of immune genes in males. Contrary to previous published work, we found that immune gene expression was readily induced in flies upon exposure to P. aeruginosa and that the two sexes responded in a similar manner. Lastly, we found that selection altered the expression of genes in males alone and only in the presence of infection. Together our findings suggest a superior immune response in male Drosophila. Wolbachia pipientis is an obligate intracellular bacterium capable of spreading itself through populations by manipulating the reproduction of its hosts. The Wolbachia strain wMelPop, which reduces longevity in D. melanogaster, has been introduced into the Dengue virus mosquito vector, Aedes aegypti, as a strategy to reduce disease transmission. The infecting Wolbachia halve the lifespan of the mosquito and induce numerous behavioral and physiological abnormalities including heightened locomotor activity and an age dependent reduction in blood feeding success. In my third study (chapter 4), we aimed to understand the mechanism underpinning these changes and hence chose to explore how Wolbachia may be interacting with the insect’s nervous and muscle tissue. Because wMelPop over-replicates in Drosophila, first we measured the bacterial density in A. aegypti. We found that there was a relationship between some of the feeding associated defects in the mosquito and the density of Wolbachia in the nervous and muscle tissue. Next, we carried out a series whole genome profiling experiments based on the head and muscle tissues to identify mosquito pathways affected by the microbe. Key findings that may relate to the phenotypes of interest include increased expression of genes relating to muscle contraction and synthesis of the neurotransmitter dopamine. Other novel findings that may not relate directly to the phenotypes of interest include evidence of a strong local tissue based immune response and widespread changes in expression of mosquito methylation and acetylation associated genes. We then used then amplification of inter-methylated sites (AIMS test) to obtain DNA fingerprints representative of the methylome of A. aegypti infected and uninfected with wMelPop. The presence of wMelPop caused hypermethylation in loci where they were not methylated in uninfected mosquitoes.

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.