Дисертації з теми "Host - microbial interaction"

Most plant pathogens are specialists where only few plant species are susceptible, while all other plants are resistant. Unraveling the mechanisms behind this can thus provide valuable information for breeding or engineering crops with durable disease resistance. A group of Pseudomonas syringae strains with different host ranges while still closely related were thus chosen for comparative study. We confirmed their close phylogenetic relationship. We found evidence supporting that these strains recombined during evolution. The Arabidopsis thaliana and tomato pathogen P. syringae pv. tomato (Pto) DC3000 was found to be an atypical tomato strain, distinct from the typical Pto strains commonly isolated in the field that do not cause disease in A. thaliana, such as Pto T1. Comparing A. thaliana defense responses to DC3000 and T1, we found that T1 is eliciting stronger responses than DC3000. T1 is likely lacking Type III effector genes necessary to suppress plant defense. To test this, we sequenced the genomes of strains that cause and do not cause disease in A. thaliana. Comparative genomics revealed candidate effector genes responsible for this host range difference. Effector genes conserved in strains pathogenic in A. thaliana were expressed in T1 to test whether they would allow T1 to growth better in A. thaliana. Surprisingly, most of them reduced T1 growth. One of the effectors, HopM1, was of particular interest because it is disrupted in typical Pto strains. Although HopM1 has known virulence function in A. thaliana, HopM1 reduced T1 growth in both A. thaliana and tomato. HopM1 also increased the number of bacterial specks but reduced their average size in tomato. Our data suggest that HopM1 can trigger defenses in these plants. Additionally, transgenic detritivore Pseudomonas fluorescens that can secrete HopM1 shows dramatically increased growth in planta. The importance of genetic background of the pathogen for the functions of individual effectors is discussed. T1 cannot be manipulated to become an A. thaliana pathogen by deleting or adding individual genes. We now have a list of genes that can be studied in the future for the molecular basis of host range determination.
Ph. D.

Obesity has emerged as one of the major global socioeconomic healthcare burdens at present. Bariatric surgery, especially Roux-en-Y gastric bypass (RYGB) has been largely utilised to treat individual suffering from morbid obesity. In the current study, a hyperinsulinemic obese Zucker rat model was employed to study two different weight loss approaches, RYGB and caloric restriction. To understand the host metabolic-microbial cross-talk, the two major analytical platforms including nuclear magnetic resonance (NMR) spectroscopy and 16S rRNA gene Illumina MiSeq sequencing were used in companion with multivariate statistical analysis to extract useful information from data with high complexity. The aim of this study was to 1) characterise the genotype-associated metabolic and microbial fingerprints; 2) investigate the dynamic changes in biofluids from RYGB-treated or caloric restriction-treated obese Zucker rats; 3) investigate genotype-related, RYGBinduced or caloric restriction-induced metabolic profiles and microbial shifts of the luminal contents; and 4) investigate the statistical correlation between metabolites and gut microbiota following either of the weight loss treatment. Metabolic observations of portal vein and peripheral blood plasma profiles in both obese and lean Zucker rats indicated the phenotype-independent absorption of short-chain fatty acids (SCFAs), choline and trimethylamine (TMA). However, phenotype-specific urinary host-microbial co-metabolites were revealed, suggesting distinct gut microbial metabolic activities in lean and obese Zucker rats. Furthermore, metabolic alterations induced by the RYGB surgery included the enhanced production of neuroactive metabolites, branchedchain amino acid (BCAA) catabolism, aromatic amino acid metabolism with lower lipogenesis and SCFA production. Even though caloric restriction demonstrated some health benefit-related biochemical and microbial markers, its effectiveness as a resolution for metabolic syndrome, especially type-2 diabetes mellitus has not been observed in this study in spite of the significant weight reduction.

Interactions of the Streptococcus anginosus group (SAG) with the extracellular matrix (ECM) and subsequent effects on host connective tissue and bacterial cells is proposed to be important in the initial establishment of dentoalveolar infections. The aim of this thesis was to investigate the interactions of the SAG with small leucine-rich proteoglycans (SLRPs), decorin and biglycan, from periodontal tissues and recombinant decorin and biglycan. Additional aims were to investigate the subsequent effects of the SAG on cellular components within host tissues and the influence of ECM components on bacterial phenotype. Using surface plasmon resonance, this study indicated that both commensal and pathogenic strains of the SAG interact with SLRPs but there was preferential binding toward the dermatan sulphate-substituted decorin and biglycan present in gingival tissues. In addition, commensal and pathogenic SAG isolates were shown to influence periodontal ligament (PDL) and endothelial ECM responses, including cell growth and PG synthesis. The effects on the different cell types illustrates the complexity of disease caused by the SAG, and helps to highlight the complicated roles decorin and biglycan play within the ECM. This study has also shown that potential virulence factors of the SAG, including degradative enzymes, are up-regulated following exposure to ECM components derived from PDL cells, potentially causing destruction of the host ECM and possibly inhibiting remodelling of the ECM. Overall, this thesis provides valuable information on host-SAG interactions, highlighting complex roles for SAG and SLRPs in the establishment of periapical abscesses.

Les interactions entre les membres d'une communauté microbienne peuvent être un moyen d'adaptation dans l'environnement. Parmi les nombreuses interactions qui ont lieu dans un écosystème et qui joue un rôle majeur sur la diversité et la dynamique des populations microbiennes est celui des virus procaryotes et leurs hôtes. Les virus peuvent également arbitrer le transfert de matériel génétique entre les procaryotes (transduction), qui pourrait être un mécanisme d'adaptation rapide. Afin de déterminer l'impact potentiel des virus et la transduction, nous avons besoin d'une meilleure compréhension de la dynamique des interactions entre virus et leurs hôtes dans l'environnement. Les données sur les virus de l'environnement sont rares, et les méthodes pour le suivi de leurs interactions avec les procaryotes sont nécessaires. Clustered regularly interspaced short palindromic repeats (CRISPRs), qui contiennent des séquences virales dans les génomes bactériens, pourraient aider à documenter l'histoire des interactions virus-hôte dans l'environnement. Ainsi, cette thèse vise à explorer les interactions virus-hôte dans un environnement donné à travers du séquences CRISPR.Les virus de la cryosphère sont considérés comme abondantes, très actif et avec de larges gammes d'hôtes. Ces caractéristiques pourraient faire de la transduction virale, un facteur clé pour l’adaptation microbienne dans ces environnements. Des métagénomes publics créés à partir des environnements avec une gamme de températures différents ont été examinés. De cette manière, certaines dynamiques d'interactions virus-hôte se sont révélées comme ayant une corrélation avec la température. Un flux de travail a ensuite été développé pour créer un réseau reliant les virus et leurs hôtes en utilisant des séquences CRISPR obtenus à partir de données métagénomiques de la glace des glaciers et du sol de l'Arctique. La création de réseaux d'infection à traves du CRISPRs a fourni une nouvelle perspective sur les interactions virus-hôte. En outre, nous avons cherché des événements de transduction dans les données métagénomiques par la recherche de séquences virales contenant de l'ADN microbien. L’analyse indiquée que les bactériophages du Ralstonia pourraient être des agents de transduction dans la glace des glaciers de l'Arctique
Interactions between the members of a microbial community can be a means of adaptation in the environment. Among the many interactions that take place in an ecosystem and have been seen to play a major role on microbial diversity and population dynamics is that of prokaryotic viruses and their hosts. Viruses can also mediate the transfer of genetic material between prokaryotes (transduction), which could be a mechanism for rapid adaptation. In order to determine the potential impact of viruses and transduction, we need a better understanding of the dynamics of interactions between viruses and their hosts in the environment. Data on environmental viruses are scarce, and methods for tracking their interactions with prokaryotes are needed. Clustered regularly interspaced short palindromic repeats (CRISPRs), which contain viral sequences in bacterial genomes, might help document the history of virus-host interactions in the environment. Thus, this thesis aimed to explore virus-host interactions in a given environment through CRISPRs. Viruses in the cryosphere have been seen to be abundant, highly active and with broad host ranges. These characteristics could make viral transduction a key driver of adaptation in these environments. Public metagenomes created from environments over a range of temperatures were examined through sequence and CRISPR analysis. In this fashion, certain virus-host interaction dynamics were found to have a correlation with temperature. A workflow was then developed to create a network linking viruses and their hosts using CRISPR sequences obtained from metagenomic data from Arctic glacial ice and soil. The creation of CRISPR-based infection networks provided a new perspective on virus-host interactions in glacial ice. Moreover, we searched for transduction events in metagenomic data by looking for viral sequences containing microbial DNA. Further analysis of the viral sequences in the CRISPRs indicated that Ralstonia phages might be agents of transduction in Arctic glacial ice

Plants and animals are characterized by intimate, enduring, often indispensable, and always complex associations with microbes. Therefore, it should come as no surprise that when the genome of a eukaryote is sequenced, a medley of bacterial sequences are produced as well. These sequences can be highly informative about the interactions between the eukaryote and its bacterial cohorts; unfortunately, they often comprise a vanishingly small constituent within a heterogeneous mixture of microbial and host sequences. Genomic analyses typically avoid the bacterial sequences in order to obtain a genome sequence for the host. Metagenomic analysis typically avoid the host sequences in order to analyze community composition and functional diversity of the bacterial component. This dissertation describes the development of a novel approach at the intersection of genomics and metagenomics, aimed at the extraction and characterization of bacterial sequences from heterogeneous sequence data using phylogenomic and bioinformatic tools. To achieve this objective, three interoperable workflows were constructed as modular computational pipelines, with built-in checkpoints for periodic interpretation and refinement. The MetaMiner workflow uses 16S small subunit rDNA analysis to enable the systematic discovery and classification of bacteria associated with a host genome sequencing project. Using this information, the ReadMiner workflow comprehensively extracts, assembles, and characterizes sequences that belong to a target microbe. Finally, AssemblySifter examines the genes and scaffolds of the eukaryotic genome for sequences associated with the target microbe. The combined information from these three workflows is used to systemically characterize a bacterial target of interest, including robust estimation of its phylogeny, assessment of its signature profile, and determination of its relationship to the associated eukaryote. This dissertation presents the development of the described methodology and its application to three eukaryotic genome projects. In the first study, the genomic sequences of a single, known endosymbiont was extracted from the genome sequencing data of its host. In the second study, a highly divergent endosymbiont was characterized from the assembled genome of its host. In the third study, genome sequences from a novel bacterium were extracted from both the raw sequencing data and assembled genome of a eukaryote that contained significant amounts of sequence from multiple competing bacteria. Taken together, these results demonstrate the usefulness of the described approach in singularly disparate situations, and strongly argue for a sophisticated, multifaceted, supervised approach to the characterization of host-associated microbes and their interactions.
Ph. D.

Les virus du sol sont capables d'influencer la structure de la communauté microbienne et le fonctionnement de l'écosystème en affectant l'abondance des cellules hôtes par lyse et par leurs caractéristiques à transférer des gènes entre les hôtes. Bien que notre compréhension sur la diversité et la fonction virales s’améliore, la connaissance des interactions hôte-virus dans le sol reste limitée. Pour mieux comprendre les interactions hôte-virus, un gradient du sol à long terme manipulé par le pH dans lequel la communauté microbienne change à travers, a été étudié. Les principaux objectifs de cette thèse ont consisté à (1) déterminer l'influence de la structure de la communauté microbienne et du pH du soil sur les virus par séquençage d’ADN haut-débit (Chapitre II), (2) déterminer l’infectivité des populations virales à partir de niches de sol co-localisées et non co-localisées avec son hôte grâce à une approche de l’essai de plaque combinée à un séquençage hybride (Chapitre III), (3) identifier les populations virales infectant des groupes fonctionnels microbiens spécifiques du sol, en particulier les méthanotrophes (Chapitre IV) et les nitrifiants (Chapitre V ), à l’aide d’une sonde isotopique stable à l'ADN. Nos premiers résultats ont montré que la structure de la communauté virale change selon le pH du sol, ce qui souligne que la communauté virale est étroitement liée aux populations hôtes. L’analyse de CRISPR systèmes a révélé des interactions virus-hôte dynamiques, avec le nombre et la taille des CRISPR systèmes distincts selon le soil à pH contrasté. L’analyse taxonomique de cette CRISPR systèmes suggère que les virus jouent un rôle essentiel dans la composition et de la fonction de la communauté procaryote du sol. Les processus co-évolutionnaires entre l'hôte (le système de restriction-modification et le CRISPR-Cas système) et les populations virales co-localisées (la mutation d’une séquence espaceur « spacer » et la méthyltransférase codée par le virus) fournissent des preuves de l'adaptation locale et que les interactions virus-hôte jouent un rôle important dans la susceptibilité d'un hôte à l'infection et par conséquent la régulation des populations bactériennes du sol. L'ADN-SIP-métagénomique ciblant des groupes fonctionnels microbiens spécifiques a permis l’analyse des populations hôte-virus individuelles. Le suivi du flux de carbone à travers les populations procaryotes et virales a révélé des interactions actives entre les virus et les hôtes méthanotrophes et nitrifiants, et les préférences de niches de pH du sol. Notre étude a montré une preuve de transfert horizontal de gènes et des gènes métaboliques auxiliaires codés par le virus, indiquant que les virus contribuent de manière significative aux cycles biogéochimiques dans le sol, tels que le carbone (les gènes qui codent pour les familles GH, peptidases et la sous-unité C de méthane monooxygénase particulaire), et l'azote (les gènes qui codent pour la nitrogénase et le cytochrome cd1-nitrite réductase). Dans l’ensemble, ces résultats ont montré que les virus du sol sont des régulateurs importants des communautés microbiennes par la lyse spécifique de l’hôte et des interactions dynamiques virus-hôte
Soil viruses have potential to influence microbial community structure and subsequent ecosystem functioning by directly affecting the abundance of host cells by lysis and through their ability to transfer genes between hosts. Although our understanding of soil viral diversity and functioning has increased, the role of viruses and their interactions with prokaryotes in soil is limited. To gain a better understanding of virus-host interactions in soil, a long-term pH-manipulated soil gradient, which microbial community structure changes across, was investigated. The main objectives of this thesis were to 1) determine the influence of microbial community structure and soil pH on viruses using metagenomics and viromics (Chapter II), 2) determine the infectivity of soil viral populations from co-localized and foreign pH soil niches using a plaque assay approach combined with hybrid metagenomics sequencing (Chapter III) and 3) identify virus populations infecting specific soil microbial functional groups, specifically methanotrophs (Chapter IV) and nitrifiers (Chapter V), using DNA stable isotope probing combined with metagenomic deep sequencing. Viral community structure was found to change with soil pH, demonstrating that viral communities are tightly linked to host populations, but also may have narrow host ranges. Analysis of clustered regularly interspaced short palindromic repeats (CRISPR) arrays revealed dynamic virus-host interactions, with the number and size of CRISPR arrays distinct across contrasting pH soil. Profiling of the host-virus linkages between soil pH, suggests that viruses play a critical role in shaping the composition and function of the soil prokaryotic community. Surprisingly, greater infectivity of a host bacterium by virus populations was found when viruses and host bacterium were not co-localized in the same pH soil. Coevolutionary processes between the host and virus populations, such as restriction modification/virus-encoded methyltransferase and CRISPR-Cas system/spacer mutation, provide evidence for local adaptation, and that virus-bacterial host interactions play an integral part in the susceptibility of a host to infection and consequently in the regulation of soil bacterial populations. Targeting specific microbial functional groups via stable isotope probing allowed analysis of individual host-virus populations. Tracking carbon flow through prokaryotic and viral populations revealed active interactions between viruses and methanotroph and nitrifier hosts, and soil pH niche preferences. Evidence of horizontal gene transfer and virus-encoded auxiliary metabolic genes, such as glycoside hydrolase families, peptidases, particulate methane monooxygenase subunit C (pmoC), nitrogenase (nifH) and cytochrome cd1-nitrite reductase, supports that viruses are significant contributors to host functioning and carbon and nitrogen cycling in soil. Overall, this work demonstrated that soil viruses are important regulators of microbial communities through specific host lysis and dynamic virus-host interactions

Les amibes libres sont des microorganismes unicellulaires eucaryotes dont l'écologie au sein des réseaux d'eau potable est mal connue. Les amibes libres représentent un enjeu de santé publique, du fait de leur capacité à favoriser la présence de bactéries potentiellement pathogènes, parmi lesquelles des mycobactéries.Une campagne de prélèvement menée sur le réseau d'eau potable de Paris a permis d'évaluer la diversité des amibes libres et de leur microbiome bactérien, par pyroséquençage ciblant les gènes ribosomaux (16S et 18S). Ces analyses ont suggéré la prédominance des genres Acanthamoeba, Vermamoeba, Echinamoeba et Protacanthamoeba. Le microbiome des amibes a révélé une grande diversité bactérienne, dominée par Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas et Pseudoxanthomonas. L'intégration des paramètres physicochimiques a permis de suggérer l'importance de l'origine de l'eau, la température, le pH et la concentration en chlore dans la dynamique des populations amibiennes. Une endosymbiose originale entre V. vermiformis et des bactéries du phylum TM6 a également été mise en évidence.Les amibes ont été fréquemment co-isolées avec des mycobactéries dans le réseau, principalement les espèces M. llatzerense et M. chelonae. Des expériences d'infection chez A. castellanii ont permis d'observer la capacité de ces mycobactéries à survivre et croitre en présence d'amibes. Par génomique comparative et analyses transcriptomiques, plusieurs facteurs de virulence, conservés entre M. llatzerense, M. chelonae et M. tuberculosis, ont été identifiés et sont surexprimés au cours de l'infection. Ces données suggérent leur implication dans la résistance à la prédation amibienne.L'ensemble de ces travaux a permis d'améliorer la connaissance des populations amibiennes et de leur microbiome au sein du réseau d'eau potable, apportant des éléments supplémentaires concernant leur implication dans la survie et la persistance des mycobactéries
Free-living amoebae are unicellular eukaryotes whose ecology in drinking water networks remains poorly understood. They may represent a public health concern, because of their ability to favour the presence of potentially pathogenic bacteria, among which are mycobacteria.A sampling scheme based on Paris drinking water network allowed identifying the diversity of both freeliving amoebae and their bacterial microbiome, using ribosomal RNA targeted pyrosequencing. These analyses indicated the major presence of Acanthamoeba, Vermamoeba, Echinamoeba and Protacanthamoeba genera. The microbiome was highly diverse and dominated by Pseudomonas, Stenotrophomonas, Bradyrhizobium, Sphingomonas and Pseudoxanthomonas. The coupling of physicochemical parameters to this analysis allowed underlining the importance of water origin, temperature, pH and chlorine concentration in shaping amoebal populations. Also an original endosymbiosis between V. vermiformis and a bacterium of the TM6 phylum was described. Free-living amoebae were frequently co-isolated with mycobacteria in the water network, mainly M. llatzerense and M. chelonae species. Infection experiments on A. castellanii illustrated the capacity of these species to resist and grow in presence of amoebae. Through genomics and transcriptomics approaches, several virulence factors, conserved between M. llatzerense, M. chelonae and M. tuberculosis were identified, and found to be upregulated during infection experiments. These results suggest their involvement in mycobacterial resistance to amoebal predation.Altogether, this work helped to better understand the ecology of free-living amoebae and their microbiome in drinking water networks, as well as the role of free-living amoebae in the survival and persistence of mycobacteria in such environments

Il est désormais admis que le microbiote intestinal joue un rôle prépondérant dans la santé de son hôte humain. Le co-métabolisme hôte-microbiote produit un très grand nombre de biomolécules intégrées au sein d’axes métaboliques complexes. De ce fait, le microbiote intestinal est considéré comme un organe endocrine à part entière. Bien que de nombreuses études se soient attachées à la caractérisation fonctionnelle spécifique de certaines molécules, les études envisageant plus globalement les relations métaboliques entre l’hôte et son microbiote intestinal restent rares. Parmi les nombreux facteurs influençant la composition et l’activité métabolique du microbiote intestinal, l’alimentation joue un rôle prépondérant. Toutefois, les relations entre l’alimentation usuelle et le microbiote intestinal n’ont pas été complètement élucidées. La compréhension des facteurs modulant le microbiote intestinal est un enjeu majeur des recherches actuelles, car des liens entre le microbiote intestinal et de nombreuses pathologies (troubles gastro-intestinaux, cardio-métaboliques, neuropsychiatriques, etc.) ont été suggérés. Dans ce contexte, nous avons utilisé une approche épidémiologique pour caractériser les associations entre la composition du microbiote intestinal d’une part et le métabolisme systémique et l’alimentation usuelle de l’hôte d’autre part, au sein de la population Milieu Intérieur (N=1 000). Enfin, dans la cohorte prospective NutriNet-Santé (N≈160 000), nous avons analysé les associations entre la consommation de fibres d’une part et le risque de maladies chroniques et le microbiote intestinal d’autre part.Nos résultats décrivent des associations spécifiques entre les caractéristiques du microbiote intestinal et certaines composantes du métabolisme de l’hôte, et suggèrent un rôle important de l’axe intestin-rein. De plus, des associations inverses entre la diversité du microbiote intestinal et la consommation d’aliments caractéristiques du régime occidental ont été détectées. Enfin, nos travaux confirment que la consommation de fibres est associée à une réduction du risque de maladies chroniques, dans un contexte où un nombre croissant d’études suggère une implication du microbiote intestinal dans de tels effets
It is now admitted that the gut microbiota plays a key role in the health status of its human host. It is indeed fully recognized as an endocrine organ producing biologically active molecules which are integrated within human metabolism. However, comprehensive studies characterizing host-gut microbial metabolic relationships remain scarce. Numerous factors have been shown to exert a modulatory impact on the gut microbiota. Notably, diet is supposed to be a major driver, but the relationships between usual diet and the gut microbiota are not fully elucidated yet. Furthermore, many studies have suggested the implication of the gut microbiota in a wide range of disease states, such as gastrointestinal, cardio-metabolic, neuropsychiatric, etc. disorders. Thus, understanding the factors influencing the gut microbiota constitutes an active area of research. In this context, we adopted an epidemiological approach to investigate one of the largest population-based samples so far (Milieu Intérieur population, N=1,000). We notably assessed the associations between gut microbiota composition on one hand and the systemic metabolism and the usual diet of the host on the other. Finally, in the NutriNet-Santé cohort (N≈160,000), we investigated the associations between the intake of dietary fibers and the risk of a variety of chronic diseases, and described how dietary fibers are associated with the gut microbiota.Overall, our results suggest that gut bacterial features are specifically associated with certain components of the systemic metabolism of the host, and we hypothesize a substantial role of the gut-kidney axis. Besides, negative associations between food items for which a limited consumption is generally recommended (i.e. processed foods) and gut microbial features were detected. Additionally, we confirm robust inverse associations between the consumption of dietary fibers and several major chronic diseases. Mounting evidence suggests that such effects could be mediated by the gut microbiota

Microbial succession, composition and ecological distribution within the gastro-intestinal tract are critical areas of study since commensal bacteria have been shown to affect animal health and development. A series of experiments were conducted to determine whether altered microbial succession in neonatal animals would modulate the development and health of pigs later in life. An initial experiment in conventional pigs was conducted to establish the early postnatal microbial succession profile and to identify early colonizing bacterial species. Culture-independent analysis of digesta and mucosal microbiota showed distinct variation between the proximal and distal gastro-intestinal tract (GIT) indicating that fecal or distal gut profiles cannot be used to predict succession in the upper GIT. Temporally, Clostridium spp. were found to be most prevalent in the GIT microbiota of the neonatal pig up to 0.5 d of age, accompanied by a high abundance of Escherichia and Shigella spp. These genera were transiently displaced by Streptococcus spp. followed by a preponderance of Lactobacillus spp. between 3 and 20 d of age. Subsequently, a “snatch-farrow” model was employed to modulate early postnatal microbial succession and investigate the effects on postweaning microbial composition. Pigs were collected into sterile towels directly from the vaginal canal and transferred to a sterile isolator environment for the first 4 days. Pigs were either inoculated with sow feces or not at 1 d of age resulting in significant differences in fecal microbial profile at 4 days of age, prior to removal from isolators. Analysis using terminal restriction fragment length polymorphisms (TRFLP) of intestinal microbiota at 28 d of age did not show significant clustering or variation in diversity indices for either group during the 4-d postnatal isolator phase. However, enumeration of selected taxa using quantitative PCR did indicate significant treatment differences in postweaning microbiota. Despite these results, this approach was rejected for further use as the protocol provided only moderate control of early postnatal colonization and variation and unpredictability of the timing of natural farrowing contributed to significant litter effects. Finally, a gnotobiotic monoassociation model was used investigate the effects of modulating early postnatal microbial succession on postweaning physiology, microbial composition and mucosal gene expression. Twenty-four cesarean-section derived piglets were monoassociated for the first 4 days of life with either L. mucosae (L), S. infantarius (S), C. perfringens (C) or E. coli (E). Pigs from treatments E and L animals showed the highest growth rate during the conventional rearing period (7-28 d of age). Monoassociation with different bacterial species during the first 4 d of life resulted in significant changes in postweaning microbial composition in small intestine and colon as assessed by quantitative PCR, although TRFLP did not identify unique clustering by treatment or variation in diversity. L. mucosae was the only inoculant species with significant variation, with a reduction in the colonic mucosa at 28 days of age. Monoassociation with L. mucosae was also associated with increased nutrition related gene expression in small intestine. Pigs monoassociated with E. coli had low expression of microbial sensing (TLR2 and 4), NFkappaB complex genes and mucins at 28 d of age. This study clearly showed that controlled early microbial succession in neonatal pigs altered post-weaning commensal microbiota composition, postweaning physiology and host gene expression in small and large intestine. The findings suggest the importance of peri-natal management and feeding strategies in promoting postweaning health and performance.

Chronic inflammatory disorder is one of the leading causes of death worldwide. Association between pathogenic infection and inflammation governs tissue homeostasis, which relies on extensive crosstalk among signaling networks. Interestingly, vitamin A deficiency is known to effectuate several aberrations in innate and adaptive immune responses and play a central role in governing fate of diseases. In this regard, our current study involves exploration of regulatory role of vitamin A metabolite – Retinoic Acid (RA) in modulating host-microbial interaction to mitigate inflammatory disorders. In this context, thesis delineates regulatory effects of RA during C. albicans induced delayed wound healing and pathogenesis. In this regard, our observation with RA treatment showed significant rescue of C. albicans induced wound healing delay. Data was corroborated with skewed anti-inflammatory response, as proinflammatory genes (Cxcl1, Tnf and IL-6) were suppressed and excessive activation of anti-inflammatory M2 macrophages genes (Retnl, Cxc3r1 and Ucp1) was observed. BMP signaling which was established previously for its significant contribution to wound delay, was abrogated upon treatment with RA. Mechanistically, RA treatment showed enhanced phagocytic uptake of C. albicans through targeted inhibition of an actin-binding protein, cofilin. It governs such effect through inhibition of C.albicans induced BMP signaling which was further found to crosstalk with down-stream effectors of Rho/Rac GTPases. Thus, current work explores the beneficial effects of RA in orchestrating tissue homeostasis. Septic arthritis is a joint inflammatory disorder caused by bacterial invasion to cartilages which eventually disseminate to the bones. The disease progression is driven by macrophage infiltration into the associated synovial joints. However, the factors mediating such inflammatory dysfunction remain obscure. In our current study, we have uncovered a critical role for Aurora kinase A driven activation of mTOR-WNT signalling axis, which acts as a key regulator of pro-inflammatory cytokine expression in joint synovium. With the limited treatment modalities, utilization of vitamins therapy was explored as a potential interface for the development of efficacious adjuncts against pathogen-driven responses. Interestingly, we found that prophylactic treatment of RA, rescued inflammatory damage and helped in preservation of articular cartilage and bone architecture. Mechanistically, adapalene treatment inhibited WNT signalling with a concomitant activation of HIPPO signalling, generating alternatively activated macrophages. Along these lines, silencing of MST1/2 attenuated the ability of adapalene to induce pro-resolving mediators. This resulted in significant decrease of synovial inflammation and septic arthritis development. Thus, the current work uncovered the potential therapeutic effects of vitamin A metabolite contributing to suppression of hyper-inflammation and tissue repair. A possible correlation between activity of RA and disease burden in host was observed. On one side potential contribution of Hippo signalling was found to govern anti-inflammatory effects of RA, which suppressed S.aureus induced septic arthritis. On the other hand, a significant inhibition of BMP signalling as attributed to RA treatment resulted in enhanced wound healing during C.albicans infection.

The epithelial cell layer that lines the intestine creates a barrier, largely mediated by the tight junction (TJ) apparatus, which serves as a first line of protection from the contents in the lumen containing an enormous number of microbes. Cellular microbiology, the study of microbial-host interactions, is used to describe mechanisms that play a role in the way epithelial cells regulate barrier properties in the context of bacterial colonization. The research in this thesis had three aims: (1) to characterize the effects of candidate pathogenic bacteria on the epithelial barrier, (2) to determine if a beneficial microbe (a probiotic bacterium) could ameliorate the deleterious effects of a pathogenic infection on this barrier, and (3) to extend the investigation of probiotic mechanisms in the context of pro-inflammatory cytokine-mediated barrier dysfunction. In the first part of this thesis, two undercharacterized bacterial species purported to cause diarrheal illness, Escherichia albertii and Hafnia alvei, were employed in a polarized epithelial infection model with outcome measures including transepithelial electrical resistance (TER), macromolecular permeability, TJ protein immunofluorescence staining, and immunoblotting. A well characterized pathogen, enterohemorrhagic Escherichia coli (EHEC), serotype O157:H7, was used as a positive control to demonstrate deleterious effects on TJs. All of the bacteria tested decreased TER, but the effects on TJs and TJ protein expression were specific to the bacterial strain tested and the epithelial model cell line used. The second component of this thesis investigated how probiotics confer beneficial effects on epithelial barrier function. A probiotic bacterium, Lactobacillus rhamnosus GG (LGG), was employed to effectively block EHEC O157:H7 adherence to epithelial cells and prevent the ability of the pathogen to induce characteristic attaching-effacing lesions on epithelial cell surfaces. LGG ameliorated the pathogenic effects on barrier function normally induced by EHEC O157:H7, including prevention of decreased TER, increased permeability to a dextran probe, and rearrangement of tight junction architecture. The third section elucidated the role of LGG in the prevention of barrier disruption due to pro-inflammatory cytokine stimuli (IFN-γ and TNF-α). Using a polarized epithelial (Caco-2bbe) cell model, LGG treatment largely prevented cytokine-induced decreases in TER and TJ disruption. Furthermore, LGG suppressed the secretion of the chemokines interleukin-8 (CXCL-8) and eotaxin-1 (CCL-11), and the activation of NF-κB. Preliminary experimentation demonstrated a role for mitogen-activated protein kinases, with pharmacologic inhibition of extracellular signal related kinase (ERK-1/2) abolishing the protective effects of LGG. Taken together, the findings presented in this thesis demonstrate how cellular microbiology models can be used to study host-microbial interactions, giving insight as to how the intestinal epithelium regulates barrier function; characterizing enteropathogenic candidates, and the diversity in responses to these bacteria that is dependent on both the bacterial strain and the epithelial cell line tested; and elucidating the mechanisms of probiotic action to reduce the deleterious effects of infection and inflammation.

Mycobacterium are important human pathogens and their strength lies in establishing acute infections, latent infections as well as co-existing with other dreadful infectious agents like HIV. The success of mycobacterium infection often relies in its ability to evade immune-surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immune regulatory molecules. In this scenario, the role of pattern recognition receptors (PRRs) in orchestrating host immune responses assumes central importance. Of the PRRs, the Toll-like receptors (TLRs) or intracellular surveillance receptors such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) govern key immune-surveillance mechanisms in recognition as well as control of mycobacterial or viral infections. The first part of this study illustrates the role of SHH signaling in macrophage induced neutrophil recruitment during mycobacterial infections. The present investigation demonstrates that, in response to mycobacterium infection, macrophages displayed robust activation of TLR2 dependent SHH signaling. By utilizing the well-documented experimental air pouch model, we show that the ability of pathogenic mycobacterium infected macrophages to recruit polymorph nuclear leukocytes (PMNs) like neutrophils to the infected site was dependent on SHH signaling. The activated SHH signaling differentially regulated the expression of proteolytic enzymes, MMP-9 and MMP-12 that would contribute to PMN migration. Interestingly, SHH-responsive krüppel-like family (KLF) of transcription factors, KLF4 and KLF5 were found to modulate these chemokine effectors to regulate neutrophil recruitment. Subsequent chapters describe novel functions of SHH signaling during RIG-I mediated anti-viral immunity and RIG-I mediated modulation of TLR2 anti-inflammatory signature in mycobacteria infected macrophages. In this perspective, we demonstrate that RIG-I ligand robustly induces the activation of SHH signaling via the phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. Furthermore, we show that the sustained inhibition of PKA-GSK-3β-SUFU negative regulatory axis upon RIG-I engagement with 5'3pRNA is critical for the activation of SHH signaling. Gain or loss of function studies implicate the necessity of RIG-I triggered MAVS-TBK1 canonical axis in the inhibition of PKA-GSK-3β-SUFU negative regulatory axis that contributes to SHH signaling activation. The RIG-I activated SHH signaling drives the production of anti-viral type 1 interferons leading to the inhibition Japanese encephalitis virus (JEV) replication. Further, RIG-I-mediated anti-viral type 1 interferon production and subsequent control of viral replication suggested the involvement of two transcriptional factors, IRF3 and YY1 in the response along a SHH axis. Further, mounting evidence clearly depicts a significant cross talk among the molecular events initiated by given TLRs and RLRs like RIG-I. Clearly, these studies present an interesting challenge in delineating the events during polymicrobial infection of host immune cells like macrophages or DCs. Altogether, our results improve our understanding of mycobacteria associated confections’ and may add significantly to the current knowledge of the delicate balance that determines a successful mycobacterial infection.

Mycobacterium are important human pathogens and their strength lies in establishing acute infections, latent infections as well as co-existing with other dreadful infectious agents like HIV. The success of mycobacterium infection often relies in its ability to evade immune-surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immune regulatory molecules. In this scenario, the role of pattern recognition receptors (PRRs) in orchestrating host immune responses assumes central importance. Of the PRRs, the Toll-like receptors (TLRs) or intracellular surveillance receptors such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) govern key immune-surveillance mechanisms in recognition as well as control of mycobacterial or viral infections. The first part of this study illustrates the role of SHH signaling in macrophage induced neutrophil recruitment during mycobacterial infections. The present investigation demonstrates that, in response to mycobacterium infection, macrophages displayed robust activation of TLR2 dependent SHH signaling. By utilizing the well-documented experimental air pouch model, we show that the ability of pathogenic mycobacterium infected macrophages to recruit polymorph nuclear leukocytes (PMNs) like neutrophils to the infected site was dependent on SHH signaling. The activated SHH signaling differentially regulated the expression of proteolytic enzymes, MMP-9 and MMP-12 that would contribute to PMN migration. Interestingly, SHH-responsive krüppel-like family (KLF) of transcription factors, KLF4 and KLF5 were found to modulate these chemokine effectors to regulate neutrophil recruitment. Subsequent chapters describe novel functions of SHH signaling during RIG-I mediated anti-viral immunity and RIG-I mediated modulation of TLR2 anti-inflammatory signature in mycobacteria infected macrophages. In this perspective, we demonstrate that RIG-I ligand robustly induces the activation of SHH signaling via the phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. Furthermore, we show that the sustained inhibition of PKA-GSK-3β-SUFU negative regulatory axis upon RIG-I engagement with 5'3pRNA is critical for the activation of SHH signaling. Gain or loss of function studies implicate the necessity of RIG-I triggered MAVS-TBK1 canonical axis in the inhibition of PKA-GSK-3β-SUFU negative regulatory axis that contributes to SHH signaling activation. The RIG-I activated SHH signaling drives the production of anti-viral type 1 interferons leading to the inhibition Japanese encephalitis virus (JEV) replication. Further, RIG-I-mediated anti-viral type 1 interferon production and subsequent control of viral replication suggested the involvement of two transcriptional factors, IRF3 and YY1 in the response along a SHH axis. Further, mounting evidence clearly depicts a significant cross talk among the molecular events initiated by given TLRs and RLRs like RIG-I. Clearly, these studies present an interesting challenge in delineating the events during polymicrobial infection of host immune cells like macrophages or DCs. Altogether, our results improve our understanding of mycobacteria associated confections’ and may add significantly to the current knowledge of the delicate balance that determines a successful mycobacterial infection.

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with a highly virulent C. neoformans strain (H99 Stud) have a lower fungal burden and live longer compared to wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. For this reason, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D-/- mice after C. neoformans infection. Post-infection, mice lacking SP-D had reduced eosinophil infiltration and IL-5 in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were used to assess the role these innate immune mediators play during the host response to C. neoformans. IL-5 overexpressing mice had increased pulmonary eosinophilia and were more susceptible to C. neoformans infection as compared to WT mice. Furthermore, the response to C. neoformans infection in SP-D-/- mice could be restored to that of WT mice by increasing IL-5 and eosinophils, via crossing the IL-5 transgene onto the SP-D-/- background. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.

Dissertation

Eastern black walnut (Juglans nigra L.) ranks among the most highly valued timber species in the central hardwood forest and across the world. This valuable tree fills a critical role in native ecosystems as a mast bearing pioneer on mesic sites. Along with other Juglans spp. (Juglandaceae), J. nigra is threatened by thousand cankers disease (TCD), an insect-vectored disease first described in 2009. TCD is caused by the bark beetle Pityophthorus juglandis Blackman (Corthylini) and the phytopathogenic fungus Geosmithia morbida Kol. Free. Ut. & Tiss. (Bionectriaceae). Together, the P. juglandis-G. morbida complex has expanded from its historical range in southwest North America throughout the western United States (U.S.) and Europe. This range expansion has led to widespread mortality among naïve hosts J. nigra and J. regia planted outside their native distributions.

The severity of TCD was previously observed to be highest in urban and plantation environments and outside of the host native range. Therefore, the objective of this work was to provide information on biotic and abiotic environmental factors that influence the severity and impact of TCD across the native and non-native range of J. nigra and across different climatic and management regimes. This knowledge would enable a better assessment of the risk posed by TCD and a basis for developing management activities that impart resilience to natural systems. Through a series of greenhouse-, laboratory- and field-based experiments, environmental factors that affect the pathogenicity and/or survival of G. morbida in J. nigra were identified, with a focus on the microbiome, climate, and opportunistic pathogens. A number of potentially important interactions among host, vector, pathogen and the rest of the holobiont of TCD were characterized. The holobiont is defined as the whole multitrophic community of organisms—including J. nigra, microinvertebrates, fungi and bacteria—that interact with one another and with the host.

Our findings indicate that interactions among host, vector, pathogen, secondary pathogens, novel microbial communities, and novel abiotic environments modulate the severity of TCD in native, non-native, and managed and unmanaged contexts. Prevailing climatic conditions favor reproduction and spread of G. morbida in the western United States due to the effect of wood moisture content on fungal competition. The microbiome of soils, roots, and stems of trees and seedlings grown outside the host native range harbor distinct, lower-diversity communities of bacteria and fungi compared to the native range, including different communities of beneficial or pathogenic functional groups of fungi. The pathogen G. morbida was also associated with a distinct community of microbes in stems compared to G. morbida-negative trees. The soil microbiome from intensively-managed plantations facilitated positive feedback between G. morbida and a disease-promomting endophytic Fusarium solani species complex sp. in roots of J. nigra seedlings. Finally, the nematode species Bursaphelenchus juglandis associated with P. juglandis synergizes with G. morbida to cause foliar symptoms in seedlings in a shadehouse; conversely, experiments and observations indicated that the nematode species Panagrolaimus sp. and cf. Ektaphelenchus sp. could suppress WTB populations and/or TCD outbreaks.

In conclusion, the composition, function, and interactions within the P. juglandis and J. nigra holobiont play important roles in the TCD pathosystem. Managers and conservationists should be aware that novel associations outside the host native range, or in monocultures, intensive nursery production, and urban and low-humidity environments may favor progression of the disease through the effects of associated phytobiomes, nematodes, and climatic conditions on disease etiology. Trees in higher diversity, less intensively managed growing environments within their native range may be more resilient to disease. Moreover, expatriated, susceptible host species (i.e., J. nigra) growing in environments that are favorable to novel pests or pest complexes (i.e., the western U.S.) may provide connectivity between emergent forest health threats (i.e., TCD) and native host populations (i.e., J. nigra in its native range).