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RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re-exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells.

Salmonella enterica serovar Typhimurium (Salmonella) is a Gram-negative bacterial pathogen capable of causing both gastroenteritis and bacteraemia in human hosts. During infection, Salmonella invokes a complex network of virulence factors, regulatory systems, and metabolic pathways to promote immune evasion, sometimes demanding antibiotic treatment for resolution. Unfortunately, antibiotic resistance has reached critical levels in this and other pathogens, necessitating the discovery of new anti-infective targets and treatment options. Herein, we have sought to exploit the dynamic interactions between Salmonella and the host immune system to identify new, conditionally active anti-Salmonella therapies. In chapter 2, we aim to identify chemical compounds that are selectively antimicrobial against intracellular Salmonella, and discover that the anxiolytic drug metergoline inhibits Salmonella survival in cultured macrophages and systemically infected mice. In chapter 3, we screen for anti-virulence compounds that target regulatory signaling in Salmonella, and characterize the inhibitory activity of methyl-3,4-dephostatin, which perturbs SsrA/B and PmrB/A signaling and enhances sensitivity to colistin in vitro and in vivo. In chapter 4, we identify several host-directed compounds that modulate macrophage immunity and investigate their ability to attenuate a multidrug resistant Salmonella infection. Together, the work presented in this thesis demonstrates the potential for drug screening in infection-relevant conditions to identify new anti-infectives with non-traditional targets. / Thesis / Doctor of Philosophy (PhD)

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京都大学 / 新制・課程博士 / 博士(農学) / 甲第24675号 / 農博第2558号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5456(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Controlling pathogenic presence in broilers has become a priority in the poultry industry to prevent economic losses due to disease and infection, as well as the possible contamination of chicken products. The use of antibiotics reduces the incidence of infections; however, their removal from production initiated the search for suitable alternatives. Probiotic ineed supplements have been widely evaluated as alternatives. Probiotic use has improved broiler performance, reduced pathogenic loads, and stimulated the immune system at later life stages. However, there is still a gap in protection during the first weeks after the chick hatches. The in ovo supplementation of probiotics has the potential of promoting early health benefits and protect the chick against pathogens after hatch. In the present study, the in ovo inoculation of different probiotic species was evaluated. It was determined that the inoculation of higher concentrations of E. faecium (107 cfu/50µL) into the egg improves growth performance and intestinal morphology compared to lower doses (105 and 106 cfu/50µL). It was also determined that not all B. subtilis serotypes are safe for in ovo inoculation, even if recognized as safe for use in feed, due to a high reduction in hatchability. However, certain B. subtilis are safe for in ovo inoculation and regulate the gut microflora through modulations in coliforms and aerobic bacteria after hatch. Lastly, the in ovo inoculation of different Lactobacillus strains does not affect hatchability or growth performance. However, different Lactobacillus species stimulated cytokine production even during the first week of hatch. The bursa of Fabricius morphology was modulated through an increase in follicular area, which could possibly induce higher antibody production against incoming pathogenic challenges. These results indicate that the in ovo inoculation of probiotic bacteria can induce earlier benefits to broiler health through early changes in gut microflora, as well as early stimulation in the immune system. The early protection provided through the in ovo inoculation of probiotics combined with the protection obtained through the administration of probiotics in feed could potentially result in overall healthier broilers and therefore improved performance.

Cellular metabolic and immune pathways can be acted upon by diverse molecular factors. Some examples include small molecules, regulatory proteins or RNAs, intermediary metabolites and hormones. These factors can also be introduced or induced by pathogens during infections. Indeed, it is known that complex interplay exists between metabolism and immunity. However, the ways in which these interactions occur, and the nature of the players are active subjects of research. Herein, three different studies are presented which investigate the roles of three distinct modulators of metabolism and/or immunity. Firstly, a natural product produced by a pathogenic fungus is shown to activate the aryl hydrocarbon receptor and induce the expression of xenobiotic metabolizing enzymes. Secondly, the modulation of lipid metabolism by an immunometabolic antiviral microRNA, microRNA-185, is deconvoluted using activity-based protein profiling (ABPP), transcriptomic and lipidomic analysis. This study also identifies a novel enzymatic target of microRNA-185 which can be targeted pharmacologically to reduce hepatitis C virus infectivity. Finally, a third study investigates the link between a poorly characterized enzyme, lysophospholipase-like 1 (LYPLAL1), and hepatic glucose metabolism using a specific activity-based probe. Overall, the work presented in this thesis makes use of various molecular and chemical biology methods to probe pathways which are acted upon by structurally diverse factors to improve our understanding of host-pathogen interactions and metabolism.

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