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Complex Bacterial Consortia Reprogram the Colitogenic Activity of Enterococcus Faecalis in a Gnotobiotic Mouse Model of Chronic Immune-Mediated Colitis

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Complex Bacterial Consortia Reprogram the Colitogenic Activity of Enterococcus Faecalis in a Gnotobiotic Mouse Model of Chronic Immune-Mediated Colitis TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Ernährung und Immunologie Complex bacterial consortia reprogram the colitogenic activity of Enterococcus faecalis in a gnotobiotic mouse model of chronic immune-mediated colitis Isabella Brigitta Lengfelder Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Siegfried Scherer Prüfer der Dissertation: 1. Prof. Dr. Dirk Haller 2. Prof. Dr. Wolfgang Liebl Die Dissertation wurde am 01.07.2019 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 11.10.2019 angenommen. I ABSTRACT ABSTRACT Inflammatory bowel diseases (IBD) are associated with compositional and functional changes of the intestinal microbiota, but specific contributions of individual bacteria to chronic intestinal inflammation remain unclear. Enterococcus faecalis is a resident member of the human intestinal core microbiota that has been linked to the pathogenesis of IBD and induces chronic colitis in susceptible monoassociated IL10-deficient (IL-10-/-) mice. In this study, we characterized the colitogenic activity of E. faecalis as part of a simplified microbial consortium based on human strains (SIHUMI). RNA sequencing analysis of E. faecalis isolated from monoassociated wild type and IL-10-/- mice identified 408 genes including 14 genes of the ethanolamine utilization (eut) locus to be significantly upregulated in response to inflammation. Despite considerable upregulation of eut genes, deletion of ethanolamine utilization (∆eutVW) had no impact on E. faecalis colitogenic activity in monoassociated IL-10-/- mice. However, replacement of the E. faecalis wild type bacteria by a ∆eutVW mutant in SIHUMI-colonized IL-10-/- mice resulted in exacerbated colitis, suggesting protective functions of E. faecalis ethanolamine utilization in complex bacterial communities. To better understand E. faecalis gene response in the presence of other microbes, we purified wild-type E. faecalis cells from the colon content of SIHUMI-colonized wild type and IL10-/- mice using immuno-magnetic separation and performed RNA sequencing. Transcriptional profiling revealed that the bacterial environment reprograms E. faecalis gene expression in response to inflammation, with the majority of regulated genes not being shared between monocolonized and SIHUMI conditions. While in E. faecalis monoassociation a general bacterial stress response could be observed, expression of E. faecalis genes in SIHUMI-colonized mice was characterized by up- regulation of genes involved in growth and replication. Interestingly, in mice colonized with SIHUMI lacking E. faecalis enhanced inflammation was observed in comparison to SIHUMI-colonized mice, supporting the hypothesis that E. faecalis ethanolamine metabolism protects against colitis in complex consortia. In conclusion, this study demonstrates that complex bacterial consortia interactions reprogram the gene expression profile and colitogenic activity of the opportunistic pathogen E. faecalis towards a protective function. II ZUSAMMENFASSUNG ZUSAMMENFASSUNG Chronisch entzündliche Darmerkrankungen gehen mit kompositionellen und funktionellen Veränderungen der Darmmikrobiota einher, allerdings ist der spezifische Beitrag einzelner Bakterien zum entzündlichen Geschehen weitgehend unklar. Enterococcus faecalis ist ein residentes Mitglied der menschlichen Darmkernmikrobiota, das mit der Pathogenese chronisch entzündlicher Darmerkrankungen in Verbindung gebracht wird und chronische Kolitis in suszeptiblen monoassoziierten IL10-defizienten (IL-10-/-) Mäusen induziert. In dieser Studie haben wir die kolitogene Aktivität von E. faecalis als Teil eines vereinfachten mikrobiellen Konsortiums basierend auf menschlichen Stämmen (SIHUMI) charakterisiert. Eine RNA-Sequenzierungsanalyse von E. faecalis Zellen, die aus monoassoziierten Wildtyp- und IL-10-/--Mäusen isoliert wurden, identifizierte 408 Gene, darunter 14 Gene des Ethanolaminverwertungs-Locus (eut), die als Reaktion auf Entzündungen signifikant hochreguliert werden. Trotz erheblicher Hochregulierung der eut-Gene hatte die Deletion der Ethanolaminverwertung (∆eutVW) keinen Einfluss auf die kolitogene Aktivität von E. faecalis in monoassoziierten Mäusen. Der Austausch des E. faecalis Wildtyp-Stammes durch eine ∆eutVW- Mutante in SIHUMI-kolonisierten IL-10-/--Mäusen führte jedoch zu einer Verschlimmerung der Entzündung. Dies deutet auf eine protektive Funktion der Ethanolaminverwertung durch E. faecalis in einer komplexen Gemeinschaft hin. Um die Genexpression von E. faecalis in Gegenwart anderer Mikroben besser zu verstehen, haben wir Wildtyp-E. faecalis-Zellen aus dem Darminhalt von SIHUMI-kolonisierten Wildtyp- und IL-10-/-- Mäusen mittels immunomagnetischer Trennung isoliert und mittels RNA-Sequenzierung analysiert. Die transkriptionelle Analyse ergab, dass das bakterielle Umfeld die Genexpression von E. faecalis in Reaktion auf Entzündungen neu programmiert. Nur ein kleiner Teil der regulierten Gene waren sowohl in E. faecalis in Monoassoziation, als auch in Co-Kolonisation mit dem SIHUMI Konsortium parallel reguliert. Während bei monoassoziiertem E. faecalis eine allgemeine Stressreaktion beobachtet werden konnte, war das Expressionsprofil von E. faecalis in SIHUMI kolonisierten Mäusen durch Gene gekennzeichnet, die an Wachstum und Replikation beteiligt sind. Interessanterweise führte die Kolonisation von IL-10-/- Mäusen mit SIHUMI unter Ausschluss von E. faecalis zu einer Verschlimmerung des inflammatorischen Phänotyps. Dies unterstützt die Hypothese, dass E. faecalis als Mitglied eines komplexen Konsortiums durch die Verwertung von Ethanolamin eine protektive Funktion in suszeptiblen IL-10-/- Mäusen vermittelt. Abschließend zeigt diese Studie, dass Interaktionen mit einem komplexen bakteriellen Konsortium das Genexpressionsprofil und die kolitogene Aktivität des opportunistischen Pathogens E. faecalis in Richtung einer protektiven Aktivität umprogrammieren. III TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT .................................................................................................................................... II ZUSAMMENFASSUNG .................................................................................................................. III TABLE OF CONTENTS ................................................................................................................... IV 1. INTRODUCTION ........................................................................................................................ 1 1.1 IBD – a multifactorial chronic disease associated with a westernized lifestyle ............................ 1 1.2 Physiologic Microbe-Host Interactions ......................................................................................... 2 1.2 Importance of microbe-host interactions in IBD pathogenesis .................................................... 4 1.2.1 Host genetic defects in the containment of intestinal microbes ........................................... 5 1.2.2 Dysbiosis of the intestinal microbiota .................................................................................... 6 1.3 Animal models of experimental colitis: Microbe-Host interactions ........................................... 10 1.4 E. faecalis as model organism for microbe-host interactions in IBD .......................................... 12 1.5 Ethanolamine utilization in E. faecalis ........................................................................................ 13 1.6 Major facilitator superfamily transporter of E. faecalis .............................................................. 14 1.7 Aim of the study .......................................................................................................................... 15 3. MATERIAL & METHODS .......................................................................................................... 16 3.1 Supplier of experimental material .............................................................................................. 16 3.2. Bacterial strains and cultivation ................................................................................................. 17 3.3 Animal experiments – Ethical statement and housing ................................................................ 18 3.4 Animal experiments – Experimental design ................................................................................ 18 3.5 Generation of the mfs deletion mutant ...................................................................................... 19 3.6 Preparation of electrocompetent E. faecalis cells ...................................................................... 21 3.7 Preparation of bacterial lysates .................................................................................................. 22 3.8 Bacterial DNA extraction ............................................................................................................. 22 3.9 Bacterial community analysis via quantitative real-time PCR (qPCR) assay ............................... 22 3.10 Droplet Digital PCR .................................................................................................................... 24 3.11 Bacterial RNA
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