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Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1–4. Since Clade 5 strains are highly prevalent in agricultural animals and a frequent cause of zoonotic infections, these strains may have evolved phenotypes that distinguish them from Clade 1–4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1–4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1–4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr- ON state. Interestingly, Clade 5 strains with a bias towards the cmr- ON state shifted to a largely cmr- OFF state during murine infection, suggesting that the cmr- OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron, we selectively modulated the levels of the bile acid tauro-β-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales. The microbiome, the collection of bacteria that live in and on human bodies, has a strong influence on how well the body works. However, the diversity of the microbiome makes it difficult to untangle exactly how it has these effects. For example, it is poorly understood how the hundreds of species of bacteria that live in the gut affect metabolism – the chemical processes that make life possible. But they are known to influence how metabolic diseases like diabetes and obesity develop. When we eat a meal, the body releases compounds called bile acids to help to digest the food. Once the bile acids reach the colon, the bacteria residing there use enzymes to chemically modify the compounds. Imbalances in the resulting pool of over 50 different bile acids may accelerate how quickly people develop metabolic disorders. It is not clear, however, which bile acids have helpful or harmful effects on metabolism. Yao et al. first identified a selective version of a prevalent gut bacterial enzyme called a bile salt hydrolase. This enzyme was then deleted from a common gut bacterium using genetic tools. Finally, Yao et al. colonized mice lacking any bacteria (i.e., germ-free mice) with either the original bacterium or the hydrolase-deleted bacterium. Mice colonized with the hydrolase-deleted bacteria gained less weight on a high fat diet and had lower levels of fat in their blood and liver. These mice also shifted to burning fats instead of carbohydrates for energy. The changes in the bile acid pool produced in mice colonized with hydrolase-deleted bacteria did not only affect metabolism. Yao et al. found differences in the activity of genes important for other biological processes as well, such as those that control circadian rhythms and immune responses. Further research is needed to investigate whether limiting the activity of the bile salt hydrolase enzyme has similar effects in humans. If so, developing drugs or probiotics that target the enzyme could lead to new treatments for people with metabolic diseases like obesity and fatty liver disease. Investigating the biological effects of other bacterially modified bile acids may identify other possible treatments as well.

Phenotypic heterogeneity refers to the ability of clonal populations of the same species to display distinct phenotypes despite experiencing the same environment. Bacteria exhibit phenotypic heterogeneity across diverse cellular processes, which can provide competitive fitness advantages both within the host and against surrounding microbiota. However, visualizing phenotypic heterogeneity at single-cell resolution within dense microbial communities is technically challenging. Here, we present a method for visualizing this heterogeneity by combining spectrally compatible reporters to track the spatial distribution of gene expression in individual bacterial cells in the mammalian gut. Using toxin gene expression in Clostridioides difficile as a model for visualizing phenotypic heterogeneity, we demonstrate that, while C. difficile primarily occupies the lumen, a subpopulation of C. difficile associates with the colonic epithelium independent of toxin production. We further show that heterogeneity in C. difficile toxin gene expression is independent of location in the gut and that a toxin gene overexpressing mutant unexpectedly forms filamentous cells during the acute phase of infection. Thus, our reporter system provides quantitative, single-cell resolution of bacterial behavior within the intact gut environment and establishes a broadly applicable platform for investigating phenotypic heterogeneity in dense microbial communities.

Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment, and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortium function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us in deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits. In nature, microbes interact antagonistically, neutrally, or beneficially. To shed light on the effects of positive interactions in microbial consortia, we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wild-type consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments, including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments. IMPORTANCE Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment, and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortium function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us in deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits. Author Video : An author video summary of this article is available.

The role of dysbiosis in food allergy (FA) remains unclear. We found that dysbiotic fecal microbiota in FA infants evolved compositionally over time and failed to protect against FA in mice. Infants and mice with FA had decreased IgA and increased IgE binding to fecal bacteria, indicative of a broader breakdown of oral tolerance than hitherto appreciated. Therapy with Clostridiales species impacted by dysbiosis, either as a consortium or as monotherapy with Subdoligranulum variabile, suppressed FA in mice as did a separate immunomodulatory Bacteroidales consortium. Bacteriotherapy induced expression by regulatory T (Treg) cells of the transcription factor ROR-γt in a MyD88-dependent manner, which was deficient in FA infants and mice and ineffectively induced by their microbiota. Deletion of Myd88 or Rorc in Treg cells abrogated protection by bacteriotherapy. Thus, commensals activate a MyD88/ROR-γt pathway in nascent Treg cells to protect against FA, while dysbiosis impairs this regulatory response to promote disease.

is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible switch sequence, with chaining strains exhibiting a bias to the -ON state. Interestingly, Clade 5 strains with a bias towards the -ON state shifted to a largely -OFF state during murine infection, suggesting that the -OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

Building upon discourse completion task (DCT) research examining men’s responses to a fat talk prompt by a male friend, this study examined how men respond to a DCT with a muscle talk prompt. Adult men (N = 110) completed an online survey including measures of their body talk engagement and DCTs depicting male dyads of similar and different body types (e.g., normal-normal, normal-overweight, normal-muscular, normal-thin). For consistency, the respondent was depicted with a “normal\" body type. The pattern of responses to the DCT dyads varied across the different speaker body types and differed compared to prior research using a fat prompt. Deflections and expansion requests were addressed more to the muscular speaker; whereas validations were given more toward normal and overweight speakers. Two sub-themes were added to the codebook. More research is needed to further understand what influences male friends’ body talk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In nature, microbes interact antagonistically, neutrally or beneficially. To shed light on the effects of positive interactions in microbial consortia we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wildtype consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments.

Visualizing phenotypic heterogeneity at single-cell resolution within dense microbial communities is technically challenging. Here, we present a method for visualizing this heterogeneity by combining spectrally compatible reporters to track the spatial distribution of gene expression in individual bacterial cells in the mammalian gut. Using toxin gene expression in Clostridioides difficile as a model for visualizing phenotypic heterogeneity, we demonstrate that, while C. difficile primarily occupies the lumen, a subpopulation of C. difficile associates with the colonic epithelium independent of toxin production. The approach further revealed that heterogeneity in C. difficile toxin gene expression is independent of location in the gut and unexpectedly showed that a toxin gene over-expressing mutant forms filamentous cells during the acute phase of infection. Thus, our reporter system provides quantitative, single-cell resolution of bacterial behavior within the intact gut environment and establishes a broadly applicable platform for investigating phenotypic heterogeneity in dense microbial communities.