Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

Electrostatic Interactions Dictate Bile Salt Hydrolase Substrate Preference.

The human intestines are colonized by trillions of microbes, comprising the gut microbiota, which produce diverse small molecule metabolites and modify host metabolites, such as bile acids, that regulate host physiology. Biosynthesized in the liver, bile acids are conjugated with glycine or taurine and secreted into the intestines, where gut microbial bile salt hydrolases (BSHs) deconjugate the amino acid to produce unconjugated bile acids that serve as precursors for secondary bile acid metabolites. Among these include a recently discovered class of microbially conjugated bile acids (MCBAs), wherein alternative amino acids are conjugated onto bile acids. To elucidate the metabolic potential of MCBAs, we performed detailed kinetic studies to investigate the preference of BSHs for host-conjugated bile acids and MCBAs. We identified a BSH that exhibits positive cooperativity uniquely for MCBAs containing an aromatic side chain. Further molecular modeling and phylogenetic analyses indicated that the BSH preference for aromatic MCBAs is due to a substrate-specific cation-π interaction and is predicted to be widespread among human gut microbial BSHs.

Shedding Light on Bacterial Physiology with Click Chemistry.

Bacteria constitute a major lifeform on this planet and play numerous roles in ecology, physiology, and human disease. However, conventional methods to probe their activities are limited in their ability to visualize and identify their functions in these diverse settings. In the last two decades, the application of click chemistry to label these microbes has deepened our understanding of bacterial physiology. With the development of a plethora of chemical tools that target many biological molecules, it is possible to track these microorganisms in real-time and at unprecedented resolution. Here, we review click chemistry, including bioorthogonal reactions, and their applications in imaging bacterial glycans, lipids, proteins, and nucleic acids using chemical reporters. We also highlight significant advances that have enabled biological discoveries that have heretofore remained elusive.

Chemoproteomic Approaches for Unraveling Prokaryotic Biology.

Bacteria are ubiquitous lifeforms with important roles in the environment, biotechnology, and human health. Many of the functions that bacteria perform are mediated by proteins and enzymes, which catalyze metabolic transformations of small molecules and modifications of proteins. To better understand these biological processes, chemical proteomic approaches, including activity-based protein profiling, have been developed to interrogate protein function and enzymatic activity in physiologically relevant contexts. Here, chemoproteomic strategies and technological advances for studying bacterial physiology, pathogenesis, and metabolism are discussed. The development of chemoproteomic approaches for characterizing protein function and enzymatic activity within bacteria remains an active area of research, and continued innovations are expected to provide breakthroughs in understanding bacterial biology.

Microbial tryptophan metabolites regulate gut barrier function via the aryl hydrocarbon receptor.

Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis, are associated with dysbiosis of the gut microbiome. Emerging evidence suggests that small-molecule metabolites derived from bacterial breakdown of a variety of dietary nutrients confer a wide array of host benefits, including amelioration of inflammation in IBDs. Yet, in many cases, the molecular pathways targeted by these molecules remain unknown. Here, we describe roles for three metabolites-indole-3-ethanol, indole-3-pyruvate, and indole-3-aldehyde-which are derived from gut bacterial metabolism of the essential amino acid tryptophan, in regulating intestinal barrier function. We determined that these metabolites protect against increased gut permeability associated with a mouse model of colitis by maintaining the integrity of the apical junctional complex and its associated actin regulatory proteins, including myosin IIA and ezrin, and that these effects are dependent on the aryl hydrocarbon receptor. Our studies provide a deeper understanding of how gut microbial metabolites affect host defense mechanisms and identify candidate pathways for prophylactic and therapeutic treatments for IBDs.

Engineered Th17 Cell Differentiation Using a Photoactivatable Immune Modulator.

T helper 17 (Th17) cells, an important subset of CD4+ T cells, help to eliminate extracellular infectious pathogens that have invaded our tissues. Despite the critical roles of Th17 cells in immunity, how the immune system regulates the production and maintenance of this cell type remains poorly understood. In particular, the plasticity of these cells or their dynamic ability to trans-differentiate into other CD4+ T cell subsets remains mostly uncharacterized. Here, we report a synthetic immunology approach using a photoactivatable immune modulator (PIM) to increase Th17 cell differentiation on demand with spatial and temporal precision to help elucidate this important and dynamic process. In this chemical strategy, we developed a latent agonist that upon photochemical activation releases a small-molecule ligand that targets the aryl hydrocarbon receptor (AhR) and ultimately induces Th17 cell differentiation. We used this chemical tool to control AhR activation with spatiotemporal precision within cells and to modulate Th17 cell differentiation on demand using UV light illumination. We envision that this approach will enable an understanding of the dynamic functions and behaviors of Th17 cells in vivo during immune responses and in mouse models of inflammatory disease.

The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition.

Given the trillions of microbes that inhabit the mammalian intestines, the host immune system must constantly maintain a balance between tolerance to commensals and immunity against pathogens to avoid unnecessary immune responses against otherwise harmless bacteria. Misregulated responses can lead to inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. The mechanisms by which the immune system maintains this critical balance remain largely undefined. Here, we demonstrate that the short-chain fatty acid n-butyrate, which is secreted in high amounts by commensal bacteria, can modulate the function of intestinal macrophages, the most abundant immune cell type in the lamina propria. Treatment of macrophages with n-butyrate led to the down-regulation of lipopolysaccharide-induced proinflammatory mediators, including nitric oxide, IL-6, and IL-12, but did not affect levels of TNF-α or MCP-1. These effects were independent of toll-like receptor signaling and activation of G-protein-coupled receptors, two pathways that could be affected by short-chain fatty acids. In this study, we provide several lines of evidence that suggest that these effects are due to the inhibition of histone deacetylases by n-butyrate. These findings elucidate a pathway in which the host may maintain tolerance to intestinal microbiota by rendering lamina propria macrophages hyporesponsive to commensal bacteria through the down-regulation of proinflammatory effectors.

Chemoproteomic profiling of substrate specificity in gut microbiota-associated bile salt hydrolases.

The gut microbiome possesses numerous biochemical enzymes that biosynthesize metabolites that impact human health. Bile acids comprise a diverse collection of metabolites that have important roles in metabolism and immunity. The gut microbiota-associated enzyme that is responsible for the gateway reaction in bile acid metabolism is bile salt hydrolase (BSH), which controls the host's overall bile acid pool. Despite the critical role of these enzymes, the ability to profile their activities and substrate preferences remains challenging due to the complexity of the gut microbiota, whose metaproteome includes an immense diversity of protein classes. Using a systems biochemistry approach employing activity-based probes, we have identified gut microbiota-associated BSHs that exhibit distinct substrate preferences, revealing that different microbes contribute to the diversity of the host bile acid pool. We envision that this chemoproteomic approach will reveal how secondary bile acid metabolism controlled by BSHs contributes to the etiology of various inflammatory diseases.

Chemoproteomic profiling of substrate specificity in gut microbiota-associated bile salt hydrolases.

The gut microbiome possesses numerous biochemical enzymes that biosynthesize metabolites that impact human health. Bile acids comprise a diverse collection of metabolites that have important roles in metabolism and immunity. The gut microbiota-associated enzyme that is responsible for the gateway reaction in bile acid metabolism is bile salt hydrolase (BSH), which controls the host's overall bile acid pool. Despite the critical role of these enzymes, the ability to profile their activities and substrate preferences remains challenging due to the complexity of the gut microbiota, whose metaproteome includes an immense diversity of protein classes. Using a systems biochemistry approach employing activity-based probes, we have identified gut microbiota-associated BSHs that exhibit distinct substrate preferences, revealing that different microbes contribute to the diversity of the host bile acid pool. We envision that this chemoproteomic approach will reveal how secondary bile acid metabolism controlled by BSHs contributes to the etiology of various inflammatory diseases.

Copper-free click chemistry in living animals.

Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as "Cu-free click chemistry," for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac(4)ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.

Electrostatic Interactions Dictate Bile Salt Hydrolase Substrate Preference.

The human intestines are colonized by trillions of microbes, comprising the gut microbiota, which produce diverse small molecule metabolites and modify host metabolites, such as bile acids, that regulate host physiology. Biosynthesized in the liver, bile acids are conjugated with glycine or taurine and secreted into the intestines, where gut microbial bile salt hydrolases (BSHs) deconjugate the amino acid to produce unconjugated bile acids that serve as precursors for secondary bile acid metabolites. Among these include a recently discovered class of microbially-conjugated bile acids (MCBAs), wherein alternative amino acids are conjugated onto bile acids. To elucidate the metabolic potential of MCBAs, we performed detailed kinetic studies to investigate the preference of BSHs for host- and microbially-conjugated bile acids. We identified a BSH that exhibits positive cooperativity uniquely for MCBAs containing an aromatic sidechain. Further molecular modeling and phylogenetic analyses indicated that BSH preference for aromatic MCBAs is due to a substrate-specific cation-π interaction and is predicted to be widespread among human gut microbial BSHs.

Activity-based protein profiling in microbes and the gut microbiome.

Activity-based protein profiling (ABPP) is a powerful chemical approach for probing protein function and enzymatic activity in complex biological systems. This strategy typically utilizes activity-based probes that are designed to bind a specific protein, amino acid residue, or protein family and form a covalent bond through a reactivity-based warhead. Subsequent analysis by mass spectrometry-based proteomic platforms that involve either click chemistry or affinity-based labeling to enrich for the tagged proteins enables identification of protein function and enzymatic activity. ABPP has facilitated elucidation of biological processes in bacteria, discovery of new antibiotics, and characterization of host-microbe interactions within physiological contexts. This review will focus on recent advances and applications of ABPP in bacteria and complex microbial communities.

Dopamine receptor D2 confers colonization resistance via gut microbial metabolites.

The gut microbiome plays major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea, and acute renal failure (hemolytic uremic syndrome)4,5. Although gut microbes can provide colonization resistance by outcompeting some pathogens or modulating host defense provided by the gut barrier and intestinal immune cells, this phenomenon remains poorly understood. Emerging evidence suggests that small-molecule metabolites produced by the gut microbiota may mediate this process6. Here, we show that tryptophan (Trp)-derived metabolites produced by the gut bacteria protect the host against Citrobacter rodentium, a murine AE pathogen widely used as a model for EHEC infection7,8, by activation of the host neurotransmitter dopamine receptor D2 (DRD2) within the intestinal epithelium. We further find that these Trp metabolites act through DRD2 to decrease expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Previously identified mechanisms of colonization resistance either directly affect the pathogen by competitive exclusion or indirectly by modulation of host defense mechanisms9,10, so our results delineate a noncanonical colonization resistance pathway against AE pathogens featuring an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization within the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches for improving gut health and treating gastrointestinal infections, which afflict millions globally.

BSH-TRAP: Bile salt hydrolase tagging and retrieval with activity-based probes.

Bile acids are important molecules that participate in digestion and regulate many host physiological processes, including metabolism and inflammation. Primary bile acids are biosynthesized from cholesterol in the liver, where they are conjugated to glycine and taurine before secretion into the intestines. A small fraction of these molecules remain in the gut, where they are modified by a microbial enzyme, bile salt hydrolase (BSH), which deconjugates the glycine and taurine groups. This deconjugation precedes all subsequent biotransformation in the intestines, including regioselective dehydroxylation and epimerization reactions, to produce numerous secondary bile acids. Thus, BSH is considered the gatekeeper enzyme of secondary bile acid metabolism, and, as a result, it controls the overall bile acid composition in the host. Despite the critical role that BSH plays in bile acid metabolism, there exist few tools to probe its activity in complex biological mixtures. In this chapter, we describe a chemoproteomic approach termed BSH-TRAP (bile salt hydrolase tagging and retrieval with activity-based probes) that enables visualization and identification of BSH activity in bacteria. Here, we describe application of BSH-TRAP to cultured bacterial strains and the gut microbes derived from mice. We envision that BSH-TRAP could be used to profile changes in BSH activity and identify novel BSH enzymes in complex biological samples, such as the gut microbiome.

External validation of the Madrid Acute Kidney Injury Prediction Score.

BACKGROUND: The Madrid Acute Kidney Injury Prediction Score (MAKIPS) is a recently described tool capable of performing automatic calculations of the risk of hospital-acquired acute kidney injury (HA-AKI) using data from from electronic clinical records that could be easily implemented in clinical practice. However, to date, it has not been externally validated. The aim of our study was to perform an external validation of the MAKIPS in a hospital with different characteristics and variable case mix. METHODS: This external validation cohort study of the MAKIPS was conducted in patients admitted to a single tertiary hospital between April 2018 and September 2019. Performance was assessed by discrimination using the area under the receiver operating characteristics curve and calibration plots. RESULTS: A total of 5.3% of the external validation cohort had HA-AKI. When compared with the MAKIPS cohort, the validation cohort showed a higher percentage of men as well as a higher prevalence of diabetes, hypertension, cardiovascular disease, cerebrovascular disease, anaemia, congestive heart failure, chronic pulmonary disease, connective tissue diseases and renal disease, whereas the prevalence of peptic ulcer disease, liver disease, malignancy, metastatic solid tumours and acquired immune deficiency syndrome was significantly lower. In the validation cohort, the MAKIPS showed an area under the curve of 0.798 (95% confidence interval 0.788-0.809). Calibration plots showed that there was a tendency for the MAKIPS to overestimate the risk of HA-AKI at probability rates ˂0.19 and to underestimate at probability rates between 0.22 and 0.67. CONCLUSIONS: The MAKIPS can be a useful tool, using data that are easily obtainable from electronic records, to predict the risk of HA-AKI in hospitals with different case mix characteristics.

Integrating electronic health data records to develop and validate a predictive model of hospital-acquired acute kidney injury in non-critically ill patients.

BACKGROUND: Models developed to predict hospital-acquired acute kidney injury (HA-AKI) in non-critically ill patients have a low sensitivity, do not include dynamic changes of risk factors and do not allow the establishment of a time relationship between exposure to risk factors and AKI. We developed and externally validated a predictive model of HA-AKI integrating electronic health databases and recording the exposure to risk factors prior to the detection of AKI. METHODS: The study set was 36 852 non-critically ill hospitalized patients admitted from January to December 2017. Using stepwise logistic analyses, including demography, chronic comorbidities and exposure to risk factors prior to AKI detection, we developed a multivariate model to predict HA-AKI. This model was then externally validated in 21 545 non-critical patients admitted to the validation centre in the period from June 2017 to December 2018. RESULTS: The incidence of AKI in the study set was 3.9%. Among chronic comorbidities, the highest odds ratios (ORs) were conferred by chronic kidney disease, urologic disease and liver disease. Among acute complications, the highest ORs were associated with acute respiratory failure, anaemia, systemic inflammatory response syndrome, circulatory shock and major surgery. The model showed an area under the curve (AUC) of 0.907 [95% confidence interval (CI) 0.902-0.908), a sensitivity of 82.7 (95% CI 80.7-84.6) and a specificity of 84.2 (95% CI 83.9-84.6) to predict HA-AKI, with an adequate goodness-of-fit for all risk categories (χ2 = 6.02, P = 0.64). In the validation set, the prevalence of AKI was 3.2%. The model showed an AUC of 0.905 (95% CI 0.904-0.910), a sensitivity of 81.2 (95% CI 79.2-83.1) and a specificity of 82.5 (95% CI 82.2-83) to predict HA-AKI and had an adequate goodness-of-fit for all risk categories (χ2 = 4.2, P = 0.83). An online tool (predaki.amalfianalytics.com) is available to calculate the risk of AKI in other hospital environments. CONCLUSIONS: By using electronic health data records, our study provides a model that can be used in clinical practice to obtain an accurate dynamic and updated assessment of the individual risk of HA-AKI during the hospital admission period in non-critically ill patients.

A strategy for the selective imaging of glycans using caged metabolic precursors.

Glycans can be imaged by metabolic labeling with azidosugars followed by chemical reaction with imaging probes; however, tissue-specific labeling is difficult to achieve. Here we describe a strategy for the use of a caged metabolic precursor that is activated for cellular metabolism by enzymatic cleavage. An N-azidoacetylmannosamine derivative caged with a peptide substrate for the prostate-specific antigen (PSA) protease was converted to cell-surface azido sialic acids in a PSA-dependent manner. The approach has applications in tissue-selective imaging of glycans for clinical and basic research purposes.

Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses.

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

Chemical Mechanisms of Colonization Resistance by the Gut Microbial Metabolome.

The gut microbiome, the collection of 100 trillion microorganisms that resides in the intestinal lumen, plays major roles in modulating host physiology. One well-established function of the gut microbiota is that of colonization resistance or the ability of the microbial collective to protect the host against enteric pathogens. Although evidence suggests that these microbes may outcompete some pathogens, there remains a lack of mechanistic understanding that underlies this competitive exclusion. In recent years, there has been great interest in small-molecule metabolites that are produced by the gut microbiota and in understanding how these molecules regulate host-pathogen interactions. In this review, we briefly summarize these findings by focusing on several classes of metabolites that mediate this important process. Understanding these host-microbe interactions in the gut may lead to identification of potential candidates for the development of prophylactics and therapeutics for many infectious diseases that are impacted by the gut microbiome.