Dietary protein from different sources escapes host digestion and is differentially modified by the microbiota.

Protein is an essential macronutrient and variations in its source and quantity have been shown to impact long-term health outcomes. Differential health impacts of dietary proteins from various sources are likely driven by differences in their digestibility by the host and subsequent availability to the intestinal microbiota. However, our current understanding regarding the fate of dietary proteins from different sources in the gut, specifically how component proteins within these sources interact with the host and the gut microbiota, is limited. To determine which dietary proteins are efficiently digested by the host and which proteins escape host digestion and are used by the gut microbiota, we used high-resolution mass spectrometry to quantify the proteins that make up different dietary protein sources before and after digestion in germ-free and conventionally raised mice. Contrary to expectation, we detected proteins from all sources in fecal samples of both germ-free and conventional mice suggesting that even protein sources with a high digestive efficiency make it in part to the colon where they can serve as a substrate for the microbiota. Additionally, we found clear patterns where specific component proteins of the dietary protein sources were used as a preferred substrate by the microbiota or were not as accessible to the microbiota. We found that specific proteins with functions that could impact host health and physiology were differentially enriched in germ-free or conventionally raised mice. These findings reveal large differences in the fate of dietary protein from various sources in the gut that could explain some of their differential health impacts.

Eubacterium rectale detoxification mechanism increases resilience of the gut environment.

Lachnospiraceae members were highly detected in dysbiotic IL-10 KO mice that displayed similar physiological outcomes as control mice. Lachnospiraceae is a highly diverse family of microbes that have been shown to display both commensal and pathogenic characteristics in the colon environment. We investigated the impact of genetic variation in five Lachnospiraceae strains on lowering cellular inflammation and ROS levels. Cell free spent media (CFSM) from Eubacterium rectale resulted in lowered ROS, and nitric oxide levels in stressed colon cells. We demonstrated through an array of multi-omics and molecular techniques that glutathione (GSH) biosynthesized by E. rectale was able to alleviate host ROS damage. We further showed downregulation of cell stress and immune response genes by host RNA sequencing, which is evidence that E. rectale microbial products promote recovery and alleviate ROS stress.

Dietary protein source strongly alters gut microbiota composition and function.

The source of protein in a persons diet affects their total life expectancy. However, the mechanisms by which dietary protein sources differentially impact human health and life expectancy are poorly understood. Dietary choices have major impacts on the composition and function of the intestinal microbiota that ultimately mediate host health. This raises the possibility that health outcomes based on dietary protein sources might be driven by interactions between dietary protein and the gut microbiota. In this study, we determine the effects of seven different sources of dietary protein on the gut microbiota in mice. We apply an integrated metagenomics-metaproteomics approach to simultaneously investigate the effects of these dietary protein sources on the gut microbiotas composition and function. The protein abundances measured by metaproteomics can provide microbial species abundances, and evidence for the phenotype of microbiota members on the molecular level because measured proteins allow us to infer the metabolic and physiological processes used by a microbial community. We showed that dietary protein source significantly altered the species composition and overall function of the gut microbiota. Different dietary protein sources led to changes in the abundance of microbial amino acid degrading proteins and proteins involved in the degradation of glycosylations on dietary protein. In particular, brown rice and egg white protein increased the abundance of amino acid degrading enzymes and egg white protein increased the abundance of bacteria and proteins usually associated with the degradation of the intestinal mucus barrier. These results show that dietary protein source can change the gut microbiotas metabolism, which could have major implications in the context of gut microbiota mediated diseases.

Limitation of amino acid availability by bacterial populations during enhanced colitis in IBD mouse model.

IMPORTANCE: Inflammatory bowel disease is associated with an increase in Enterobacteriaceae and Enterococcus species; however, the specific mechanisms are unclear. Previous research has reported the associations between microbiota and inflammation, here we investigate potential pathways that specific bacteria populations use to drive gut inflammation. Richie et al. show that these bacterial populations utilize an alternate sulfur metabolism and are tolerant of host-derived immune-response products. These metabolic pathways drive host gut inflammation and fuel over colonization of these pathobionts in the dysbiotic colon. Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize essential host nutrients.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Resources to Facilitate Use of the Altered Schaedler Flora (ASF) Mouse Model to Study Microbiome Function.

Animals colonized with a defined microbiota represent useful experimental systems to investigate microbiome function. The altered Schaedler flora (ASF) represents a consortium of eight murine bacterial species that have been used for more than 4 decades where the study of mice with a reduced microbiota is desired. In contrast to germ-free mice, or mice colonized with only one or two species, ASF mice show the normal gut structure and immune system development. To further expand the utility of the ASF, we have developed technical and bioinformatic resources to enable a systems-based analysis of microbiome function using this model. Here, we highlighted four distinct applications of these resources that enable and improve (i) measurements of the abundance of each ASF member by quantitative PCR; (ii) exploration and comparative analysis of ASF genomes and the metabolic pathways they encode that comprise the entire gut microbiome; (iii) global transcriptional profiling to identify genes whose expression responds to environmental changes within the gut; and (iv) discovery of genetic changes resulting from the evolutionary adaptation of the microbiota. These resources were designed to be accessible to a broad community of researchers that, in combination with conventionally-reared mice (i.e., with complex microbiome), should contribute to our understanding of microbiome structure and function. IMPORTANCE Improved experimental systems are needed to advance our understanding of how the gut microbiome influences processes of the mammalian host as well as microbial community structure and function. An approach that is receiving considerable attention is the use of animal models that harbor a stable microbiota of known composition, i.e., defined microbiota, which enables control over an otherwise highly complex and variable feature of mammalian biology. The altered Schaedler flora (ASF) consortium is a well-established defined microbiota model, where mice are stably colonized with 8 distinct murine bacterial species. To take better advantage of the ASF, we established new experimental and bioinformatics resources for researchers to make better use of this model as an experimental system to study microbiome function.

Perspective: Simple State Communities to Study Microbial Interactions: Examples and Future Directions.

Microbial interactions in natural environments are intricately complex. High numbers and rich diversity of microorganisms, along with compositional heterogeneities complicate the cause. It is essential to simplify these complex communities to understand the microbial interactions. We proposed a concept of "simple state community," which represents a subset of microbes and/or microbial functions of the original population that is necessary to build a stable community. By combining microbial culturing and high-throughput sequencing, we can better understand microbe-microbe and microbe-host interactions. To support our proposed model, we used carbon-based and nitrogen-based media to capture the simple state communities. We used 16S rRNA amplicon sequencing and assigned taxonomic identity to the bacterial populations before and after simple state communities. We showed that simple state communities were a subset of the original microbial communities at both phyla and genera level. We further used shotgun metagenomics to gain insights into the functional potential of the assembled simple state communities. Our proposed model supported the goal of simplifying the complex communities across diverse systems to provide opportunity to facilitate comprehension of both the structure and function of the subset communities. Further applications of the concept include the high-throughput screening of simple state communities using the BIOLOG® system and continuous culturing (Chemostat). This concept has the potential to test diverse experimental hypotheses in simplified microbial communities, and further extend that knowledge to answer the overarching questions at a more holistic level.