Mucosal or systemic microbiota exposures shape the B cell repertoire.

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.

Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes.

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-ICc12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes.

Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals.

Bacterial viruses (known as "phages") shape the ecology and evolution of microbial communities, making them promising targets for microbiome engineering. However, knowledge of phage biology is constrained because it remains difficult to study phage transmission dynamics within multi-member communities and living animal hosts. We therefore created "Phollow": a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Combining Phollow with optically transparent zebrafish enabled us to directly visualize phage outbreaks within the vertebrate gut. We observed that virions can be rapidly taken up by intestinal tissues, including by enteroendocrine cells, and quickly disseminate to extraintestinal sites, including the liver and brain. Moreover, antibiotics trigger waves of interbacterial transmission leading to sudden shifts in spatial organization and composition of defined gut communities. Phollow ultimately empowers multiscale investigations connecting phage transmission to transkingdom interactions that have the potential to open new avenues for viral-based microbiome therapies.

What Lies Beneath? Taking the Plunge into the Murky Waters of Phage Biology.

The sequence revolution revealed that bacteria-infecting viruses, known as phages, are Earth's most abundant biological entities. Phages have far-reaching impacts on the form and function of microbial communities and play a fundamental role in ecological processes. However, even well into the sequencing revolution, we have only just begun to explore the murky waters around the phage biology iceberg. Many viral reads cannot be assigned to a culturable isolate, and reference databases are biased toward more easily collectible samples, which likely distorts our conclusions. This minireview points out alternatives to mapping reads to reference databases and highlights innovative bioinformatic and experimental approaches that can help us overcome some of the challenges in phage research and better decipher the impact of phages on microbial communities. Moving beyond the identification of novel phages, we highlight phage metabolomics as an important influencer of bacterial host cell physiology and hope to inspire the reader to consider the effects of phages on host metabolism and ecosystems at large. We encourage researchers to report unassigned/unknown sequencing reads and contigs and to continue developing alternative methods to investigate phages within sequence data.

High throughput sequencing provides exact genomic locations of inducible prophages and accurate phage-to-host ratios in gut microbial strains.

BACKGROUND: Temperate phages influence the density, diversity and function of bacterial populations. Historically, they have been described as carriers of toxins. More recently, they have also been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. Despite recent advances in studying prophages using non-targeted sequencing approaches, methodological challenges in identifying inducible prophages in bacterial genomes and quantifying their activity have limited our understanding of prophage-host interactions. RESULTS: We present methods for using high-throughput sequencing data to locate inducible prophages, including those previously undiscovered, to quantify prophage activity and to investigate their replication. We first used the well-established Salmonella enterica serovar Typhimurium/p22 system to validate our methods for (i) quantifying phage-to-host ratios and (ii) accurately locating inducible prophages in the reference genome based on phage-to-host ratio differences and read alignment alterations between induced and non-induced prophages. Investigating prophages in bacterial strains from a murine gut model microbiota known as Oligo-MM12 or sDMDMm2, we located five novel inducible prophages in three strains, quantified their activity and showed signatures of lateral transduction potential for two of them. Furthermore, we show that the methods were also applicable to metagenomes of induced faecal samples from Oligo-MM12 mice, including for strains with a relative abundance below 1%, illustrating its potential for the discovery of inducible prophages also in more complex metagenomes. Finally, we show that predictions of prophage locations in reference genomes of the strains we studied were variable and inconsistent for four bioinformatic tools we tested, which highlights the importance of their experimental validation. CONCLUSIONS: This study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity. The ability to generate such quantitative information will be critical in helping us to gain better insights into the factors that determine phage activity and how prophage-bacteria interactions influence our microbiome and impact human health. Video abstract.

miR-802 regulates Paneth cell function and enterocyte differentiation in the mouse small intestine.

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.

Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice.

The microbiota confers colonization resistance, which blocks Salmonella gut colonization1. As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella's AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen-microbiota-host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process.

Microbiota stability in healthy individuals after single-dose lactulose challenge-A randomized controlled study.

BACKGROUND AND AIMS: Lactulose is a common food ingredient and widely used as a treatment for constipation or hepatic encephalopathy and a substrate for hydrogen breath tests. Lactulose is fermented by the colon microbiota resulting in the production of hydrogen (H2). H2 is a substrate for enteropathogens including Salmonella Typhimurium (S. Typhimurium) and increased H2 production upon lactulose ingestion might favor the growth of H2-consuming enteropathogens. We aimed to analyze effects of single-dose lactulose ingestion on the growth of intrinsic Escherichia coli (E. coli), which can be efficiently quantified by plating and which share most metabolic requirements with S. Typhimurium. METHODS: 32 healthy volunteers (18 females, 14 males) were recruited. Participants were randomized for single-dose ingestion of 50 g lactulose or 50 g sucrose (controls). After ingestion, H2 in expiratory air and symptoms were recorded. Stool samples were acquired at days -1, 1 and 14. We analyzed 16S microbiota composition and abundance and characteristics of E. coli isolates. RESULTS: Lactulose ingestion resulted in diarrhea in 14/17 individuals. In 14/17 individuals, H2-levels in expiratory air increased by ≥20 ppm within 3 hours after lactulose challenge. H2-levels correlated with the number of defecations within 6 hours. E. coli was detectable in feces of all subjects (2 x 10(2)-10(9) CFU/g). However, the number of E. coli colony forming units (CFU) on selective media did not differ between any time point before or after challenge with sucrose or lactulose. The microbiota composition also remained stable upon lactulose exposure. CONCLUSION: Ingestion of a single dose of 50 g lactulose does not significantly alter E. coli density in stool samples of healthy volunteers. 50 g lactulose therefore seems unlikely to sufficiently alter growth conditions in the intestine for a significant predisposition to infection with H2-consuming enteropathogens such as S. Typhimurium (www.clinicaltrials.gov NCT02397512).