The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut.

Chronic stress disrupts microbiota-gut-brain axis function and is associated with altered tryptophan metabolism, impaired gut barrier function, and disrupted diurnal rhythms. However, little is known about the effects of acute stress on the gut and how it is influenced by diurnal physiology. Here, we used germ-free and antibiotic-depleted mice to understand how microbiota-dependent oscillations in tryptophan metabolism would alter gut barrier function at baseline and in response to an acute stressor. Cecal metabolomics identified tryptophan metabolism as most responsive to a 15-min acute stressor, while shotgun metagenomics revealed that most bacterial species exhibiting rhythmicity metabolize tryptophan. Our findings highlight that the gastrointestinal response to acute stress is dependent on the time of day and the microbiome, with a signature of stress-induced functional alterations in the ileum and altered tryptophan metabolism in the colon.

The impact of acute and chronic stress on gastrointestinal physiology and function: a microbiota-gut-brain axis perspective.

The physiological consequences of stress often manifest in the gastrointestinal tract. Traumatic or chronic stress is associated with widespread maladaptive changes throughout the gut, although comparatively little is known about the effects of acute stress. Furthermore, these stress-induced changes in the gut may increase susceptibility to gastrointestinal disorders and infection, and impact critical features of the neural and behavioural consequences of the stress response by impairing gut-brain axis communication. Understanding the mechanisms behind changes in enteric nervous system circuitry, visceral sensitivity, gut barrier function, permeability, and the gut microbiota following stress is an important research objective with pathophysiological implications in both neurogastroenterology and psychiatry. Moreover, the gut microbiota has emerged as a key aspect of physiology sensitive to the effects of stress. In this review, we focus on different aspects of the gastrointestinal tract including gut barrier function as well as the immune, humoral and neuronal elements involved in gut-brain communication. Furthermore, we discuss the evidence for a role of stress in gastrointestinal disorders. Existing gaps in the current literature are highlighted, and possible avenues for future research with an integrated physiological perspective have been suggested. A more complete understanding of the spatial and temporal dynamics of the integrated host and microbial response to different kinds of stressors in the gastrointestinal tract will enable full exploitation of the diagnostic and therapeutic potential in the fast-evolving field of host-microbiome interactions.

Meeting report of the sixth annual tri-service microbiome consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing amongst consortium members, which includes collaborators in academia and industry. The 6th Annual TSMC Symposium was a hybrid meeting held in Fairlee, Vermont on 27-28 September 2022 with presentations and discussions centered on microbiome-related topics within seven broad thematic areas: (1) Human Microbiomes: Stress Response; (2) Microbiome Analysis & Surveillance; (3) Human Microbiomes Enablers & Engineering; (4) Human Microbiomes: Countermeasures; (5) Human Microbiomes Discovery - Earth & Space; (6) Environmental Micro & Myco-biome; and (7) Environmental Microbiome Analysis & Engineering. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the activities and outcomes from the 6th annual TSMC symposium.

Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor.

Bile acids play an important role in digestion and human health, are found throughout the gastrointestinal tract, and are excreted in feces. Therefore, bile acids are promising biomarkers for monitoring health and detecting fecal contamination in water sources. Here, we engineered a bile acid sensor by expressing the transcription factor BreR, a TetR-like repressor from Vibrio cholorae, in Escherichia coli. The sensor was further optimized by screening a promoter library. To further characterize the BreR sensor and increase its utility, we moved expression to a cell-free expression (CFE) system, resulting in an approximately 3 orders of magnitude increase in deoxycholic acid sensitivity. We next optimized this sensor to detect bile acids in fecal water, wastewater, and serum and transferred the CFE sensor to a paper-based assay to enhance fieldability.

Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

Exploring Changes in the Host Gut Microbiota During a Controlled Human Infection Model for Campylobacter jejuni.

Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 105 colony-forming units of C. jejuni; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections.

Meeting report of the fourth annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations. The annual TSMC symposium is designed to enable information sharing between DoD scientists and leaders in the field of microbiome science, thereby keeping DoD consortium members informed of the latest advances within the microbiome community and facilitating the development of new collaborative research opportunities. The 2020 annual symposium was held virtually on 24-25 September 2020. Presentations and discussions centered on microbiome-related topics within four broad thematic areas: (1) Enabling Technologies; (2) Microbiome for Health and Performance; (3) Environmental Microbiome; and (4) Microbiome Analysis and Discovery. This report summarizes the presentations and outcomes of the 4th annual TSMC symposium.

In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, mono- or co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic's persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.

Epidemiology and associated microbiota changes in deployed military personnel at high risk of traveler's diarrhea.

Travelers' diarrhea (TD) is the most prevalent illness encountered by deployed military personnel and has a major impact on military operations, from reduced job performance to lost duty days. Frequently, the etiology of TD is unknown and, with underreporting of cases, it is difficult to accurately assess its impact. An increasing number of ailments include an altered or aberrant gut microbiome. To better understand the relationships between long-term deployments and TD, we studied military personnel during two nine-month deployment cycles in 2015-2016 to Honduras. To collect data on the prevalence of diarrhea and impact on duty, a total of 1173 personnel completed questionnaires at the end of their deployment. 56.7% reported reduced performance and 21.1% reported lost duty days. We conducted a passive surveillance study of all cases of diarrhea reporting to the medical unit with 152 total cases and a similar pattern of etiology. Enteroaggregative E. coli (EAEC, 52/152), enterotoxigenic E. coli (ETEC, 50/152), and enteropathogenic E. coli (EPEC, 35/152) were the most prevalent pathogens detected. An active longitudinal surveillance of 67 subjects also identified diarrheagenic E. coli as the primary etiology (7/16 EPEC, 7/16 EAEC, and 6/16 ETEC). Eleven subjects were recruited into a nested longitudinal substudy to examine gut microbiome changes associated with deployment. A 16S rRNA amplicon survey of fecal samples showed differentially abundant baseline taxa for subjects who contracted TD versus those who did not, as well as detection of taxa positively associated with self-reported gastrointestinal distress. Disrupted microbiota was also qualitatively observable for weeks preceding and following the incidents of TD. These findings illustrate the complex etiology of diarrhea amongst military personnel in deployed settings and its impacts on job performance. Potential factors of resistance or susceptibility can provide a foundation for future clinical trials to evaluate prevention and treatment strategies.

Meeting report of the third annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Evaluation of Probiotics for Warfighter Health and Performance.

The probiotic industry continues to grow in both usage and the diversity of products available. Scientific evidence supports clinical use of some probiotic strains for certain gastrointestinal indications. Although much less is known about the impact of probiotics in healthy populations, there is increasing consumer and scientific interest in using probiotics to promote physical and psychological health and performance. Military men and women are a unique healthy population that must maintain physical and psychological health in order to ensure mission success. In this narrative review, we examine the evidence regarding probiotics and candidate probiotics for physical and/or cognitive benefits in healthy adults within the context of potential applications for military personnel. The reviewed evidence suggests potential for certain strains to induce biophysiological changes that may offer physical and/or cognitive health and performance benefits in military populations. However, many knowledge gaps exist, effects on health and performance are generally not widespread among the strains examined, and beneficial findings are generally limited to single studies with small sample sizes. Multiple studies with the same strains and using similar endpoints are needed before definitive recommendations for use can be made. We conclude that, at present, there is not compelling scientific evidence to support the use of any particular probiotic(s) to promote physical or psychological performance in healthy military personnel. However, plausibility for physical and psychological health and performance benefits remains, and additional research is warranted. In particular, research in military cohorts would aid in assessing the value of probiotics for supporting physical and psychological health and performance under the unique demands required of these populations.

Gut-brain axis serotonergic responses to acute stress exposure are microbiome-dependent.

BACKGROUND: Understanding the mechanisms underpinning the response to acute stress is critical for determining how this can be modulated in both health and disease and across sexes. Stress can markedly alter the microbiome and gut-brain axis signaling with the serotonergic system being particularly sensitive to acute stress. As the impact of acute stress on regional serotonergic dynamics in the gut-brain axis and the contribution of the microbiome to this are poorly appreciated, we used microbiota-deficient mice to assess whether the serotonergic response to acute stress exposure is microbiome dependent. METHODS: Adult male and female conventional, germ-free, and colonized germ-free mice underwent a single acute stressor and samples were harvested immediately or 45 minutes following stress. Serotonin and related metabolites and serotonergic gene expression were determined. KEY RESULTS: Our data clearly show the microbiota influenced gastrointestinal serotonergic response to acute stress in a sex- and region-dependent manner. Male-specific poststress increases in colonic serotonin were absent in germ-free mice but normalized following colonization. mRNA serotonergic gene expression was differentially expressed in colon and ileum of germ-free mice on a sex-dependent basis. Within the frontal cortex, absence of the microbiome altered basal serotonin, its main metabolite 5-hydroxyindoleacetic acid, and prevented stress-induced increases in serotonin turnover. CONCLUSIONS AND INFERENCES: The gut microbiome influences the set points of the brain and gastrointestinal serotonergic systems and affected their response to acute stress in a sex- and region-dependent manner.

A Pilot Study of the Effect of Deployment on the Gut Microbiome and Traveler's Diarrhea Susceptibility.

Traveler's diarrhea (TD) is a recurrent and significant issue for many travelers including the military. While many known enteric pathogens exist that are causative agents of diarrhea, our gut microbiome may also play a role in TD susceptibility. To this end, we conducted a pilot study of the microbiome of warfighters prior to- and after deployment overseas to identify marker taxa relevant to TD. This initial study utilized full-length 16S rRNA gene sequencing to provide additional taxonomic resolution toward identifying predictive taxa.16S rRNA analyses of pre- and post-deployment fecal samples identified multiple marker taxa as significantly differentially abundant in subjects that reported diarrhea, including Weissella, Butyrivibrio, Corynebacterium, uncultivated Erysipelotrichaceae, Jeotgallibaca, unclassified Ktedonobacteriaceae, Leptolinea, and uncultivated Ruminiococcaceae. The ability to identify TD risk prior to travel will inform prevention and mitigation strategies to influence diarrhea susceptibility while traveling.

The role of the microbiota in acute stress-induced myeloid immune cell trafficking.

There has been a growing recognition of the involvement of the gastrointestinal microbiota in the development of stress-related disorders. Acute stress leads to activation of neuroendocrine systems, which in turn orchestrate a large-scale redistribution of innate immune cells. Both these response systems are independently known to be primed by the microbiota, even though much is still unclear about the role of the gastrointestinal microbiota in acute stress-induced immune activation. In this study, we investigated whether the microbiota influences acute stress-induced changes in innate immunity using conventionally colonised mice, mice devoid of any microbiota (i.e. germ-free, GF), and colonised GF mice (CGF). We also explored the kinetics of stress-induced immune cell mobilisation in the blood, the spleen and mesenteric lymph nodes (MLNs). Mice were either euthanised prior to stress or underwent restraint stress and were then euthanised at various time points (i.e. 0, 45- and 240-minutes) post-stress. Plasma adrenaline and noradrenaline levels were analysed using ELISA and immune cell levels were quantified using flow cytometry. GF mice had increased baseline levels of adrenaline and noradrenaline, of which adrenaline was normalised in CGF mice. In tandem, GF mice had decreased circulating levels of LY6Chi and LY6Cmid, CCR2+ monocytes, and granulocytes, but not LY6C-, CX3CR1+ monocytes. These deficits were normalised in CGF mice. Acute stress decreased blood LY6Chi and LY6Cmid, CCR2+ monocytes while increasing granulocyte levels in all groups 45 min post-stress. However, only GF mice showed stress-induced changes in LY6Chi monocytes and granulocytes 240 min post-stress, indicating impairments in the recovery from acute stress-induced changes in levels of specific innate immune cell types. LY6C-, CX3CR1+ monocytes remained unaffected by stress, indicating that acute stress impacts systemic innate immunity in a cell-type-specific manner. Overall, these data reveal novel cell-type-specific changes in the innate immune system in response to acute stress, which in turn are impacted by the microbiota. In conclusion, the microbiota influences the priming and recovery of the innate immune system to an acute stressor and may inform future microbiota-targeted therapeutics aimed at modulating stress-induced immune activation in stress-related disorders.

Amplifying Riboswitch Signal Output Using Cellular Wiring.

If fieldable riboswitch-based biological sensors are to fulfill their potential, it is necessary to increase their signal output. Here we report a novel modular amplification system using a riboswitch to initiate signaling between a sensing strain and a reporter strain of E. coli. A quorum sensing signaling molecule biologically wires the sensing and reporter strains together. The amplification circuit increased the amount of fluorescence generated on ligand binding compared to when the riboswitch controlled fluorescence expression directly. This had the corollary effect of increasing the sensitivity of the system, and allowed riboswitch-based reporting in E. coli strains that did not produce a detectable output when the riboswitch directly controlled reporter expression. The amplification circuit also reduced the time required to detect a signal output. The modularity of this amplification system coupled with the achievable increases in output can advance the development of riboswitches and biological sensors.

Riboswitch-Based Reversible Dual Color Sensor.

Riboswitches are RNA-based "sensors" that utilize chemically induced structural changes in the 5'-untranslated region of mRNA to regulate expression of downstream genes. Coupling a specific riboswitch with a reporter gene system translates chemical detection by the cell into a quantifiable reporter protein signal. For the majority of reporter gene systems, the readout signal is only expressed in the presence of the target analyte. This makes it difficult to determine the viability and localization of the uninduced biosensor when it is used for "real-word" applications. To address this problem, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). Thus, the sensor is always fluorescent, but its color is determined by detection of a specific analyte. We demonstrate that the recombinase-based dual-color reporter can be successfully applied to monitor the activation of a theophylline synthetic riboswitch that was used as our model system. To show the feasibility of the FimE recombinase-based system to serve as a reporter for monitoring activation of multiple synthetic riboswitches and, therefore, expand the applicability of the system, we tested a number of previously developed synthetic riboswitches responsive to different analytes. We show that the dual-color reporter system can be successfully used to monitor activation of M6 and M6″ riboswitches responsive to ammeline and pyrimido[4,5-d]pyrimidine-2,4-diamine, respectively, and a 2,4,6-trinitrotoluene-responsive riboswitch developed in this study. We also demonstrate that the system can be reversed by HbiF recombinase-mediated fimS inversion to the initial state of the fluorescent reporter, creating a resettable and reusable cell-based sensor.

Integrating and amplifying signal from riboswitch biosensors.

Biosensors offer a built-in energy supply and inherent sensing machinery that when exploited correctly may surpass traditional sensors. However, biosensor systems have been hindered by a narrow range of ligand detection capabilities, a relatively low signal output, and their inability to integrate multiple signals. Integration of signals could increase the specificity of the sensor and enable detection of a combination of ligands that may indicate environmental or developmental processes when detected together. Amplifying biosensor signal output will increase detector sensitivity and detection range. Riboswitches offer the potential to widen the diversity of ligands that may be detected, and advances in synthetic biology are illuminating myriad possibilities in signal processing using an orthogonal parts-based engineering approach. In this chapter, we describe the design, building, and testing of a riboswitch-based Boolean logic AND gate in bacteria, where an output requires the activation of two riboswitches, and the biological circuitry required to amplify the output of the AND gate using natural extracellular bacterial communication signals to "wire" cells together.

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

Elucidation of small RNAs that activate transcription in bacteria.

Small non-coding RNA (sRNA) control of gene expression has been shown to play a prominent role in genetic regulation. While the majority of identified bacterial sRNAs exert their control at the translational level, a few examples of bacterial sRNAs that inhibit transcription have also been identified. Using an engineered combinatorial RNA library, we have elucidated bacterial sRNAs that activate transcription of a target gene in E. coli to varying degrees. Mutation of the strongest activator modified its activation potential. Our results suggest that transcriptional activation of our target gene results from recruitment of the bacterial RNA polymerase complex to the promoter region. These data, coupled with the malleability of RNA, provide a context to define synthetic control of genes in bacteria at the transcriptional level.

Identification and molecular characterization of a complement C3 molecule in a lophotrochozoan, the Hawaiian bobtail squid Euprymna scolopes.

Examination of the EST database of the light organ of the Hawaiian bobtail squid Euprymna scolopes revealed a sequence with similarity to complement C3. RACE yielded the full open reading frame of this protein. Analysis of the resultant sequence revealed that Es-C3 (E. scolopes-C3) has conserved residues and domains known to be critical for C3 function. The gene encoding C3 was expressed in all tissues tested, indicating that its expression is widely distributed throughout the animal's body. Immunocytochemistry using an antibody against Es-C3 revealed that the protein is produced principally in the apical surfaces of epithelial cells. The finding of the gene encoding C3 in this mollusk extends the occurrence of this molecule to the lophotrochozoans, demonstrating that complement genes occur in all major branches of the animal kingdom.