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京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24093号 / 社医博第124号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 古川 壽亮, 教授 村井 俊哉, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. / Thesis / Doctor of Philosophy (PhD)

<p>The glycopeptide antibiotic vancomycin is used as front line treatment for serious Gram-positive infections and resistance to this drug is widespread. Three genes are essential for resistance, <em>vanHAX</em>, which are controlled by a two-component regulatory system VanR and VanS. Here, glycopeptide resistance is found to be ancient and diverse in the environment. A <em>vanA</em> open reading frame from 30 000 yr old DNA was identified and the enzyme was shown to be as functional as comparable to modern day VanA homologs. In the environment resistance is found to be diverse and widespread. For example, the organism <em>Desulfitobacterium hafniense</em> Y51 VanH was shown as non-essential in conferring inducible resistance. Furthermore in the glycopeptide producer <em>Amycolatopsis balhimycina</em> harboring the classic <em>vanHAX,</em> a functional VanA homolog is described as an orphan gene outside of any recognizable gene cassette .</p> <p>Glycopeptides are natural products made by members of the Actinomycete family and are modified by different types of tailoring enzymes. Of particular interest is the glycopeptide A47934, which is ‘aglyco’, and sulfated. The sulfotransferase StaL will transfer not only a sulfate group to A47934, but a sulfamide and fluorosulfonate group. Focusing on additional tailoring enzymes, the biosynthetic cluster of the sulfated glycopeptide UK68597 was sequenced. This cluster has provided a resource for glycopeptide tailoring enzymes for use to modify the A47934 backbone. Sulfation was the first focus and the substrate promiscuity of StaL was explored to expand the chemical diversity A47934 and vancomycin. This work has led to the discovery that glycopeptide sulfation will antagonize the activation and expression of <em>vanHAX</em>. A new sulfated vancomycin derivative was created with this antagonizing activity in the clinical pathogen <em>Enterococcus faecium</em> of the VanB phenotype. Implications of these results and the further use of tailoring enzymes to modify glycopeptides to antagonize resistance will be discussed.</p> / Doctor of Philosophy (PhD)

Increasing evidence from both animal and human research implicates prenatal infection in the development of long-lasting disruptions in immune function of offspring, including an increased risk of infection and allergic disorders, such as asthma. Infection, specifically during early life, also has been repeatedly associated with subsequent risk of asthma in childhood. Moreover, antibiotic exposure, during both the pre- and post-natal period, has been linked with several of these immune-related health outcomes. The aim of the current study was to investigate the role of maternal infection during pregnancy in relation to the development of offspring infection and subsequent asthma risk. Further, we considered the confounding factors of pre- and postnatal antibiotic exposure in the context of offspring infection and asthma inception. Participants (N=2062) were pregnant women and their offspring prospectively enrolled in a longitudinal birth cohort study with follow-up data through childhood. Extensive health information, including data on illness episodes and antibiotic exposures, was obtained from medical records for both mothers and their offspring. Results indicated that second trimester prenatal infection was associated with an increased risk of offspring infection from birth through age five. Both offspring infection and antibiotic exposure from birth through age five were significantly associated with a diagnosis of asthma in children at age five. Offspring infection was found to mediate the relationship between mothers’ second trimester infection and offspring asthma. Sensitivity analyses suggested that our results primarily were due to infections and not antibiotic use, given that findings were replicated when restricting analyses to samples of mothers and offspring without antibiotic exposure. These findings suggest fetal origins of offspring infection and asthma risk. / Psychology