Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

No description available.

Immunological heterogeneity is the norm in most free-living vertebrate populations, creating a diverse and challenging landscape for pathogens to replicate and transmit. This dissertation work sought to determine sources of immunological heterogeneity, as well as the consequences of this heterogeneity on pathogen fitness and evolution. A major source of heterogeneity in free-living host populations is the degree of exposure to a pathogen, as well as a host's history of exposure to a pathogen, which can create variation in standing immunity. We sought to determine the role of exposure heterogeneity on host susceptibility and immunity to secondary infection, and the influence of this heterogeneity on pathogen fitness and virulence evolution in a wild bird-bacterium system. We first determined that exposure level has a significant effect on host susceptibility to infection, severity of disease and infection, as well as immunity produced to secondary infection. Subsequently, we tested whether exposure history, and the immunity formed from this previous exposure, altered the within-host fitness advantage to virulent pathogens. We determined that previous low-level repeat exposure, which wild hosts likely encounter while foraging, produces a within-host environment which greatly favors more virulent pathogens. While within-host processes are vital for understanding and interpreting the evolutionary pressures on a pathogen, the ultimate metric of pathogen fitness is transmission. We therefore tested whether exposure history altered the transmission potential of a host and whether prior host exposure selected for more virulent pathogens. The transmission potential of a host significantly decreased with previous exposure, and high levels of previous exposure selected for more virulent pathogens. While we anticipated selection to be strongest at low-levels of exposure based on our previous results, we found here that high doses of prior exposure resulted in the strongest transmission advantage to virulence. This study also provided insight into the nuanced nature of transmission, which our results indicate is determined both by the degree of within-host pathogen replication as well as host disease severity. Together, our findings underscore the importance of exposure level and exposure history in natural populations in determining susceptibility, immunity and pathogen virulence evolution. / Doctor of Philosophy / Infectious diseases disrupt and threaten all life on this planet. To better anticipate and understand why some diseases are more harmful than others, it is vital that we consider the natural variability that exists in animal populations. A major source of variation in populations that experience disease is exposure level to a pathogen, as well as the history of exposure to a pathogen, which can alter an individual’s protection against future exposures. We sought to determine the role of exposure level on the likelihood of an individual contracting an infection, their protection from future infections, and the influence this has on pathogen evolution in a wild bird-bacterium system. We determined that exposure level has a significant effect on the likelihood an individual has of becoming infected, how severe the infection became, as well as how protected they were from future infections. Subsequently, we tested whether exposure history, and the immunity formed from previous exposure, altered the ability of pathogen strains that cause different levels of harm to replicate. We determined that previous low-level exposure, which hosts likely encounter in the wild, creates a level of immunity that favors more harmful strains of the pathogen. While understanding what happens within a host is important, the ultimate metric for predicting whether more or less harmful types of pathogens will persist is the ability of each pathogen type to spread from one host to another. We therefore tested whether exposure history altered the spread potential of a host and whether previous exposure preferentially favored the spread of more harmful pathogens. The spread potential of a host was much lower if that host had previously been exposed to the pathogen, and high levels of previous exposure in hosts only allowed the more harmful pathogen types to spread. We also found that a host’s spread potential was the result of both how much pathogen they had in their body, as well as how inflamed their affected tissues were. Together, our results indicate that natural variation in prior exposure to pathogens, which is common in all animal populations, including humans, can favor more harmful pathogen types.

Des études antérieures démontrent que les descendants de peuples européens et africains présentent des différences de susceptibilité à certaines maladies infectieuses. Ces différences suggèrent des variations interpopulationnelles de la réponse immunitaire qui résultent probablement de l’adaptation de ces individus aux pathogènes de leur environnement. Nous avons caractérisé la réponse immunitaire chez des descendants de peuples européens et africains à des infections bactériennes. Nous avons infecté des macrophages dérivés de monocytes de 30 Américains d’origine africaine (Africains) et de 31 Américains d’origine européenne (Européens) avec les pathogènes intracellulaires Listeria monocytogenes et Salmonella typhimurium pendant 4 heures, puis nous avons mesuré le niveau d’expression pangénomique des cellules infectées et non infectées par séquençage de l’ARNm. Nous avons estimé le niveau de contrôle de l’infection par les macrophages à 2, 4 et 24 heures post-infection en évaluant le taux de survie des bactéries. Nous avons observé que les Africains présentent significativement moins de bactéries intracellulaires après 4 et 24 heures que les Européens, suggérant que les Africains contrôlent mieux les infections bactériennes. Nous avons identifié des différences interpopulationnelles dans le niveau de sécrétion des cytokines et dans le niveau d’expression de certains gènes, ce qui suggère que les Africains modulent une réponse inflammatoire plus forte que les Européens. Nous avons démontré que plusieurs de ces gènes ont subi des évènements de sélection positive récents seulement chez les Européens. Notre étude a identifié plusieurs gènes candidats susceptibles d’influencer le cours des infections bactériennes chez les humains. Nos résultats indiquent que les différences dans la progression des maladies infectieuses entre les populations européennes et africaines seraient le résultat de la sélection naturelle. / Previous studies demonstrate that people of African and European ancestry differ in their susceptibility to certain infectious diseases. Differences in infection progression between these populations suggest inter-population variation in the immune response, possibly caused by adaptation to the pathogens of their historical environments. Here, we characterize the immune response of people of African and European ancestry to bacterial infections. Monocyte-derived macrophages from 30 African Americans (Africans) and 31 European Americans (Europeans) were infected with the intracellular pathogens Listeria monocytogenes and Salmonella typhimurium for 4 hours and whole genome gene expression of infected and non-infected cells was measured by RNA-sequencing. Macrophage control of bacterial infection at 2, 4 and 24 hours was assessed by culturing infected cell lysate and counting colony-forming units to approximate bacterial survival rate. We found that macrophages derived from Africans presented fewer intracellular bacteria after 4 and 24 hours than Europeans, suggesting that Africans better control intracellular bacterial infections. Concordant with this observation, we identified inter-population differences in cytokine secretion and gene expression that might explain this pattern of increased infection control in Africans. Interestingly, several of those differences indicate that Africains have a stronger pro-inflammatory response than Europeans. We show that several of these genes appear to have been subject to recent selection in the Europeans population alone. We also identify multiple candidate genes that may affect the course of infection in these populations. Overall, our findings suggest that differences in infectious disease progression observed in Africans and in Europeans may be the outcome of natural selection.

Les populations de bivalves exploités subissent régulièrement des épizooties qui affaiblissent voire déciment les stocks, et qui peuvent avoir des conséquences majeures pour l’aquaculture. Ces maladies, dues à des virus, bactéries, ou parasites, se développent particulièrement au printemps et en été. Ces périodes de l’année offrent également des conditions propices aux efflorescences de micro-algues toxiques, dont des dinoflagellés du genre Alexandrium. Ainsi, le risque de co-occurrence d’efflorescences d’Alexandrium sp. et de maladies infectieuses chez les bivalves est élevé. Or, ces micro-algues synthétisent et excrètent des neurotoxines et des composés cytotoxiques responsables d’altérations physiologiques chez les bivalves. L’objectif de cette thèse est d’évaluer les effets combinés d’une exposition à Alexandrium sp. et d’une infection par des agents pathogènes sur la physiologie des bivalves, à travers l’étude de différentes interactions tripartites bivalve – pathogène – Alexandrium sp. Les résultats de ce travail indiquent que différents profils de réponse existent en fonction des espèces impliquées dans ces interactions. Ainsi, une exposition à Alexandrium sp. peut augmenter le taux d’infection par des agents pathogènes chez des bivalves ou au contraire le diminuer. Les réponses hémocytaires associées peuvent traduire l’implication des défenses immunitaires dans ces modulations hôte-pathogène. De plus, l’exposition à des agents pathogènes peut interférer avec le processus d’accumulation de toxines algales dans les tissus des bivalves, illustrant la complexité de ces interactions. Ces résultats, associés à l’observation de lésions tissulaires chez les bivalves peuvent traduire l’altération des activités de nutrition (filtration, digestion…). Ce travail de thèse apporte une meilleure compréhension de l’implication des efflorescences toxiques dans le développement des maladies touchant les bivalves d’intérêt commercial, mais également de l’implication de l’environnement biotique des bivalves sur l’accumulation de phycotoxines réglementées. / Bivalve populations undergo regular epidemics that weaken or decimate exploited stocks and thus limit aquaculture. These diseases are caused mainly by viruses, bacteria or parasites, and occur primarily during spring and summer. This period of the year also provides favorable conditions for toxic dinoflagellate blooms, including species of the genus Alexandrium. Thus, the risk of Alexandrium sp. blooms and infectious diseases co-occurring in bivalves is high. However, these micro-algae synthesize and excrete toxins and cytotoxic compounds responsible for physiological changes in bivalves and could lead to an immuno-compromised status.The objective of this thesis is to evaluate the combined effects on bivalve physiology of exposure to the toxic dinoflagellate, Alexandrium sp., and infection by pathogens, through the study of different bivalve - pathogen - Alexandrium sp. tripartite interactions. The results of this work highlight the species-specific nature of these impacts.Thus, exposure to Alexandrium catenella reduces the herpesviruses infection in oyster Crassostrea gigas, whereas the dinoflagellate A. fundyense increases the susceptibility of C. virginica oyster to the parasite Perkinsus marinus, probably via immuno-suppression, as suggested by the partial inhibition of hemocyte responses. Additionally, the effect of a toxic algal bloom on oyster susceptibility to opportunistic diseases when exposed to a new microbial environment (simulating a transfer) was evaluated. Hemocyte responses to a changing microbial environment were suppressed by exposure to A. catenella, although no new bacterial infection was detected.Finally, exposure to pathogens or to a new microbial environment interferes with the processes by which oysters exposed to A. catenella accumulate algal toxins, illustrating the complexity of these interactions. These results provide a better understanding of the involvement of toxic algal blooms in the development of diseases affecting commercial bivalve species, but also of the involvement of the bivalve biotic environment in the accumulation of regulated toxins.

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.

Darwin a révélé au monde que les espèces vivantes ne cessent jamais d’évoluer, mais les mécanismes moléculaires de cette évolution restent le sujet de recherches intenses. La biologie systémique propose que les relations entre génotype, environnement et phénotype soient sous-tendues par un ensemble de réseaux moléculaires dynamiques au sein de la cellule, mais l’organisation de ces réseaux demeure mystérieuse. En combinant des concepts établis en biologie évolutive et systémique avec la cartographie d’interactions protéiques et l’étude des méthodologies d’annotation de génomes, j’ai développé de nouvelles approches bioinformatiques qui ont en partie dévoilé la composition et l’organisation des systèmes cellulaires de trois organismes eucaryotes : la levure de boulanger, le nématode Caenorhabditis elegans et la plante Arabidopsis thaliana. L’analyse de ces systèmes m’a conduit à proposer des hypothèses sur les principes évolutifs des systèmes biologiques. En premier lieu, je propose une théorie selon laquelle la traduction fortuite de régions intergéniques produirait des peptides sur lesquels la sélection naturelle agirait pour aboutir occasionnellement à la création de protéines de novo. De plus, je montre que l’évolution de protéines apparues par duplication de gènes est corrélée avec celle de leurs profils d’interactions. Enfin, j’ai mis en évidence des signatures de la co-évolution ancestrale hôte-pathogène dans l’organisation topologique du réseau d‘interactions entre protéines de l’hôte. Mes travaux confortent l’hypothèse que les systèmes moléculaires évoluent, eux aussi, de manière darwinienne. / Darwin exposed to the world that living species continuously evolve. Yet the molecular mechanisms of evolution remain under intense research. Systems biology proposes that dynamic molecular networks underlie relationships between genotype, environment and phenotype, but the organization of these networks is mysterious. Combining established concepts from evolutionary and systems biology with protein interaction mapping and the study of genome annotation methodologies, I have developed new bioinformatics approaches that partially unveiled the composition and organization of cellular systems for three eukaryotic organisms: the baker’s yeast, the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana. My analyses led to insights into the evolution of biological systems. First, I propose that the translation of peptides from intergenic regions could lead to de novo birth of new protein-coding genes. Second, I show that the evolution of proteins originating from gene duplications and of their physical interaction repertoires are tightly interrelated. Lastly, I uncover signatures of the ancestral host-pathogen co-evolution in the topology of a host protein interaction network. My PhD work supports the thesis that molecular systems also evolve in a Darwinian fashion.

À différentes échelles de temps, le but de ma thèse a été de comprendre l'évolution des virus entomopathogènes à travers l’étude de la diversification et de l’adaptation génomique de grands virus à ADN d’insectes. Dans un premier temps, j’ai pu estimer les âges de diversifications des baculovirus et des nudivirus, et proposer un scénario de coévolution à long terme entre ces virus et leurs hôtes insectes. Puis, me plaçant sur une échelle de temps moindre, j’ai montré que les hôtes insectes sont le facteur principal de la diversification des baculovirus, et de façon surprenante, j’ai également observé que l'environnement biotique de ces virus, c’est-à-dire les plantes hôtes des insectes, joue un rôle central dans leur évolution. Dans un second temps, des mutations ponctuelles ont pu être reliées à l’adaptation locale de populations différentiées du baculovirus SeMNPV. Enfin, l’étude de l'adaptation génomique convergente entre les entomopoxvirus et les baculovirus a mis en évidence que les transferts horizontaux de gènes sont une source importante de variabilité pour les grands virus à ADN, pour l'adaptation aux mêmes niches écologiques. Les gènes et les mécanismes identifiés dans ce travail de thèse apportent des éléments nouveaux pour comprendre comment les génomes sont façonnés par l’écologie. / At different timescales, the purpose of my PhD was to understand insect virus evolution through the study of the genomic diversification and adaptation of insect large DNA viruses. Firstly, I was able to estimate the ages of baculovirus and nudivirus diversifications, and to propose a long-term coevolutionary scenario between these viruses and their insect hosts. Then, on a narrower timescale, I showed that insect hosts are the major factor in baculovirus diversification, and surprisingly, I also observed that the virus biotic environment, i.e. insect host plants, plays a central role in their evolution. Secondly, punctual mutations have been linked to the local adaptation of differentiated populations of the baculovirus SeMNPV. Finally, the study of convergent genomic adaptation between entomopoxviruses and baculoviruses highlighted that horizontal gene transfers are an important source of variability for large DNA viruses, for the adaption to the same ecological niches. Genes and mechanisms identified in this PhD work provide new insights to understand how genomes are shaped by ecology.

Afin de déjouer les défenses immunitaires de l’hôte et créer les niches nécessaires à leur survie, les bactéries pathogènes mettent on œuvre de nombreux mécanismes ciblant les voies de signalisation de la cellule hôte. L’un de ces mécanismes repose sur la sécrétion de protéines bactériennes dans les cellules cibles afin de moduler directement leurs réseaux de signalisation. Cependant, les signaux, les senseurs et les effecteurs impliqués dans ces régulations sont encore peu ou mal connus. La détection de l’environnement dans la cellule hôte lors de l'infection est l’élément clé d’une réponse adaptée, et les systèmes de signalisation basés sur les mécanismes de phosphorylation sont indispensables à l'adaptation hôte-pathogène. L’aspect innovant de ce projet repose sur l’étude du rôle des Ser/Thr kinases et phosphatases sécrétées lors des interactions hôte-pathogène, modifiant ainsi la réponse globale de l’hôte durant l’infection. Pendant ma thèse, j’ai tout d’abord étudié le rôle d’une nouvelle protéine kinase bactérienne identifiée chez Coxiella burnetii, nommée CstK (Coxiella serine threonine Kinase). C. burnetii, l’agent étiologique de la zoonose appelée fièvre Q, modifie les défenses de la cellule hôte, permettant sa réplication dans des vacuoles spécifiques à l’intérieur de la cellule hôte. Par ailleurs, la sécrétion d’un grand nombre d’effecteurs bactériens est indispensable au détournement du phagosome par Coxiella. Nous ainsi avons démontré que cette potentielle protéine kinase, identifiée in silico dans le génome de C. burnetii, est capable de s’autophosphoryler et par conséquent possède une activité kinase. De plus, nous avons identifié différentes protéines spécifiques de la cellule hôte interagissant avec CstK à l'aide du modèle amibe Dictyostelium discoideum, un phagocyte professionnel eucaryote, permettant des études génétiques et biochimiques. Dans la deuxième partie de mon projet, je me suis intéressée au rôle d’une probable protéine sécrétée, la tyrosine phosphatase PtpA, durant l’infection par Staphylococcus aureus. Bien connue dans les hôpitaux, où elle est responsable de nombreuses maladies nosocomiales, cette bactérie possède un grand nombre de facteurs de virulence, responsables d’infections variées, et l’apparition exponentielle de souches multi-résistantes en font un problème majeur. Ce pathogène est capable d’envahir et de persister dans un grand nombre de types cellulaires différents chez l’Homme, en sécrétant des protéines effectrices qui vont moduler les réponses cellulaires. Nous avons démontré que PtpA était sécrétée durant la phase de croissance bactérienne, et pu déterminer que PtpA possédait une activité tyrosine phosphatase, régulée par la tyrosine kinase CapA1B2 de S. aureus. Enfin, en utilisant le modèle D. discoideum, nous avons pu identifier des protéines de l’hôte qui interagissent avec PtpA, mais leur rôle dans l’infection n’est pas encore connu. / Bacterial pathogens have developed diverse strategies towards host signalling pathways, in order to subvert the immune response and/or create permissive niches for their survival. One such strategy is based on the secretion of bacterial signalling proteins into the target host cells, thereby directly modulating the status of host signalling networks. Because the mechanisms involved are largely intractable to most in vivo analyses, very little is known about the signals, sensors, and effectors mediating these adaptations. Sensing the host environment is a key component to execute appropriate developmental programs, and the eukaryotic-like phosphosignaling systems in prokaryotes are emerging as equally important regulatory systems as the well-known eukaryotic systems, but the study of their functions is still in its infancy. The innovative aspect of this project resides in the study of the emerging role of secreted Ser/Thr kinases and phosphatases in the control of host-pathogen interactions thus modifying the global host response during infection. During my thesis, I first investigated the role of a novel bacterial protein kinase identified in Coxiella burnetii that we named CstK (Coxiella serine threonine Kinase). C. burnetii, the etiological agent of the emerging zoonosis Q fever, subverts host cell defenses, permitting its intracellular replication in specialized vacuoles within host cells. Secretion of a large number of bacterial effectors into host cell is absolutely required for rerouting the Coxiella phagosome. We demonstrated that this putative protein kinase identified by in silico analysis of the C. burnetii genome is able to autophosphorylate and undergoes in vitro phosphorylation. Moreover, we identified specific host cell proteins interacting with CstK, by the use of the model amoeba Dictyostelium discoideum, an eukaryotic professional phagocyte amenable to genetic and biochemical studies. In the second part of my project, I was interested in the role of a putative secreted protein tyrosine phosphatase (PtpA) during Staphylococcus aureus infection. Well-known in hospital-acquired diseases, this bacteria produces multiple virulence factors that lead to various severe diseases, and the increase of multi-resistant strains is a major concern. This pathogen has the ability to invade and persist in a number of different human host cell types, secreting effector proteins to modulate cellular responses. Here we demonstrated that PtpA is secreted during the bacterial growth. We also determined that PtpA presents a tyrosine phosphatase activity that is regulated by the tyrosine protein kinase CapA1B2 of S. aureus. At last, using the D. discoideum model, we identified some host proteins that interact with PtpA, but their link with infection still remain to be studied.