Gaelle Quere

Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes

Resident gut microbes co-exist with transient bacteria to form the gut microbiota. Despite increasing evidence suggesting a role for transient microbes on gut microbiota function, the interplay between resident and transient members of this microbial community is poorly defined. We aimed to determine the extent to which a host’s autochthonous gut microbiota influences niche permissivity to transient bacteria using a fermented milk product (FMP) as a vehicle for five food-borne bacterial strains. Using conventional and gnotobiotic rats and gut microbiome analyses (16S rRNA genes pyrosequencing and reverse transcription qPCR), we demonstrated that the clearance kinetics of one FMP bacterium, Lactococcus lactis CNCM I-1631, were dependent on the structure of the resident gut microbiota. Susceptibility of the resident gut microbiota to modulation by FMP intervention correlated with increased persistence of L. lactis. We also observed gut microbiome configurations that were associated with altered stability upon exposure to transient bacteria. Our study supports the concept that allochthonous bacteria have transient and subject-specific effects on the gut microbiome that can be leveraged to re-engineer the gut microbiome and improve dysbiosis-related diseases.

Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons

Significance Microbes hold promise as an inflammatory bowel disease (IBD) therapy. Lactococcus lactis, which has not been appreciated as a beneficial microbe, attenuated colitis in three preclinical mouse IBD models. Neither colonization nor an intact bacterium throughout the colon per se was required. Rather, host lysozyme-mediated lysis in an inflamed colon led to L. lactis’s release of its superoxide dismutase, which was necessary for its colitis-attenuating and oxidative stress-reducing activity. Overall, these findings unveil a mechanism by which a bacterium offers benefits to the host but requires the host for targeted release of this beneficial activity. Furthermore, because L. lactis is generally regarded as safe, it represents an opportunity for rapid bench-to-bedside testing in IBD. Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet−/− Rag2−/− mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631’s beneficial effect in the T-bet−/− Rag2−/− model. Similar effects were obtained in two additional colonic inflammation models, Il10−/− mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2−) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA’s extracytoplasmic O2− scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA’s bioaccessibility.

141 Lactococcus Lactis CNCM I-1631 Targets Oxidative Stress to Ameliorate Inflammation in Early-Onset Experimental Colitis

Background&Aims:Circadian rhythms govern a wide array of intestinal epithelial functions in mammals, including nutrient absorption, microbial sensing, migration, and self-renewal. In the absence of coordinating signals generated by the central pacemaker activity of the suprachiasmatic nucleus, mammalian cells derived from peripheral tissues typically require an external cue such as a serum shock or steroid pulse to synchronize circadian rhythms in culture. Building on our recent finding of robust circadian rhythms in mouse small intestinal organoids (enteroids) derived from PERIOD2:LUCIFERASE (PER2:LUC) mice, we tested the capacity of PER2::LUC enteroids to synchronize circadian rhythms in the absence of these external cues.Methods:We generated PER2::LUC jejunal enteroids and maintained them in an incubating luminometer to measure real time oscillations of PER2 abundance. Enteroid experiments were performed under typical culture conditions in the presence or absence of MatrigelTM. Prior to bioluminescent recordings, populations of enteroids were subjected to a 2-hour 50% serum shock vs. a dexamethasone pulse to synchronize clock vs. no external synchronization. Separately, unsynchronized enteroids were incubated in the presence or absence of steroid receptor antagonist RU-486. Results: Serum-shocked and dexamethasone-treated enteroids displayed synchronized circadian rhythms of PER2 abundance immediately upon recording. The amplitude and persistence of these rhythms were increased in enteroids embedded in MatrigelTM vs. those suspended in media. In the absence of a serum shock or dexamethasone pulse, enteroids spontaneously developed synchronized circadian rhythms within 12 hours of recording. RU486 treatment prior to bioluminescent recording significantly delayed the onset of this spontaneous synchronization and dampened the amplitude of PER2 oscillations by 2to 6-fold. Conclusions: PERIOD2::LUCIFERASE enteroids autonomously synchronize circadian rhythms, providing further evidence of a tightly regulated peripheral clock intrinsic to the intestinal epithelium. Additional studies addressing the role of glucocorticoids in coordinating circadian rhythms in enteroids and in vivo should yield novel insights regarding circadian regulation of gut homeostasis and stem cell dynamics and pharmacologic effects of glucocorticoids on the intestinal epithelium.