In Silico Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Clostridium difficile Toxin A
Xiao X, Sarma S, Menegatti S, Crook N, Magness ST, Hall CK
ACS Chem Biol. 2022 Jan 21;17(1):118-128 PMID: 34965093
Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.
Genome-wide CRISPR Knockout Screen Identified PLAC8 as an Essential Factor for SADS-CoVs Infection
Tse LV, Meganck RM, Araba KC, Yount BL, Shaffer KM, Hou YJ, Munt JE, Adams LE, Wykoff JA, Morowitz JM, Dong S, Magness ST, Marzluff W, Gonzalez LM, Ehre C, Baric RS
Natl Acad Sci U S A . 2022 May 3;119(18):e2118126119 PMID: 35476513
Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.
A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure
Ok MT, Liu J, Bliton RJ, Hinesley CM, San Pedro EET, Breau KA, Gomez-Martinez I, Burclaff JR, Magness ST
Am J Physiol Gastrointest Liver Physiol. 2023 Feb 7. PMID: 36749911
Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdA/B toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude post-basal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.
Genome Sequence of Citrobacter freundii AMC0717, Isolated from the Intestinal Lumen of an 11-Year-Old Organ Donor
Marsh AJ, Chandrashekhar K, Ng S, Roach J, Magness ST, Azcarate-Peril MA
Microbiol Resour Announc. 2020 Nov 12;9(46):e00995-20 PMID: 33184159
Eubacterium callanderi AMC0717 was isolated from the mucosa of the transverse colon of an 11-year-old organ donor. This strain contains genes putatively encoding short-chain fatty acids (SCFAs), exopolysaccharide (EPS), and several B vitamins.
Genome Sequence of Citrobacter freundii AMC0703, Isolated from the Intestinal Lumen of an 11-Year-Old Organ Donor
Marsh AJ, Chandrashekhar K, Ng S, Roach J, Magness ST, Azcarate-Peril MA
Microbiol Resour Announc. 2020 Nov 12;9(46):e00994-20 PMID: 33184158
Citrobacter freundii AMC0703 was isolated from the intestinal mucosa of an 11-year-old organ donor. Genome analysis revealed the presence of multiple factors potentially aiding in pathogenicity, including fimbriae, flagella, and genes encoding resistance to fluoroquinolones, cephamycin, fosfomycin, and aminocoumarin.
Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum Infection
Ferguson SH, Foster DM, Sherry B, Magness ST, Nielsen DM, Gookin JL
Cell Mol Gastroenterol Hepatol. 2019;8(1):1-20. Epub 2019 Mar 5. PMID: 30849550
BACKGROUND & AIMS: The epithelial response is critical for intestinal defense against Cryptosporidium, but is poorly understood. To uncover the host strategy for defense against Cryptosporidium, we examined the transcriptional response of intestinal epithelial cells (IECs) to C parvum in experimentally infected piglets by microarray. Up-regulated genes were dominated by targets of interferon (IFN) and IFN-λ3 was up-regulated significantly in infected piglet mucosa. Although IFN-λ has been described as a mediator of epithelial defense against viral pathogens, there is limited knowledge of any role against nonviral pathogens. Accordingly, the aim of the study was to determine the significance of IFN-λ3 to epithelial defense and barrier function during C parvum infection. METHODS: The significance of C parvum-induced IFN-λ3 expression was determined using an immunoneutralization approach in neonatal C57BL/6 mice. The ability of the intestinal epithelium to up-regulate IFN-λ2/3 expression in response to C parvum infection and the influence of IFN-λ2/3 on epithelial defense against C parvum invasion, intracellular development, and loss of barrier function was examined using polarized monolayers of a nontransformed porcine-derived small intestinal epithelial cell line (IPEC-J2). Specifically, changes in barrier function were quantified by measurement of transepithelial electrical resistance and transepithelial flux studies. RESULTS: Immunoneutralization of IFN-λ2/3 in C parvum-infected neonatal mice resulted in a significantly increased parasite burden, fecal shedding, and villus blunting with crypt hyperplasia during peak infection. In vitro, C parvum was sufficient to induce autonomous IFN-λ3 and interferon-stimulated gene 15 expression by IECs. Priming of IECs with recombinant human IFN-λ3 promoted cellular defense against C parvum infection and abrogated C parvum-induced loss of barrier function by decreasing paracellular permeability to sodium. CONCLUSIONS: These studies identify IFN-λ3 as a key epithelial defense mechanism against C parvum infection.
IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model
Zwarycz B, Gracz AD, Rivera KR, Williamson IA, Samsa LA, Starmer J, Daniele MA, Salter-Cid L, Zhao Q, Magness ST
Cell Mol Gastroenterol Hepatol. 2018 Jul 4;7(1):1-17. eCollection 2019. PMID: 30364840
Crohn's disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn's disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal. METHODS: A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo. RESULTS: High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal-associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs. CONCLUSIONS:
Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.
Reserve Stem Cells in Intestinal Homeostasis and Injury
Bankaitis ED, Ha A, Kuo CJ, Magness ST
Gastroenterology. 2018 Nov;155(5):1348-1361. Epub 2018 Aug 15. Review. PMID: 30118745
Renewal of the intestinal epithelium occurs approximately every week and requires a careful balance between cell proliferation and differentiation to maintain proper lineage ratios and support absorptive, secretory, and barrier functions. We review models used to study the mechanisms by which intestinal stem cells (ISCs) fuel the rapid turnover of the epithelium during homeostasis and might support epithelial regeneration after injury. In anatomically defined zones of the crypt stem cell niche, phenotypically distinct active and reserve ISC populations are believed to support homeostatic epithelial renewal and injury-induced regeneration, respectively. However, other cell types previously thought to be committed to differentiated states might also have ISC activity and participate in regeneration. Efforts are underway to reconcile the proposed relatively strict hierarchical relationships between reserve and active ISC pools and their differentiated progeny; findings from models provide evidence for phenotypic plasticity that is common among many if not all crypt-resident intestinal epithelial cells. We discuss the challenges to consensus on ISC nomenclature, technical considerations, and limitations inherent to methodologies used to define reserve ISCs, and the need for standardized metrics to quantify and compare the relative contributions of different epithelial cell types to homeostatic turnover and post-injury regeneration. Increasing our understanding of the high-resolution genetic and epigenetic mechanisms that regulate reserve ISC function and cell plasticity will help refine these models and could affect approaches to promote tissue regeneration after intestinal injury.
Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells
Bhatt AP, Gunasekara DB, Speer J, Reed MI, Peña AN, Midkiff BR, Magness ST, Bultman SJ, Allbritton NL, Redinbo MR
ACS Infect Dis. 2018 Jan 12;4(1):46-52. Epub 2017 Nov 10. PMID: 29094594
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.
Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen 1(I)-promoter-GFP reporter mice
Ding S, Walton KLW, Blue RE, MacNaughton K, Magness ST, Lund PK
PLoS One. 2010 Aug 16;5(8):e12191. PMID: 22880035
Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. Methods: FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3 and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA. Results: Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin+ cells and much fewer α-SMA+ cells. GFP mRNA strongly correlated with collagen α1(I) mRNA. Conclusions: One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP
transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis.
Sry-box (SOX) transcription factors in gastrointestinal physiology and disease
Gracz AD, Magness ST
Am J Physiol Gastrointest Liver Physiol. 2011 Apr;300(4):G503-15. Epub 2011 Feb 3. Review. PMID: 21995959
The genetic mechanisms underlying tissue maintenance of the gastrointestinal tract are critical for the proper function of the digestive system under normal physiological stress. The identification of transcription factors and related signal transduction pathways that regulate stem cell maintenance and lineage allocation is attractive from a clinical standpoint in that it may provide targets for novel cell- or drug-based therapies. Sox [sex-determining region Y (Sry) box-containing] factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems and might provide valuable insight toward the understanding of these processes in endodermally derived tissues of the gastrointestinal tract. In this review, we focus on the known genetic functions of Sox factors and their roles in epithelial tissues of the esophagus, stomach, intestine, colon, pancreas, and liver. Additionally, we discuss pathological conditions in the gastrointestinal tract that are associated with a dysregulation of Sox factors. Further study of Sox factors and their role in gastrointestinal physiology and pathophysiology may lead to advances that facilitate control of tissue maintenance and development of advanced clinical therapies.
High-fat diet: bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse
Ding S, Chi MM, Scull BP, Rigby R, Schwerbrock NM, Magness ST, Jobin C, Lund PK
PLoS One. 2010 Aug 16;5(8):e12191. PMID: 20808947
BACKGROUND: Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2-16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-alpha mRNA and activation of a NF-kappaB(EGFP) reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-alpha mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-kappaB(EGFP) in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-kappaB(EGFP) in GF NF-kappaB(EGFP) mice. CONCLUSIONS/SIGNIFICANCE: Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance.