Targeted sampling of natural product space to identify bioactive natural product-like polyketide macrolides.

Polyketide or polyketide-like macrolides (pMLs) continue to serve as a source of inspiration for drug discovery. However, their inherent structural and stereochemical complexity challenges efforts to explore related regions of chemical space more broadly. Here, we report a strategy termed the Targeted Sampling of Natural Product space (TSNaP) that is designed to identify and assess regions of chemical space bounded by this important class of molecules. Using TSNaP, a family of tetrahydrofuran-containing pMLs are computationally assembled from pML inspired building blocks to provide a large collection of natural product-like virtual pMLs. By scoring functional group and volumetric overlap against their natural counterparts, a collection of compounds are prioritized for targeted synthesis. Using a modular and stereoselective synthetic approach, a library of polyketide-like macrolides are prepared to sample these unpopulated regions of pML chemical space. Validation of this TSNaP approach by screening this library against a panel of whole-cell biological assays, reveals hit rates exceeding those typically encountered in small molecule libraries. This study suggests that the TSNaP approach may be more broadly useful for the design of improved chemical libraries for drug discovery.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling.

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit, the anti-inflammatory immune system, wound healing, normal social behavior in mice, and prevents bone reabsorption. Oxytocin impacts these functions and oxytocin signaling is required for L. reuteri-mediated wound healing and social behavior; however, the events in the gut leading to oxytocin stimulation and beneficial effects are unknown. Here we report evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We find that L. reuteri facilitates oxytocin secretion from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling.

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit 1 , the anti-inflammatory immune system 2-4 , wound healing 5-7 , normal social behavior in mice 8-10 , and prevents bone reabsorption 11-17 . Each of these functions is impacted by oxytocin 18-22 , and oxytocin signaling is required for L. reuteri- mediated wound healing 5 and social behavior 9 ; however, the initiating events in the gut that lead to oxytocin stimulation and related beneficial functions remain unknown. Here we found evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We subsequently found that L. reuteri facilitates oxytocin secretion directly from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells 23 , while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri . This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbiota composition is associated with differential risk of urogenital infection. Although vaginal Lactobacillus spp. are thought to confer protection through acidification, bacteriocin production, and immunomodulation, lack of an in vivo model system that closely resembles the human vaginal microbiota remains a prominent barrier to mechanistic discovery. We performed 16S rRNA amplicon sequencing of wildtype C57BL/6J mice, commonly used to study pathogen colonization, and found that the vaginal microbiome composition varies highly both within and between colonies from three distinct vivaria. Because of the strong influence of environmental exposure on vaginal microbiome composition, we assessed whether a humanized microbiota mouse ( HMb mice) would model a more human-like vaginal microbiota. Similar to humans and conventional mice, HMb mice vaginal microbiota clustered into five community state types ( h mCST). Uniquely, HMb mice vaginal communities were frequently dominated by Lactobacilli or Enterobacteriaceae . Compared to genetically-matched conventional mice, HMb mice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia , but no differences were observed with uropathogenic E. coli . Specifically, vaginal Enterobacteriaceae and Lactobacillus were associated with the absence of uterine GBS. Anti-GBS activity of HMb mice vaginal E. coli and L. murinus isolates, representing Enterobacteriaceae and Lactobacillus respectively, were characterized in vitro and in vivo . Although L. murinus reduced GBS growth in vitro , vaginal pre-inoculation with HMb mouse-derived E. coli , but not L. murinus , conferred protection against vaginal GBS burden. Overall, the HMb mice are an improved model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens. IMPORTANCE: An altered vaginal microbiota, typically with little to no levels of Lactobacillus , is associated with increased susceptibility to urogenital infections, although mechanisms driving this vulnerability are not fully understood. Despite known inhibitory properties of Lactobacillus against urogenital pathogens, clinical studies with Lactobacillus probiotics have shown mixed success. In this study, we characterize the impact of the vaginal microbiota on urogenital pathogen colonization using a humanized microbiota mouse model that more closely mimics the human vaginal microbiota. We found several vaginal bacterial taxa that correlated with reduced pathogen levels but showed discordant effects in pathogen inhibition between in vitro and in vivo assays. We propose that this humanized microbiota mouse platform is an improved model to describe the role of the vaginal microbiota in protection against urogenital pathogens. Furthermore, this model will be useful in testing efficacy of new probiotic strategies in the complex vaginal environment.

A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis.

Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

Systems biology approach to functionally assess the Clostridioides difficile pangenome reveals genetic diversity with discriminatory power.

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

Gut Microbiota From Sjögren syndrome Patients Causes Decreased T Regulatory Cells in the Lymphoid Organs and Desiccation-Induced Corneal Barrier Disruption in Mice.

Sjögren syndrome (SS) is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption. A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in SS patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome using stools collected from sex- and age-matched healthy (n = 20), dry eye (n = 4) and SS (n = 7) subjects. The observed Operational Taxonomic Units (OTUs) and Shannon alpha diversity were significantly decreased in SS compared to healthy controls, and there was a significant inverse correlation between observed OTUs and ocular severity score. We also identified specific bacterial strains that are differentially modulated in SS vs. healthy subjects. To investigate if the differential composition of intestinal microbiome would have an impact on the immune and eye phenotype, we performed functional studies using germ-free mice colonized with human intestinal microbiota from SS patients and healthy controls. Flow cytometry analysis demonstrated reduced frequency of CD4+ FOXP3+ cells in ocular draining cervical lymph nodes (CLN) in mice colonized with SS patient intestinal microbiota 4 weeks post-colonization. We also found that offspring of SS-humanized mice also have fewer CD4+FOXP3+ cells in the CLN as well as spleen, demonstrating vertical transmission. SS-humanized mice subjected to desiccating stress exhibited greater corneal barrier disruption as compared to healthy control humanized mice under the same conditions. Taken together, these data support the hypothesis that the intestinal microbiota can modulate ocular surface health, possibly by influencing development of CD4+ FOXP3+ regulatory T cells (Tregs) in the ocular draining lymph nodes.

Gut-derived butyrate suppresses ocular surface inflammation.

Dry eye is a common ocular inflammatory disorder characterized by tear film instability and reduced tear production. There is increasing evidence that homeostasis of the ocular surface is impacted by the intestinal microbiome. We are interested in investigating the potential role of microbially produced small molecules in mediating the interaction between the intestinal microbiota and the ocular surface. One such molecule is butyrate, a short-chain fatty acid (SCFA) produced by certain members of the gut microbiota through fermentation of dietary fiber. Here we show that SCFA transporter SLC5A8 is expressed in vivo in murine conjunctival and corneal epithelium. Pre-treatment of in vitro corneal epithelial cultures or bone marrow-derived dendritic cells (BMDCs) with phenylbutyrate (PBA) reduces lipopolysaccharide-induced pro-inflammatory Tnf expression. Corneal epithelial cultures and BMDCs isolated from Slc5a8 knockout mice are unable to respond to PBA pre-treatment, suggesting that SLC5A8 is required for the protective effect of PBA. The treatment of mice undergoing desiccating stress (DS) with oral tributyrin, a prodrug form of butyrate, reduces inflammation at the ocular surface in vivo, and this effect partially requires SLC5A8. Finally, expression analysis on conjunctival tissue isolated from mice subjected to DS with and without tributyrin treatment revealed that treatment downregulated genes involved in Type I interferon signaling. Together these data support our hypothesis that SCFAs produced in the gut participate in the maintenance of ocular surface homeostasis.

RbgA ensures the correct timing in the maturation of the 50S subunits functional sites.

RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

Ribosomal subunits in bacteria assemble according to energy landscapes comprised of multiple parallel pathways. In this study, the authors identified a critical maturation step in the late assembly stages of the large 50S ribosomal subunit in bacteria. This step represents a merging point where all parallel assembly pathways of the ribosomal particles converge. At this critical step, the convergent assembly intermediate that accumulates in cells exists in a 'locked' state, and its maturation is paused. The RbgA protein acts on this critical step to 'unlock' the last maturation steps involving folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

Differentially Expressed Gene Pathways in the Conjunctiva of Sjögren Syndrome Keratoconjunctivitis Sicca.

Sjögren syndrome (SS) is an autoimmune condition that targets the salivary and lacrimal glands, with cardinal clinical signs of dry eye (keratoconjunctivitis sicca, KCS) and dry mouth. The conjunctiva of SS patients is often infiltrated by immune cells that participate in the induction and maintenance of local inflammation. The purpose of this study was to investigate immune-related molecular pathways activated in the conjunctiva of SS patients. Female SS patients (n=7) and controls (n=19) completed a series of oral, ocular surface exams. Symptom severity scores were evaluated using validated questionnaires (OSDI and SANDE). All patients fulfilled the ACR/EULAR criteria for SS and the criteria for KCS. Fluorescein and lissamine green dye staining evaluated tear-break-up time (TBUT), corneal and conjunctival disease, respectively. Impression cytology of the temporal bulbar conjunctiva was performed to collect cells lysed and subjected to gene expression analysis using the NanoString Immunology Panel. 53/594 differentially expressed genes (DEGs) were observed between SS and healthy controls; 49 DEGs were upregulated, and 4 were downregulated (TRAF5, TGFBI, KLRAP1, and CMKLRI). The top 10 DEGs in descending order were BST2, IFITM1, LAMP3, CXCL1, IL19, CFB, LY96, MX1, IL4R, CDKN1A. Twenty pathways had a global significance score greater or equal to 2. Spearman correlations showed that 29/49 upregulated DEGs correlated with either TBUT (inverse) or OSDI or conjunctival staining score (positive correlations). Venn diagrams identified that 26/29 DEGs correlated with TBUT, 5/26 DEGs correlated with OSDI, and 16/26 correlated with conjunctival staining scores. Five upregulated DEGs (CFB, CFI, IL1R1, IL2RG, IL4R) were uniquely negatively correlated with TBUT. These data indicate that the conjunctiva of SS patients exhibits a phenotype of immune activation, although some genes could be inhibitory. Some of the DEGs and pathways overlap with previous DEGs in salivary gland biopsies, but new DEGs were identified, and some of these correlated with symptoms and signs of dry eye. Our results indicate that gene analysis of conjunctiva imprints is a powerful tool to understand the pathogenesis of SS and develop new therapeutic targets.

AMiGA: Software for Automated Analysis of Microbial Growth Assays.

The analysis of microbial growth is one of the central methods in the field of microbiology. Microbial growth dynamics can be characterized by meaningful parameters, including carrying capacity, exponential growth rate, and growth lag. However, microbial assays with clinical isolates, fastidious organisms, or microbes under stress often produce atypical growth shapes that do not follow the classical microbial growth pattern. Here, we introduce the analysis of microbial growth assays (AMiGA) software, which streamlines the analysis of growth curves without any assumptions about their shapes. AMiGA can pool replicates of growth curves and infer summary statistics for biologically meaningful growth parameters. In addition, AMiGA can quantify death phases and characterize diauxic shifts. It can also statistically test for differential growth under distinct experimental conditions. Altogether, AMiGA streamlines the organization, analysis, and visualization of microbial growth assays. IMPORTANCE Our current understanding of microbial physiology relies on the simple method of measuring microbial populations' sizes over time and under different conditions. Many advances have increased the throughput of those assays and enabled the study of nonlab-adapted microbes under diverse conditions that widely affect their growth dynamics. Our software provides an all-in-one tool for estimating the growth parameters of microbial cultures and testing for differential growth in a high-throughput and user-friendly fashion without any underlying assumptions about how microbes respond to their growth conditions.

The metabolic profile of Bifidobacterium dentium reflects its status as a human gut commensal.

BACKGROUND: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium. RESULTS: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment. CONCLUSIONS: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.

Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation.

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.

Human-Derived Bifidobacterium dentium Modulates the Mammalian Serotonergic System and Gut-Brain Axis.

BACKGROUND & AIMS: The human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood. METHODS: Adult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT-receptor expression in the brain, and 5-HT-receptor-dependent behavior was evaluated using the marble burying test. RESULTS: B dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium-secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT-receptor 2a in B dentium-treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT-receptor 2a. CONCLUSIONS: These data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior.

Probiotics and the Microbiome-How Can We Help Patients Make Sense of Probiotics?

The notion of probiotics as microbes that confer health benefits has its origins in the speculative ideas that are more than a century old, yet remain largely unsubstantiated by scientific evidence. The recent advances in microbiome science have highlighted the importance of intestinal microbes in human physiology and disease pathogenesis. These developments have provided a boost to the probiotics industry, which continues to experience exponential growth driven mainly by creative marketing. Consumers, patients, and most health care providers are not able to discern the underlying science or differentiate the permitted claims that promise vague health benefits from disease-specific claims reserved for drugs. No probiotic product has been able to satisfy the regulatory requirements to be categorized as a drug, a substance intended to cure, mitigate, or prevent disease. However, patients take probiotic products in the belief that they will help to treat their intestinal or systemic diseases. Thus far, the regulators have failed to create policies that would assist to inform the public in this area. In fact, the existing regulatory regime actually creates formidable barriers to research that could provide evidence for clinical efficacy of probiotic products. We propose a potential solution to this vexing problem, where a committee created through a partnership of academia, professional organizations, and industry, but free of potential conflicts of interest, would be charged with rigorous evaluation of specific probiotic products and the evidence in support of their different claims. Companies that would submit to this process would earn the trust of consumers and healthcare providers, as well as a distinction in the marketplace.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.