Gut microbial ß-glucuronidases influence endobiotic homeostasis and are modulated by diverse therapeutics.

Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.

Diverse but desolate landscape of gut microbial azoreductases: A rationale for idiopathic IBD drug response.

Prodrugs reliant on microbial activation are widely used but exhibit a range of efficacies that remain poorly understood. The anti-inflammatory compound 5-aminosalicylic acid (5-ASA), which is packaged in a variety of azo-linked prodrugs provided to most Ulcerative Colitis (UC) patients, shows confounding inter-individual variabilities in response. Such prodrugs must be activated by azo-bond reduction to form 5-ASA, a process that has been attributed to both enzymatic and non-enzymatic catalysis. Gut microbial azoreductases (AzoRs) are the first catalysts shown to activate azo-linked drugs and to metabolize toxic azo-chemicals. Here, we chart the scope of the structural and functional diversity of AzoRs in health and in patients with the inflammatory bowel diseases (IBDs) UC and Crohn's Disease (CD). Using structural metagenomics, we define the landscape of gut microbial AzoRs in 413 healthy donor and 1059 IBD patient fecal samples. Firmicutes encode a significantly higher number of unique AzoRs compared to other phyla. However, structural and biochemical analyses of distinct AzoRs from the human microbiome reveal significant differences between prevalent orthologs in the processing of toxic azo-dyes, and their generally poor activation of IBD prodrugs. Furthermore, while individuals with IBD show higher abundances of AzoR-encoding gut microbial taxa than healthy controls, the overall abundance of AzoR-encoding microbes is markedly low in both disease and health. Together, these results establish that gut microbial AzoRs are functionally diverse but sparse in both health and disease, factors that may contribute to non-optimal processing of azo-linked prodrugs and idiopathic IBD drug responses.

Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract.

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

Quantitative Investigation of Irinotecan Metabolism, Transport, and Gut Microbiome Activation.

The anticancer drug irinotecan shows serious dose-limiting gastrointestinal toxicity regardless of intravenous dosing. Although enzymes and transporters involved in irinotecan disposition are known, quantitative contributions of these mechanisms in complex in vivo disposition of irinotecan are poorly understood. We explained intestinal disposition and toxicity of irinotecan by integrating 1) in vitro metabolism and transport data of irinotecan and its metabolites, 2) ex vivo gut microbial activation of the toxic metabolite SN-38, and 3) the tissue protein abundance data of enzymes and transporters relevant to irinotecan and its metabolites. Integration of in vitro kinetics data with the tissue enzyme and transporter abundance predicted that carboxylesterase (CES)-mediated hydrolysis of irinotecan is the rate-limiting process in the liver, where the toxic metabolite formed is rapidly deactivated by glucuronidation. In contrast, the poor SN-38 glucuronidation rate as compared with its efficient formation by CES2 in the enterocytes is the key mechanism of the intestinal accumulation of the toxic metabolite. The biliary efflux and organic anion transporting polypeptide-2B1-mediated enterocyte uptake can also synergize buildup of SN-38 in the enterocytes, whereas intestinal P-glycoprotein likely facilitates SN-38 detoxification in the enterocytes. The higher SN-38 concentration in the intestine can be further nourished by ß-d-glucuronidases. Understanding the quantitative significance of the key metabolism and transport processes of irinotecan and its metabolites can be leveraged to alleviate its intestinal side effects. Further, the proteomics-informed quantitative approach to determine intracellular disposition can be extended to determine susceptibility of cancer cells over normal cells for precision irinotecan therapy. SIGNIFICANCE STATEMENT: This work provides a deeper insight into the quantitative relevance of irinotecan hydrolysis (activation), conjugation (deactivation), and deconjugation (reactivation) by human or gut microbial enzymes or transporters. The results of this study explain the characteristic intestinal exposure and toxicity of irinotecan. The quantitative tissue-specific in vitro to in vivo extrapolation approach presented in this study can be extended to cancer cells.

The Gut Microbiota Impact Cancer Etiology through "Phase IV Metabolism" of Xenobiotics and Endobiotics.

The human gut microbiome intimately complements the human genome and gut microbial factors directly influence health and disease. Here we outline how the gut microbiota uniquely contributes to cancer etiology by processing products of human drug and endobiotic metabolism. We formally propose that the reactions performed by the gut microbiota should be classified as "Phase IV xenobiotic and endobiotic metabolism." Finally, we discuss new data on the control of cancer by the inhibition of gut microbial phase IV enzymes responsible for tumor initiation and progression.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

Garcinoic Acid Is a Natural and Selective Agonist of Pregnane X Receptor.

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.

A Rare Mutation in SPLUNC1 Affects Bacterial Adherence and Invasion in Meningococcal Disease.

BACKGROUND: Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined. METHODS: We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays. RESULTS: Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1. CONCLUSIONS: A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling.

It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial ß-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.

Gut microbial ß-glucuronidases reactivate estrogens as components of the estrobolome that reactivate estrogens.

Gut microbial ß-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. Furthermore, bacterial GUS enzymes within the gastrointestinal tract have been postulated to be a contributing factor in hormone-driven cancers. However, to date, there has been no experimental evidence to support these hypotheses. Here we provide the first in vitro analysis of the ability of 35 human gut microbial GUS enzymes to reactivate two distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide, to estrone and estradiol, respectively. We show that certain members within the Loop 1, mini-Loop 1, and FMN-binding classes of gut microbial GUS enzymes can reactivate estrogens from their inactive glucuronides. We provide molecular details of key interactions that facilitate these catalytic processes and present the structures of two novel human gut microbial GUS enzymes related to the estrobolome. Further, we demonstrate that estrogen reactivation by Loop 1 bacterial GUS enzymes can be inhibited both in purified enzymes and in fecal preparations of mixed murine fecal microbiota. Finally, however, despite these in vitro and ex vivo data, we show that a Loop 1 GUS-specific inhibitor is not capable of reducing the development of tumors in the PyMT mouse model of breast cancer. These findings validate that gut microbial GUS enzymes participate in the estrobolome but also suggest that the estrobolome is a multidimensional set of processes on-going within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins.

Targeting Regorafenib-Induced Toxicity through Inhibition of Gut Microbial ß-Glucuronidases.

Regorafenib (Stivarga) is an oral small molecule kinase inhibitor used to treat metastatic colorectal cancer, hepatocellular carcinomas, and gastrointestinal stromal tumors. Diarrhea is one of the most frequently observed adverse reactions associated with regorafenib. This toxicity may arise from the reactivation of the inactive regorafenib-glucuronide to regorafenib by gut microbial ß-glucuronidase (GUS) enzymes in the gastrointestinal tract. We sought to unravel the molecular basis of regorafenib-glucuronide processing by human intestinal GUS enzymes and to examine the potential inhibition of these enzymes. Using a panel of 31 unique gut microbial GUS enzymes derived from the 279 mapped from the human gut microbiome, we found that only four were capable of regorafenib-glucuronide processing. Using crystal structures as a guide, we pinpointed the molecular features unique to these enzymes that confer regorafenib-glucuronide processing activity. Furthermore, a pilot screen identified the FDA-approved drug raloxifene as an inhibitor of regorafenib reactivation by the GUS proteins discovered. Novel synthetic raloxifene analogs exhibited improved potency in both in vitro and ex vivo studies. Taken together, these data establish that regorafenib reactivation is exclusively catalyzed by gut microbial enzymes and that these enzymes are amenable to targeted inhibition. Our results unravel key molecular details of regorafenib reactivation in the GI tract and provide a potential pathway to improve clinical outcomes with regorafenib.

Mouse Gut Microbiome-Encoded ß-Glucuronidases Identified Using Metagenome Analysis Guided by Protein Structure.

Gut microbial ß-glucuronidase (GUS) enzymes play important roles in drug efficacy and toxicity, intestinal carcinogenesis, and mammalian-microbial symbiosis. Recently, the first catalog of human gut GUS proteins was provided for the Human Microbiome Project stool sample database and revealed 279 unique GUS enzymes organized into six categories based on active-site structural features. Because mice represent a model biomedical research organism, here we provide an analogous catalog of mouse intestinal microbial GUS proteins-a mouse gut GUSome. Using metagenome analysis guided by protein structure, we examined 2.5 million unique proteins from a comprehensive mouse gut metagenome created from several mouse strains, providers, housing conditions, and diets. We identified 444 unique GUS proteins and organized them into six categories based on active-site features, similarly to the human GUSome analysis. GUS enzymes were encoded by the major gut microbial phyla, including Firmicutes (60%) and Bacteroidetes (21%), and there were nearly 20% for which taxonomy could not be assigned. No differences in gut microbial gus gene composition were observed for mice based on sex. However, mice exhibited gus differences based on active-site features associated with provider, location, strain, and diet. Furthermore, diet yielded the largest differences in gus composition. Biochemical analysis of two low-fat-associated GUS enzymes revealed that they are variable with respect to their efficacy of processing both sulfated and nonsulfated heparan nonasaccharides containing terminal glucuronides.IMPORTANCE Mice are commonly employed as model organisms of mammalian disease; as such, our understanding of the compositions of their gut microbiomes is critical to appreciating how the mouse and human gastrointestinal tracts mirror one another. GUS enzymes, with importance in normal physiology and disease, are an attractive set of proteins to use for such analyses. Here we show that while the specific GUS enzymes differ at the sequence level, a core GUSome functionality appears conserved between mouse and human gastrointestinal bacteria. Mouse strain, provider, housing location, and diet exhibit distinct GUSomes and gus gene compositions, but sex seems not to affect the GUSome. These data provide a basis for understanding the gut microbial GUS enzymes present in commonly used laboratory mice. Further, they demonstrate the utility of metagenome analysis guided by protein structure to provide specific sets of functionally related proteins from whole-genome metagenome sequencing data.

Enterococcus faecalis Gluconate Phosphotransferase System Accelerates Experimental Colitis and Bacterial Killing by Macrophages.

Enterococcus faecalis strains are resident intestinal bacteria associated with invasive infections, inflammatory bowel diseases, and colon cancer. Although factors promoting E. faecalis colonization of intestines are not fully known, one implicated pathway is a phosphotransferase system (PTS) in E. faecalis strain OG1RF that phosphorylates gluconate and contains the genes OG1RF_12399 to OG1RF_12402 (OG1RF_12399-12402). We hypothesize that this PTS permits growth in gluconate, facilitates E. faecalis intestinal colonization, and exacerbates colitis. We generated E. faecalis strains containing deletions/point mutations in this PTS and measured bacterial growth and PTS gene expression in minimal medium supplemented with selected carbohydrates. We show that E. faecalis upregulates OG1RF_12399 transcription specifically in the presence of gluconate and that E. faecalis strains lacking, or harboring a single point mutation in, OG1RF_12399-12402 are unable to grow in minimal medium containing gluconate. We colonized germfree wild-type and colitis-prone interleukin-10-deficient mice with defined bacterial consortia containing the E. faecalis strains and measured inflammation and bacterial abundance in the colon. We infected macrophage and intestinal epithelial cell lines with the E. faecalis strains and measured intracellular bacterial survival and proinflammatory cytokine secretion. The presence of OG1RF_12399-12402 is not required for E. faecalis colonization of the mouse intestine but is associated with an accelerated onset of experimental colitis in interleukin-10-deficient mice, altered bacterial composition in the colon, enhanced E. faecalis survival within macrophages, and increased proinflammatory cytokine secretion by colon tissue and macrophages. Further studies of bacterial carbohydrate metabolism in general, and E. faecalis PTS-gluconate in particular, during inflammation may identify new mechanisms of disease pathogenesis.

Three structurally and functionally distinct ß-glucuronidases from the human gut microbe Bacteroides uniformis.

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

Enhanced biofilm prevention activity of a SPLUNC1-derived antimicrobial peptide against Staphylococcus aureus.

SPLUNC1 is a multifunctional protein of the airway with antimicrobial properties. We previously reported that it displayed antibiofilm activities against P. aeruginosa. The goal of this study was to determine whether (1) the antibiofilm property is broad (including S. aureus, another prevalent organism in cystic fibrosis); (2) the α4 region is responsible for such activity; and (3), if so, this motif could be structurally optimized as an antimicrobial peptide with enhanced activities. We used S. aureus biofilm-prevention assays to determine bacterial biomass in the presence of SPLUNC1 and SPLUNC1Δα4 recombinant proteins, or SPLUNC1-derived peptides (α4 and α4M1), using the well-established crystal-violet biofilm detection assay. The SPLUNC1Δα4 showed markedly reduced biofilm prevention compared to the parent protein. Surprisingly, the 30-residue long α4 motif alone demonstrated minimal biofilm prevention activities. However, structural optimization of the α4 motif resulted in a modified peptide (α4M1) with significantly enhanced antibiofilm properties against methicillin-sensitive (MSSA) and-resistant (MRSA) S. aureus, including six different clinical strains of MRSA and the well-known USA300. Hemolytic activity was undetectable at up to 100µM for the peptides. The data warrant further investigation of α4-derived AMPs to explore the potential application of antimicrobial peptides to combat bacterial biofilm-related infections.

Gut Microbial ß-Glucuronidase Inhibition via Catalytic Cycle Interception.

Microbial ß-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS-GlcA catalytic intermediate as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm the GUS-dependent formation of inhibitor-glucuronide conjugates by LC-MS and show that methylated piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved piperazine- and piperidine-containing drugs from many classes, including those for the treatment of depression, infection, and cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of therapeutics may impact GUS activities in the human gut.

Microbial Glucuronidase Inhibition Reduces Severity of Diclofenac-Induced Anastomotic Leak in Rats.

BACKGROUND: The non-steroidal anti-inflammatory drug diclofenac has been associated with intestinal anastomotic leakage, although the underlying pathophysiology is unclear. Previous data suggest that reactivation of biliary diclofenac metabolites by microbial ß-glucuronidases in the gut plays a role in harming the intestinal mucosa, and that microbiome-targeted glucuronidase inhibition prevents this damage. Here, the microbial glucuronidase inhibitor Inh1 was examined for its ability to reduce diclofenac-induced anastomotic leakage in rats. METHODS: Ninety male Wistar rats were allocated to five groups. In the two diclofenac groups, group DCF received diclofenac (3 mg/kg per day) and group DCF-Inh1 additionally received 800 mcg/kg per day of glucuronidase inhibitor Inh1 solution orally. In non-diclofenac groups, animals received either Inh1 (800 mcg/kg per day; group Inh1) solution, the vehicle (methylcellulose; group Veh), or no solution (group Ctrl). All solutions were provided from the day of surgery until sacrifice on day three. Plasma concentrations of diclofenac were determined. Outcomes were anastomotic leakage, leak severity, and anastomotic strength. RESULTS: Anastomotic leak rates were 89% in group DCF and 44% in group DCF-Inh1 (p = 0.006). Leak severity was reduced in group DCFic-Inh1 (p = 0.029). In non-diclofenac cohorts, mostly minor leakage signs were observed in 25% in group Ctrl, 39% in group Inh1 (0.477), and 24% in group Veh (p = 1.000). Bursting pressure and breaking strength were not significantly different. Plasma concentrations of diclofenac were not changed by Inh1. CONCLUSION: Microbial glucuronidase inhibitor reduces diclofenac-induced anastomotic leakage severity, which suggests a harmful effect of diclofenac metabolite reactivation in the gut. This finding improves the understanding of the pathogenesis of anastomotic leakage.

SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Cystic fibrosis (CF) is a common genetic disease with significantly increased mortality. CF airways exhibit ion transport abnormalities, including hyperactivity of the epithelial Na+ channel (ENaC). Short-palate lung and nasal epithelial clone 1 (SPLUNC1) is a multifunctional innate defense protein that is secreted into the airway lumen. We have previously demonstrated that SPLUNC1 binds to and inhibits ENaC to maintain fluid homeostasis in airway epithelia and that this process fails in CF airways. Despite this, how SPLUNC1 actually regulates ENaC is unknown. Here, we found that SPLUNC1 caused αγ-ENaC to internalize, whereas SPLUNC1 and ß-ENaC remained at the plasma membrane. Additional studies revealed that SPLUNC1 increased neural precursor cell-expressed developmentally down-regulated protein 4-2-dependent ubiquitination of α- but not ß- or γ-ENaC. We also labeled intracellular ENaC termini with green fluorescent protein and mCherry, and found that extracellular SPLUNC1 altered intracellular ENaC Forster resonance energy transfer. Taken together, our data indicate that SPLUNC1 is an allosteric regulator of ENaC that dissociates αßγ-ENaC to generate a new SPLUNC1-ß-ENaC complex. These data indicate a novel mode for regulating ENaC at the plasma membrane.-Kim, C. S., Ahmad, S., Wu, T., Walton, W. G., Redinbo, M. R., Tarran, R. SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells.

The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.

The role of the microbiome in cancer development and therapy.

Answer questions and earn CME/CNE The human body harbors enormous numbers of microbiota that influence cancer susceptibility, in part through their prodigious metabolic capacity and their profound influence on immune cell function. Microbial pathogens drive tumorigenesis in 15% to 20% of cancer cases. Even larger numbers of malignancies are associated with an altered composition of commensal microbiota (dysbiosis) based on microbiome studies using metagenomic sequencing. Although association studies cannot distinguish whether changes in microbiota are causes or effects of cancer, a causative role is supported by rigorously controlled preclinical studies using gnotobiotic mouse models colonized with one or more specific bacteria. These studies demonstrate that microbiota can alter cancer susceptibility and progression by diverse mechanisms, such as modulating inflammation, inducing DNA damage, and producing metabolites involved in oncogenesis or tumor suppression. Evidence is emerging that microbiota can be manipulated for improving cancer treatment. By incorporating probiotics as adjuvants for checkpoint immunotherapy or by designing small molecules that target microbial enzymes, microbiota can be harnessed to improve cancer care. CA Cancer J Clin 2017;67:326-344. © 2017 American Cancer Society.