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
   IL-10-deficient (IL-10^−/−) mice. In this study, we characterized the
   colitogenic activity of E. faecalis as part of a simplified human
   microbial consortium based on seven enteric bacterial 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 that were significantly
   up-regulated in response to inflammation. Despite considerable
   up-regulation of eut genes, deletion of ethanolamine utilization
   (ΔeutVW) had no impact on E. faecalis colitogenic activity in
   monoassociated IL-10^−/− mice. However, replacement of the E. faecalis
   wild type bacteria by a ΔeutVW 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 IL-10^−/− 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 differentially expressed 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 toward a protective
   function.

   Keywords: Enterococcus faecalis, ethanolamine utilization, gnotobiotic
   mouse models, microbial consortium, SIHUMI, IBD, IL-10 deficient mouse,
   RNA sequencing

Introduction

   Inflammatory bowel diseases (IBD) with the two dominant types Crohn's
   disease (CD) and ulcerative colitis (UC) are chronic relapsing
   inflammatory disorders affecting the distal intestine. Several factors
   appear to be involved in the pathogenesis of IBD, including genetic
   susceptibility ([39]1, [40]2) together with diverse environmental
   triggers resulting in an inappropriate T-cell mediated activation of
   immunity toward components of the intestinal microbiota ([41]3–[42]5).
   IBD is associated with compositional ([43]6–[44]10) and functional
   ([45]11, [46]12) changes of the intestinal microbiota referred to as
   dysbiosis. Dysbiotic changes associated with IBD are characterized by
   an overrepresentation of opportunistic pathogens and a loss of
   beneficial commensal organisms, indicating that the pathogenic
   potential of a dysbiotic community can be linked to certain organisms.
   Consequently, the specific contribution of individual bacteria to
   disease pathogenesis needs to be investigated in detail.

   A relevant bacterial species in the context of IBD is Enterococcus
   faecalis. E. faecalis is a Gram-positive resident member of the human
   intestinal core microbiota harboring a number of pathogenic traits,
   which explains the association of this bacterium with inflammatory
   diseases and fatal nosocomial infections ([47]13–[48]15). Relative to
   healthy volunteers, the abundance of enterococci is increased in fecal
   samples of CD patients ([49]16–[50]18). This is in line with a high
   frequency of E. faecalis housekeeping and virulence genes in CD cohorts
   ([51]19) and a high likelihood of the presence of virulence factors in
   E. faecalis isolates originating from inflamed IBD mucosa ([52]20). UC
   patients have increased numbers of mucosa-associated E. faecalis
   correlating with disease activity ([53]21) and a high E.
   faecalis-specific IgG sero-reactivity ([54]22).

   The dual characteristics of E. faecalis as core member of the human
   intestinal microbiota and as an opportunistic pathogen make this
   bacterium an ideal model to study microbe-host interactions relevant to
   the development of chronic colitis in a genetically susceptible host.
   In the IL10^tm1Cgn mouse (IL-10^−/−), which is a well-characterized
   model of human chronic colitis, monoassociation with E. faecalis
   induces severe intestinal inflammation, whereas wild type mice remain
   disease-free ([55]23–[56]26). In previous studies, we could show that
   certain bacterial structures contribute to the colitogenic activity of
   E. faecalis in monoassociated IL-10^−/− mice ([57]27, [58]28).

   Using germ-free wild type and IL-10^−/− mice monoassociated with E.
   faecalis or colonized with a colitogenic human enteric bacterial
   consortium (SIHUMI) ([59]29), we aim to unravel the functional
   relevance of E. faecalis in colitis development with regard to gene
   expression and interactions with co-colonizing bacteria. Based on RNA
   sequencing (RNA seq) analysis, we characterize the role of ethanolamine
   (EA) utilization for E. faecalis survival and colitogenic activity in a
   susceptible host.

   The intestine is a rich source of the metabolite EA, which is derived
   from phospholipid phosphatidylethanolamine of bacterial and eukaryotic
   cell membranes ([60]30, [61]31). Bacteria capable of EA catabolism can
   utilize this compound as a source of carbon and/or nitrogen to promote
   growth as well as a signal influencing virulence during host
   colonization ([62]32). EA utilization is a well-recognized property of
   diverse pathogens, for example Salmonella and enterohemorrhagic
   Escherichia coli (EHEC) ([63]33–[64]37). The only commensals known to
   carry EA utilization genes are E. faecalis and some commensal strains
   of E. coli ([65]32, [66]38–[67]41). E. faecalis catabolizes EA using
   enzymes encoded by the eut genes that are contained in a locus
   consisting of 19 genes. The eut gene expression is controlled by the
   EutVW two-component system, comprising the sensor histidine kinase EutW
   and the response regulator EutV ([68]42, [69]43). Using an E. faecalis
   eutVW double deletion mutant ([70]43), we are able to establish a novel
   correlation between E. faecalis EA utilization and an attenuation of
   the pro-inflammatory host response in the presence of a complex
   bacterial consortium.

   Using immuno-magnetic separation, we are able to purify wild type E.
   faecalis cells from the colon content of SIHUMI-colonized wild type and
   IL-10^−/− mice and analyze the gene expression in the presence of other
   bacteria. We show that E. faecalis adapts transcriptionally to the
   other co-colonizing bacteria. To unravel whether these transcriptional
   alterations have functional consequences, we colonized wild type and
   IL-10^−/− mice with the SIHUMI consortium in the presence and absence
   of E. faecalis. We determine a protective activity of E. faecalis in
   SIHUMI colonized susceptible mice, demonstrating that complex bacterial
   consortia interactions can reprogram the colitogenic activity of the
   opportunistic pathogen E. faecalis toward a protective function.

Materials and Methods

Bacterial Strains and Cultivation

   For this study we used the characterized human oral E. faecalis strain
   OG1RF and an isogenic eutVW double deletion mutant kindly provided by
   Danielle Garsin (Department of Microbiology and Molecular Genetics, The
   University of Texas Health Science Center at Houston, Houston, Texas,
   USA) ([71]43). E. faecalis strains ([72]Table 1) were cultivated in
   Brain Heart Infusion (BHI) broth or on BHI agar (BD Difco) at 37°C
   under aerobic conditions. The SIHUMI ([73]29) consortium ([74]Table 1)
   was kindly provided by R. Balfour Sartor (Division of Gastroenterology
   and Hepatology, University of North Carolina, Chapel Hill, NC, USA).
   All SIHUMI strains were cultivated under anaerobic conditions on
   Wilkins-Chalgren-Agar (WCA, Thermo Fisher Scientific) or in WCA broth
   supplemented with 0.05% (w/v) L-cystein (Carl Roth) and 0.002% (w/v)
   dithiothreitol (Sigma Aldrich). Of note, a different strain set for
   rats is also called SIHUMI and should not be mistaken ([75]46). For
   colonization of germ-free mice with the SIHUMI consortium, the
   bacterial mixture was prepared as follows. Strains were grown
   individually in Hungate tubes for 24 h. A mixture of all strains with
   an equal cell density of 1 × 10^9 cells/ml was prepared in sterile
   glass tubes, mixed 1:1 with sterile glycerol in culture medium (40%
   v/v, gassed with N[2]) and sealed with a rubber septum. The bacterial
   mixture was stored at −80°C until use.

Table 1.

   Bacterial strains used in this study.
   Strain Characteristics References