Vasoactive Intestinal Polypeptide Plays a Key Role in the Microbial-Neuroimmune Control of Intestinal Motility.

BACKGROUND & AIMS: Although chronic diarrhea and constipation are common, the treatment is symptomatic because their pathophysiology is poorly understood. Accumulating evidence suggests that the microbiota modulates gut function, but the underlying mechanisms are unknown. We therefore investigated the pathways by which microbiota modulates gastrointestinal motility in different sections of the alimentary tract. METHODS: Gastric emptying, intestinal transit, muscle contractility, acetylcholine release, gene expression, and vasoactive intestinal polypeptide (VIP) immunoreactivity were assessed in wild-type and Myd88-/-Trif-/- mice in germ-free, gnotobiotic, and specific pathogen-free conditions. Effects of transient colonization and antimicrobials as well as immune cell blockade were investigated. VIP levels were assessed in human full-thickness biopsies by Western blot. RESULTS: Germ-free mice had similar gastric emptying but slower intestinal transit compared with specific pathogen-free mice or mice monocolonized with Lactobacillus rhamnosus or Escherichia coli, the latter having stronger effects. Although muscle contractility was unaffected, its neural control was modulated by microbiota by up-regulating jejunal VIP, which co-localized with and controlled cholinergic nerve function. This process was responsive to changes in the microbial composition and load and mediated through toll-like receptor signaling, with enteric glia cells playing a key role. Jejunal VIP was lower in patients with chronic intestinal pseudo-obstruction compared with control subjects. CONCLUSIONS: Microbial control of gastrointestinal motility is both region- and bacteria-specific; it reacts to environmental changes and is mediated by innate immunity-neural system interactions. By regulating cholinergic nerves, small intestinal VIP plays a key role in this process, thus providing a new therapeutic target for patients with motility disorders.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Mouse models for bacterial enteropathogen infections: insights into the role of colonization resistance.

Globally, enteropathogenic bacteria are a major cause of morbidity and mortality.1-3 Campylobacter, Salmonella, Shiga-toxin-producing Escherichia coli, and Listeria are among the top five most commonly reported zoonotic pathogens in the European Union.4 However, not all individuals naturally exposed to enteropathogens go on to develop disease. This protection is attributable to colonization resistance (CR) conferred by the gut microbiota, as well as an array of physical, chemical, and immunological barriers that limit infection. Despite their importance for human health, a detailed understanding of gastrointestinal barriers to infection is lacking, and further research is required to investigate the mechanisms that underpin inter-individual differences in resistance to gastrointestinal infection. Here, we discuss the current mouse models available to study infections by non-typhoidal Salmonella strains, Citrobacter rodentium (as a model for enteropathogenic and enterohemorrhagic E. coli), Listeria monocytogenes, and Campylobacter jejuni. Clostridioides difficile is included as another important cause of enteric disease in which resistance is dependent upon CR. We outline which parameters of human infection are recapitulated in these mouse models, including the impact of CR, disease pathology, disease progression, and mucosal immune response. This will showcase common virulence strategies, highlight mechanistic differences, and help researchers from microbiology, infectiology, microbiome research, and mucosal immunology to select the optimal mouse model.

The stationary phase-specific sRNA FimR2 is a multifunctional regulator of bacterial motility, biofilm formation and virulence.

Bacterial pathogens employ a plethora of virulence factors for host invasion, and their use is tightly regulated to maximize infection efficiency and manage resources in a nutrient-limited environment. Here we show that during Escherichia coli stationary phase the 3' UTR-derived small non-coding RNA FimR2 regulates fimbrial and flagellar biosynthesis at the post-transcriptional level, leading to biofilm formation as the dominant mode of survival under conditions of nutrient depletion. FimR2 interacts with the translational regulator CsrA, antagonizing its functions and firmly tightening control over motility and biofilm formation. Generated through RNase E cleavage, FimR2 regulates stationary phase biology by fine-tuning target mRNA levels independently of the chaperones Hfq and ProQ. The Salmonella enterica orthologue of FimR2 induces effector protein secretion by the type III secretion system and stimulates infection, thus linking the sRNA to virulence. This work reveals the importance of bacterial sRNAs in modulating various aspects of bacterial physiology including stationary phase and virulence.

A protocol for generating germ-free Heligmosomoides polygyrus bakeri larvae for gnotobiotic helminth infection studies.

The microbes indigenous to helminth species are a major obstacle to deciphering host-parasite interactions. Repurposing a system of reversible bacterial colonization, we have generated germ-free Heligomosomoides polygyrus bakeri (Hpb) larvae that maintain the sterility of axenic mice upon infection. This protocol provides a valuable tool for controlled studies of helminth-microbiota-immune interactions.

Robust microbe immune recognition in the intestinal mucosa.

The mammalian mucosal immune system acts as a multitasking mediator between bodily function and a vast diversity of microbial colonists. Depending on host-microbial interaction type, mucosal immune responses have distinct functions. Immunity to pathogen infection functions to limit tissue damage, clear or contain primary infection, and prevent or lower the severity of a secondary infection by conferring specific long-term adaptive immunity. Responses to nonpathogenic commensal or mutualistic microbes instead function to tolerate continuous colonization. Mucosal innate immune and epithelial cells employ a limited repertoire of innate receptors to program the adaptive immune response accordingly. Pathogen versus nonpathogen immune discrimination appears to be very robust, as most individuals successfully maintain life-long mutualism with their nonpathogenic microbiota, while mounting immune defense to pathogenic microbe infection specifically. However, the process is imperfect, which can have immunopathological consequences, but may also be exploited medically. Normally innocuous intestinal commensals in some individuals may drive serious inflammatory autoimmunity, whereas harmless vaccines can be used to fool the immune system into mounting a protective anti-pathogen immune response. In this article, we review the current knowledge on mucosal intestinal bacterial immune recognition focusing on TH17 responses and identify commonalities between intestinal pathobiont and vaccine-induced TH17 responses.

Plant chemistry and food web health.

Plants are systemically relevant to our planet not only by constituting a major part of its biomass, but also because they produce a vast diversity of bioactive phytochemicals. These compounds often modulate interactions between plants and the environment, and can have substantial effects on plant consumers and their health. By taking a food web perspective, we highlight the role of bioactive phytochemicals in linking soils, plants, animals and humans and discuss their contributions to systems health. The analysis of connections among food web components revealed an underexplored potential of phytochemicals to optimize food web health and productivity.

Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions.

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

Respiratory tissue-associated commensal bacteria offer therapeutic potential against pneumococcal colonization.

Under eubiotic conditions commensal microbes are known to provide a competitive barrier against invading bacterial pathogens in the intestinal tract, on the skin or on the vaginal mucosa. Here, we evaluate the role of lung microbiota in Pneumococcus colonization of the lungs. In eubiosis, the lungs of mice were dominantly colonized by Lactobacillus murinus. Differential analysis of 16S rRNA gene sequencing or L. murinus-specific qPCR of DNA from total organ homogenates vs.broncho alveolar lavages implicated tight association of these bacteria with the host tissue. Pure L. murinus conditioned culture medium inhibited growth and reduced the extension of pneumococcal chains. Growth inhibition in vitro was likely dependent on L. murinus-produced lactic acid, since pH neutralization of the conditioned medium aborted the antibacterial effect. Finally, we demonstrate that L. murinus provides a barrier against pneumococcal colonization in a respiratory dysbiosis model after an influenza A virus infection, when added therapeutically.

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type.

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.

Biogeography of microbial bile acid transformations along the murine gut.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Publisher Correction: Engineering bacterial symbionts of nematodes improves their biocontrol potential to counter the western corn rootworm.

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

Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection.

Paneth cells promote angiogenesis and regulate portal hypertension in response to microbial signals.

BACKGROUND & AIMS: Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure. METHODS: We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). In vivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation. RESULTS: Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. In vivo endomicroscopy showed decreased vascular proliferation in the absence of PCs. CONCLUSIONS: These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension. LAY SUMMARY: Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.

Engineering bacterial symbionts of nematodes improves their biocontrol potential to counter the western corn rootworm.

The western corn rootworm (WCR) decimates maize crops worldwide. One potential way to control this pest is treatment with entomopathogenic nematodes (EPNs) that harbor bacterial symbionts that are pathogenic to insects. However, WCR larvae sequester benzoxazinoid secondary metabolites that are produced by maize and use them to increase their resistance to the nematodes and their symbionts. Here we report that experimental evolution and selection for bacterial symbionts that are resistant to benzoxazinoids improve the ability of a nematode-symbiont pair to kill WCR larvae. We isolated five Photorhabdus symbionts from different nematodes and increased their benzoxazinoid resistance through experimental evolution. Benzoxazinoid resistance evolved through multiple mechanisms, including a mutation in the aquaporin-like channel gene aqpZ. We reintroduced benzoxazinoid-resistant Photorhabdus strains into their original EPN hosts and identified one nematode-symbiont pair that was able to kill benzoxazinoid-sequestering WCR larvae more efficiently. Our results suggest that modification of bacterial symbionts might provide a generalizable strategy to improve biocontrol of agricultural pests.

In vitro and in vivo characterization of Clostridium scindens bile acid transformations.

The human gut hosts trillions of microorganisms that exert a profound influence on human biology. Gut bacteria communicate with their host by secreting small molecules that can signal to distant organs in the body. Bile acids are one class of these signaling molecules, synthesized by the host and chemically transformed by the gut microbiota. Among bile acid metabolizers, bile acid 7-dehydroxylating bacteria are commensals of particular importance as they carry out the 7-dehydroxylation of liver-derived primary bile acids to 7-dehydroxylated bile acids. The latter represents a major fraction of the secondary bile acid pool. The microbiology of this group of gut microorganisms is understudied and warrants more attention. Here, we detail the bile acid transformations carried out by the 7-dehydroxylating bacterium Clostridium scindens in vitro and in vivo. In vitro, C. scindens exhibits not only 7α-dehydroxylating capabilities but also, the ability to oxidize other hydroxyl groups and reduce ketone groups in primary and secondary bile acids. This study revealed 12-oxolithocholic acid as a major transient product in the 7α-dehydroxylation of cholic acid. Furthermore, the in vivo study included complementing a gnotobiotic mouse line (devoid of the ability to 7-dehydroxylate bile acids) with C. scindens and investigating its colonization dynamics and bile acid transformations. Using NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry), we demonstrate that the large intestine constitutes a niche for C. scindens, where it efficiently 7-dehydroxylates cholic acid to deoxycholic acid. Overall, this work reveals a novel transient species during 7-dehydroxylation as well as provides direct evidence for the colonization and growth of 7-dehydroxylating bacteria in the large intestine.

Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response.

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.