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Open Dimarzio - Dissertation Final The Pennsylvania State University The Graduate School College of Agricultural Sciences HIJACKING HOST METABOLISM WITH LACTOBACILLUS—UNDERSTANDING THE IMPLICATIONS OF BILE SALT HYDROLASE DIVERSITY A Dissertation in Food Science by Michael J. DiMarzio © 2016 Michael J. DiMarzio Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2016 The dissertation of Michael J. DiMarzio was reviewed and approved* by the following: Edward G. Dudley Associate Professor of Food Science Dissertation Advisor Chair of Committee Andrew D. Patterson Associate Professor of Molecular Toxicology Joshua D. Lambert Associate Professor of Food Science Robert F. Roberts Professor of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School ii Abstract The tremendous bacterial community which inhabits the human gastrointestinal tract is a newly appreciated intermediary for nutrient uptake and processing. Compositional variations in this bacterial community are associated with obesity, and recent evidence suggests that bacterial modification of bile acids secreted in the small intestine contributes to the regulation of fat storage. Bile salt hydrolase (BSH) activity against the bile acid tauro-beta-muricholic acid (T-β- MCA) in particular has been suggested as a critical mediator of host bile acid, glucose, and lipid homeostasis via the farnesoid X receptor (FXR) signaling pathway. Lactobacillus species are key players in this feedback loop, and their history of use as probiotic bacteria for promoting gastrointestinal health in humans makes them ideally suited for applications that exploit the bile acid regulatory feedback mechanism to control metabolism. BSH activity is widely associated with Lactobacillus species, but BSH substrate specificity for T-β-MCA had not been characterized in Lactobacillus prior to this work. Here, a strain of L. johnsonii with robust BSH activity against T-β-MCA in vitro was identified from the mouse gut microbiota. A screening assay performed on a collection of 14 probiotic strains from nine species of Lactobacillus identified BSH substrate specificity for T-β- MCA only in two of three L. johnsonii strains. Genomic analysis of these L. johnsonii strains revealed the presence of three bsh genes which are homologous to bsh genes in the human associated strain L. johnsonii NCC533. Further analysis revealed broad differences in substrate specificity even among the closely related bsh homologs, and suggests that the phylogeny of these enzymes does not closely correlate with substrate specificity. Predictive modeling was able to explain the difference in BSH activity for T-β-MCA in these homologs, and may be a useful tool for identifying additional BSHs from within the gut microbial community with the potential to affect host metabolism. This research also demonstrated the capability of transgenic BSH active bacteria to alter bile acid composition and affect FXR signaling in monocolonized germ free mice. However, these changes did not affect weight gain in the host, indicating the need for future research to understand the factors governing FXR mediated metabolic control. Additionally, studies of Lactobacillus administration to conventional and germ free mice revealed a disconnect between in vitro BSH activity and the ability of a probiotic to affect host bile acid composition. In particular, colonization of germ free mice with a strain of L. johnsonii did not result in reduced cecal T-β-MCA concentrations. Moreover, background BSH activity endogenous to the gut microbiota of conventional mice was found to almost completely reduce intestinal concentrations of T-β-MCA at baseline, leaving little potential for probiotic intervention. Follow up studies focusing on how BSH active probiotics affect the total BSH capacity of the gut microbiota are therefore needed. Finally, the biological role of BSHs in Lactobacillus is not entirely clear. BSH activity has been associated with increased colonization and intestinal persistence in several bacterial species, and is thought to provide some protection against bile acid toxicity. However, several studies have suggested BSHs comprise part of a global response to bile and acid stress, which may not be entirely consistent among strains. Isotopic Radio Outlier Analysis™ (IROA) was used to study the metabolic response of several probiotic Lactobacillus strains to bile acid stress. This approach improves metabolite identification from background noise compared to traditional metabolomics methods. Comparison of L. acidophilus NCFM, L. plantarum WCFS1, L. johnsonii LB1, and L. johnsonii NCK88 revealed differences in bile acid response among the strains, and suggests a role for amino acid metabolism in L. acidophilus NCFM. Further optimization of the IROA experimental design may uncover new roles for BSHs in Lactobacillus, and will inform improved probiotic strain selection. iii Ultimately this research discovered a strain of Lactobacillus autochthonous to the mouse intestine which exhibits clear BSH activity against T-β-MCA and offers promise for controlling host metabolism as a probiotic. These findings will guide ongoing efforts to optimize next generation probiotics for weight control in humans and agricultural animals. iv Table of Contents List of Figures ............................................................................................................................... vii List of Tables ............................................................................................................................... viii List of Abbreviations .................................................................................................................... ix Acknowledgments ........................................................................................................................ xii Chapter 1 – Statement of the Problem ......................................................................................... 1 Chapter 2 – Literature Review ..................................................................................................... 3 2.1 Introduction .................................................................................................................. 3 2.2 An overview of Lactobacillus ...................................................................................... 4 2.3 Lactobacillus associations with obesity ....................................................................... 5 2.4 Effects of Lactobacillus probiotics on weight in humans and animals ........................ 7 2.5 Colonization of Lactobacillus probiotics in the gastrointestinal tract .......................... 9 2.6 The gut microbiota, bile acid homeostasis, and host metabolism .............................. 14 2.7 Bile salt hydrolase activity in Lactobacillus probiotics ............................................. 18 2.8 Manipulating bile salt hydrolase activity to control host metabolism ........................ 23 2.9 Conclusions and research objectives .......................................................................... 26 Chapter 3 – Identification of two L. johnsonii strains with potential for metabolic control based on bile salt hydrolase activity against the potent FXR antagonist T-β- MCA .............................................................................................................................................. 28 3.1 Introduction ................................................................................................................ 28 3.2 Results ........................................................................................................................ 30 3.2.1 Isolation of mouse intestinal Lactobacillus ................................................ 30 3.2.2 Lactobacillus BSH activity against T-β-MCA ........................................... 30 3.2.3 Genomic comparison of L. johnsonii strains ............................................. 31 3.2.4 Comparison of BSH substrate specificity .................................................. 35 3.2.5 Modeling BSH substrate interactions with T-β-MCA ............................... 35 3.3 Discussion .................................................................................................................. 37 3.4 Materials and Methods ............................................................................................... 41 3.4.1 Bacterial strains and growth conditions ..................................................... 41 3.4.2 Enumeration and isolation of Lactobacillus .............................................. 42 3.4.3 Identification and characterization of Lactobacillus .................................. 42 3.4.4 DNA sequencing ........................................................................................ 43 3.4.5 BSH sequence comparisons ....................................................................... 43 3.4.6 BSH cloning ............................................................................................... 43 3.4.7 BSH activity assay ..................................................................................... 44 3.4.8 UPLC-ESI-QTOFMS analysis ................................................................... 44 3.4.9 Predictive modeling ................................................................................... 45 Chapter 4 – Effects of bile salt hydrolase producing bacteria on bile acid composition, FXR signaling,
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