Clostridium difficile Toxins A and B Decrease Intestinal SLC26A3 Protein Expression

Am J Physiol Gastrointest Liver Physiol 315: G43–G52, 2018. First published March 29, 2018; doi:10.1152/ajpgi.00307.2017. RESEARCH ARTICLE Epithelial Biology and Secretion Clostridium difficile toxins A and B decrease intestinal SLC26A3 protein expression Hayley Coffing,1 Shubha Priyamvada,1 Arivarasu N. Anbazhagan,1 Christine Salibay,3 Melinda Engevik,4 James Versalovic,4 Mary Beth Yacyshyn,5 Bruce Yacyshyn,5 Sangeeta Tyagi,1 Seema Saksena,1,2 Ravinder K. Gill,1 Waddah A. Alrefai,1,2 and Pradeep K. Dudeja1,2 1 Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; 3Department of Pathology, University of Illinois at Chicago, Chicago, Illinois; 4Department of Pathology and Immunology, Baylor College of Medicine and Department of Pathology, Texas Children’s Hospital, Houston, Texas; and 5Division of Digestive Diseases, Department. of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 2 Submitted 4 October 2017; accepted in final form 12 March 2018 Coffing H, Priyamvada S, Anbazhagan AN, Salibay C, Engevik M, Versalovic J, Yacyshyn MB, Yacyshyn B, Tyagi S, Saksena S, Gill RK, Alrefai WA, Dudeja PK. Clostridium difficile toxins A and B decrease intestinal SLC26A3 protein expression. Am J Physiol Gastrointest Liver Physiol 315: G43–G52, 2018. First published March 29, 2018; doi:10.1152/ajpgi.00307.2017.—Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhea in the United States. Although C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficileassociated diarrhea remains poorly understood. Studies have shown that a decrease in both NHE3 (Na⫹/H⫹ exchanger) and DRA (downregulated in adenoma, Cl⫺/HCO⫺ 3 exchanger), resulting in decreased electrolyte absorption, is implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and downregulated in patients with CDI, but the role of DRA in CDI remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrated that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA, as NHE3 and PAT-1 (putative anion transporter 1) protein levels were unaffected by TcdA and TcdB. Additionally, purified TcdA and TcdA ⫹ TcdB, but not TcdB, resulted in a decrease in colonic DRA protein levels in a toxigenic mouse model of CDI. Finally, patients with recurrent CDI also exhibited significantly reduced expression of colonic DRA protein. Together, these findings indicate that C. difficile toxins markedly downregulate intestinal expression of DRA which may contribute to the diarrheal phenotype of CDI. NEW & NOTEWORTHY Our studies demonstrate, for the first time, that C. difficile toxins reduce DRA protein, but not mRNA, levels in intestinal epithelial cells. These findings suggest that a downregulation of DRA may be a critical factor in C. difficile infection-associated diarrhea. chloride transport; Clostridium difficile; DRA; human CDI; toxigenic mouse model Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), Jesse Brown VA Medical Center, 820 South Damen Ave., Chicago, IL 60612 (e-mail: pkdudeja@uic.edu). INTRODUCTION Clostridium difficile infection (CDI, recently classified as Clostridioides difficile) is the leading cause of antibiotic-associated diarrhea and the most common cause of nosocomial infection in the United States (39). CDI causes a spectrum of gastrointestinal symptoms ranging from self-limiting diarrhea to severe diarrhea and in extreme cases, pseudomembranous colitis, sepsis, and death (39). The symptoms of CDI are primarily mediated through the release of two major exotoxins, TcdA and TcdB. TcdA and TcdB are large (308 and 270 kDa, respectively) glucosyltransferases that inactivate Rho family GTPases (Rho, Rac, and Cdc42) (6, 23). TcdA and TcdB are taken up via receptor-mediated endocytosis (21), translocate to the cytosol, and irreversibly inactivate Rho GTPases through the addition of a glucose moiety at Thr-35 or Thr-37, thereby preventing their active conformation (35). Inhibition of these GTPases leads to cytoskeletal disruption, intestinal epithelial cell damage, and cell death via apoptotic and necrotic mechanisms (35). Although the causative agents of CDI (TcdA and TcdB) are known, the contribution of each toxin to disease progression and the pathophysiology underlying C. difficileassociated diarrhea remains poorly understood (2, 21). Diarrheal diseases are multifactorial disorders caused by increased secretion and/or decreased absorption in the gastrointestinal tract (29, 42). The coupled operation of luminal membrane Na⫹/H⫹ and Cl⫺/HCO⫺ 3 exchangers is the predominant route for electroneutral NaCl absorption in the human ileum and colon (29). SLC9A3 (NHE3) is the key transporter involved in Na⫹ absorption in the ileum and colon (29). Two members of the SLC26 gene family, SLC26A3 and SLC26A6 (DRA and PAT-1, respectively), have been implicated in the intestinal luminal Cl⫺/HCO⫺ 3 exchange process; however, DRA is considered the critical transporter given its role in congenital chloride diarrhea (CLD), a genetic disorder characterized by profuse chloride-rich diarrhea and metabolic alkalosis (41). Moreover, knockdown of DRA is known to induce a diarrheal phenotype, similar to CLD, in mice whereas knockdown of PAT-1 does not (17). Furthermore, DRA expression is significantly reduced in animal models of inflammatory and infectious diarrhea and in patients with inflammatory bowel disease (11, 42). Thus electroneutral electrolyte absorption by http://www.ajpgi.org G43 Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. G44 C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION NHE3 and DRA represents a therapeutic target for diarrheal diseases. Previous studies have shown that purified TcdB causes internalization of NHE3 from the apical surface in various cell lines (13). Additionally, decreased NHE3 expression and function were also shown in patients with CDI (8). However, how C. difficile toxins affect DRA has not been explored. In addition to in vitro models, we also investigated the role of DRA in a toxigenic mouse model of CDI. Historically, the golden Syrian hamster model and the ileal loop model were the most commonly used animal models to study CDI (7, 10, 15, 39). However, the hamster model presents challenges; for example, animals quickly develop fulminant colitis and the ileum is the primary organ affected (3, 4). This phenotype is distinctly different from the human presentation of CDI, thus highlighting the need for more physiologically applicable models. Similarly, the ileal loop model directly administers toxins to the ileum and also includes the risks inherent to small animal surgery (7, 14). Thus our present studies utilized the intrarectal mouse model of toxin administration, a model that accurately recapitulates aspects of human CDI with the colon primarily affected (15). Finally, our studies also examined expression of DRA in human biopsies from recurrent CDI patients. Since the role of DRA in C. difficile-associated diarrhea is unclear, we examined the effects of purified C. difficile toxins on DRA expression. Our findings demonstrated that incubation with TcdA and TcdB leads to a drastic reduction in DRA protein levels in intestinal epithelial cells. Furthermore, this downregulation of DRA appeared to involve posttranscriptional mechanisms as DRA mRNA levels remained unchanged after toxin administration. Similar to our in vitro data, DRA protein levels were also significantly reduced in mice administered TcdA and TcdA ⫹ TcdB. Mice administered TcdB, however, did not show significant changes in DRA protein levels. Last, we also observed a drastic reduction in colonic DRA protein levels in patients with recurrent CDI vs. healthy subjects. Taken together, these studies show that C. difficile toxins consistently decreased DRA protein levels in various models of CDI, thus identifying DRA as a potential target for therapeutic management of CDI-associated diarrhea. MATERIALS AND METHODS C. difficile toxins. C. difficile toxins were obtained from List Biological Laboratories (Campbell, CA). Toxin A (no. 152C) was dissolved in molecular-grade water and stored at 4°C at a concentration of 100 ␮g/ml. Toxin B (no. 155L) was stored at 4°C at a concentration of 200 ␮g/ml. Cell culture. Caco2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in T-75 plastic flasks at 37°C and 5% CO2-95% air environment. Caco2 cells were grown in MEM (ATCC) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mg/l gentamicin. For this study, cells between passages 25 and 40 were used. Caco2 cells were plated on 24-well plates (Costar, Corning, NY) at a density of 2 ⫻ 104 cells/well. Fully differentiated Caco2 monolayers (10 –14 days postplating) were treated with purified C. difficile toxin A (152C, List Biological Laboratories) and/or toxin B (155L, List Biological Laboratories) for 6 –24 h at various concentrations in serum-free culture medium to assess DRA protein expression, mRNA expression, and cell cytotoxicity. T-84 cells were grown in DMEM-F12 with 10% fetal bovine serum as previously described (11). Mice. All mice used in our studies were female C57BL/6 between 10 and 12 wk of age (Jackson, Bar Harbor, ME). The instillation of toxins A and B was performed as described previously (12, 15) with minor modifications. Purified TcdA (10 ␮g), TcdB (10 ␮g), or TcdA/TcdB (5 ␮g each) toxin in 100 ␮l PBS were administered to mice. Briefly, a tube was lubricated with water-soluble personal lubricant and intrarectally inserted 3.5 cm. One-hundred microliters of solution was slowly administered while pressure was applied to the anal area to prevent leakage. The tube was then slowly removed and pressure was applied to the anal area for an additional 30 s. Mice were euthanized at 4 h postinstillation, and colonic tissues were harvested. Patient biopsies. Slides with sections from healthy and CDI patient colonic biopsies were generously provided by Dr. Mary B. Yacyshyn and Dr. Bruce Yacyshyn, Univ. of Cincinnati, Cincinnati, OH. All patients and healthy volunteers provided written, informed consent consistent with IRB approval of the University of Cincinnati Medical Center. Volunteers were considered healthy when they presented without previous or current GI symptoms, history of chronic disease, or cancer. Colon biopsies were collected, fixed in neutral buffered formalin, and paraffin-embedded. Paraffin sections were obtained from deidentified patients with recurrent CDI diagnosis (C. difficilepositive ELISA or LAMP toxin test) and no other known morbidity/ disorder as previously described (8). CDI-positive patients did not have history of inflammatory bowel disease (IBD), colostomy, cancer, small bowel obstruction, or diverticulosis. Immunofluorescence staining. Mouse distal colon samples were embedded in Optimal Cutting Temperature compound (OCT), and 5-␮m-thick sections were applied to glass slides. Sections were fixed with 4% paraformaldehyde in PBS (pH 8.5) for 15 min at room temperature and permeabilized using Nonidet P-40 in PBS for 5 min. Sections were then placed in blocking solution (2.5–5% normal goat serum) for 2 h at room temperature. Sections were then incubated with anti-DRA and anti-villin (Abcam, Cambridge, MA) antibodies in 1% NGS (normal goat serum) for 1 h at room temperature. After washing with 1% NGS, sections were incubated with secondary antibodies Alexa fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen) for 1 h, then mounted with Slowfade Gold antifade with DAPI (Invitrogen) under coverslips. Additionally, sections from patient biopsies from healthy subjects and recurrent CDI were also stained for DRA. Briefly, biopsies (as described in patient biopsies above) from the transverse colon were resected and fixed overnight at 4°C in neutral-buffered formalin and embedded in paraffin. Sections (6 –7 ␮m thick) were applied to glass slides at Univ. of Cincinnati. Immunofluorescent studies of DRA were then conducted at UIC as described above. All sections were imaged on a Carl Zeiss LSM 510 laser-scanning confocal microscope using a 20⫻ objective. RNA extraction and real-time PCR. To quantitate mRNA of DRA and other transporters, total RNA from Caco2 cells and mice colonic mucosa was extracted using RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantity and quality of total extracted RNA was verified using a Nanodrop spectrophotometer. Extracted RNA was amplified with Brilliant SYBR Green qRT-PCR Master Mix kit (Agilent Technologies, Santa Clara, CA) by using gene-specific primers for transporters. The relative mRNA levels of DRA and other transporters were expressed as fold change of control normalized to GAPDH used as an internal control gene. Protein lysates and Western blotting. Tissue lysates were prepared from mucosal scrapings of the colon and total protein was extracted using RIPA lysis buffer (Cell Signaling, Danvers, MA) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN). Mucosal scrapings were homogenized using a bullet blender and subsequent sonication (2 pulses, 30 s each). Lysates were then centrifuged at 13,000 rpm for 10 min at 4°C to remove cell debris. Caco2 cells were treated with purified TcdA and TcdB (0.1–25 ng/ml) for various times (6 h, 24 h). Control cells were treated with equal amounts of vehicle (molecular grade water). After treatment, control and toxin-treated AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION cells were washed with 1X PBS to remove residual media and lysed in 1X Cell Lysis Buffer (Cell Signaling, Danvers, MA) and 1X Protease Cocktail Inhibitor (Roche, Indianapolis, IN). Cells were further lysed by sonication (2 pulses for 30 s each), and the lysates were centrifuged at 13,000 rpm for 10 min at 4°C to remove cell debris. The supernatant of each lysate was collected and the total protein concentration was determined by the Bradford method and stored at ⫺80°C until use. Equal amounts (40 – 60 ␮g/sample) of whole cell lysates were solubilized in SDS-gel loading buffer and boiled for 8 min. Proteins were loaded on 7.5% SDS-polyacrylamide gels and transblotted to nitrocellulose membranes. After transfer, membranes were incubated in blocking buffer for 1 h (1X PBS and 5% nonfat dry milk) at room temperature with gentle agitation. The membranes were then probed with affinity purified anti-DRA antibody (1:100 dilution), anti-actin antibody (Cell Signaling, 1:30,000), anti-Rac1 antibody (BD Transduction, m102ab, 1:500), anti-total Rac1 (EMD Millipore, 1:10,000), anti-MCT-1 (Santa Cruz, H70, 1:200), anti-NHE3 (1: 6,000), and anti-PAT-1 (1:8,000) in 2.5% nonfat dry milk (1X PBS) overnight at 4°C with gentle agitation. The antibodies for NHE3 and PAT-1 were graciously provided by Dr. Chris Yun (Emory Univ.) and Dr. Peter Aronson (Yale Univ.), respectively. The membranes were washed four times with wash buffer (1X PBS ⫹ 0.1% Tween-20) for G45 5 min each. Membranes were then probed with HRP-conjugated goat anti-rabbit and anti-mouse antibodies (Santa Cruz, 1:2,000) in 2.5% nonfat dry milk (1X PBS) for 1 h at room temperature with gentle agitation. Membranes were then washed (4 ⫻ 5 min each) and visualized using Enhanced chemiluminescence detection (Bio-Rad). Statistical analyses. All data were analyzed by Prism (Prism GraphPad Software). Results are expressed as means ⫾ SE and represent the data from three to six independent experiments. Student’s t-test or one-way ANOVA with Tukey’s multiple comparison test was used for statistical analysis. P ⬍ 0.05 was considered statistically significant. RESULTS C. difficile TcdA and TcdB decreased DRA protein levels in intestinal epithelial cells. Given that NHE3 expression is altered after toxin administration (13) and in patients with CDI (8), we first investigated the effect of purified C. difficile toxins on DRA protein expression in intestinal epithelial cells. Caco2 cells treated with low doses (0.5–10 ng/ml) (16) of TcdA had a significant reduction in DRA protein levels in a dose- Fig. 1. TcdA and TcdB decreased DRA protein levels in intestinal epithelial cells. Confluent Caco2 cells (14 days postplating) were treated with purified TcdA (0.5–10 ng/ml) (or TcdB (0.1–5 ng/ml) for 6 and 24 h. DRA levels were quantified and normalized to ␤-actin. A: DRA protein levels were decreased 45– 80% in response to TcdA at 6 and 24 h (n ⫽ 5). B: TcdB also decreased DRA protein levels by 50 – 80% at 6 and 24 h (n ⫽ 5). C: decreased DRA protein levels were also seen at 24 h in confluent T84 cells (5– 6 days postplating) given TcdA and TcdB (n ⫽ 3). Differences between toxin-treated cells and control: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, ****P ⬍ 0.0001. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. G46 C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION dependent manner at both 6 and 24 h (Fig. 1A). Similarly, low doses (0.1–5 ng/ml) (5, 9) of TcdB also decreased DRA protein levels in a dose-dependent manner at both 6 and 24 h (Fig. 1B). Furthermore, these findings were not found to be cell line specific as these doses of TcdA and TcdB also decreased DRA protein expression in T84 cells as well (Fig. 1C). Additionally, we also wanted to verify that our commercially available purified toxins were efficacious at the low doses used. Using an antibody specific for the nonglucosylated form of Rac1 (25), we found that active (able to bind GTP) Rac1 levels were significantly reduced in response to both TcdA and TcdB at 6 and 24 h in a dosedependent manner (data not shown). These data verified that both purified toxins inactivated their molecular targets at the concentrations and time points utilized in our vitro studies. Toxin-mediated decrease in DRA protein was not due to increased cell death. To identify whether this marked decrease in DRA protein levels was due to increased cell death, we performed cell viability assays using lactate dehydrogenase (LDH) release as a marker. Low doses of TcdA did not induce significant LDH release from Caco2 cells at 6 or 24 h (Fig. 2A). Similarly, Caco2 cells treated with low doses of TcdB also did not release significantly more LDH at 6 or 24 h (Fig. 2B). These results demonstrated that the doses of TcdA and TcdB reduced DRA protein levels without causing significant cell death. TcdA- and TcdB-induced decrease in DRA protein in intestinal epithelial cells was specific. Given that C. difficile toxins have been shown to affect the function and localization (9, 13) of other ion transporters, we next examined whether the effects of TcdA and TcdB were specific to DRA. We found that protein levels of NHE3 (Na⫹/H⫹ exchanger 3) remained un- changed in the presence of varying doses of TcdA and TcdB at 24 h (Fig. 3A). Additionally, we observed that PAT-1 (putative anion exchanger 1) was also unaffected by TcdA and TcdB administration at 24 h (Fig. 3A). We next examined whether ion transporters other than Na⫹/H⫹ and Cl⫺/HCO⫺ 3 exchangers were affected by C. difficile toxins. We found that protein expression of monocarboxylate transporter 1 (MCT-1), a colonic butyrate transporter known to be downregulated in intestinal infections and inflammation (19, 37), was decreased only at the highest dose of TcdA but unchanged in the presence of TcdB (Fig. 3B). Thus, while TcdA may moderately affect colonic butyrate transport via MCT-1, the rapid decrease in protein expression by both toxins was specific to DRA. TcdA and TcdB had no effect on DRA mRNA expression in Caco2 cells. Given that bacterial pathogens have been shown to downregulate DRA at the transcriptional level (20), we next examined how purified toxins would affect DRA mRNA levels. Contrary to DRA protein expression, we found no difference in DRA mRNA levels in Caco-2 cells with either TcdA or TcdB (Fig. 4). Furthermore, we also investigated the effects of toxins on other intestinal ion transporters (CFTR, MCT-1, NHE2, PAT-1, NHE3, SERT) and found no significant differences (Table 1). However, in the presence of TcdA at 24 h, we observed a significant increase in MDR1, a multidrug-resistance protein responsible for efflux of xenobiotics and bacterial toxins from the intestinal mucosa (31). Additionally, TcdBtreated Caco2 cells had increased mRNA levels of IL-8, a chemokine produced by macrophages and intestinal epithelial cells during inflammatory conditions, including CDI (26).These findings indicated that while both TcdA and TcdB Fig. 2. Decrease in DRA protein was not due to increased cell death. Confluent Caco2 cells were treated with purified TcdA (0.5–10 ng/ml) (A) or TcdB (0.1–5 ng/ml) (B) for 6 and 24 h. The treatment media were collected and analyzed for lactate dehydrogenase (LDH) release. Samples were analyzed using an ELISA plate reader to measure absorbance at 492 nm. LDH levels were quantified and normalized to two controls: cell-free media (EMEM) and cells treated with 1% Triton X-100 as a positive control (n ⫽ 5); 0 indicates control (untreated) Caco2 cells. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION G47 Fig. 3. TcdA and TcdB did not alter Na⫹/H⫹ exchanger 3 (NHE3) and putative anion exchanger 1 (PAT-1) expression in Caco2 cells. Confluent Caco2 cells (14 days postplating) were treated with purified TcdA (0.5–25 ng/ml) or TcdB (0.1–10 ng/ml) for 6 and 24 h. A: NHE3 and PAT-1 levels were quantified and normalized to the housekeeping gene ␤-actin (n ⫽ 4). B: monocarboxylate transporter 1 (MCT1) levels were quantified and normalized to the housekeeping gene ␤-actin (n ⫽ 5). *P ⬍ 0.05. inactivate Rho GTPases and, in our current findings, decrease expression of DRA protein levels, each toxin is capable of inducing different signaling pathways and immune responses as seen previously (16, 23, 35). Finally, these data illustrated that the TcdA- and TcdB-mediated downregulation of DRA protein likely occurred via a posttranscriptional mechanism. TcdA and TcdA ⫹ TcdB decreased DRA protein, but not mRNA, levels in the colon of C57BL/6 mice. Although differentiated Caco2 cells, an intestinal epithelial cell line, are a well-established model for studying the effects of bacterial pathogens (11, 19) and C. difficile specifically (12, 21), we next sought out an animal model of CDI to investigate how TcdA and TcdB affected DRA expression in a more physiologically relevant model. Using a previously established intrarectal mouse model (15), we investigated the direct effects of purified toxins A and B on the colon, the primary target of C. difficile colonization and infection (3, 5, 39). Consistent with our in vitro studies, mice administered TcdA and TcdA ⫹ TcdB exhibited significantly lower DRA protein levels compared with untreated controls (Fig. 5A). This finding was further supported by immunofluorescent staining of DRA showing decreased levels of DRA protein at the apical surface of colonic sections in TcdA- and TcdA ⫹ TcdB-treated mice (Fig. 5B). Interestingly, TcdB-treated mice did not exhibit statistically significant changes in DRA protein expression at this dose and time point (Fig. 5A). To investigate the role of transcriptional modulation of DRA in the toxigenic mouse model, we also examined DRA mRNA levels in the colonic mucosa. Similar to our in vitro results shown in Fig. 4, we found that DRA mRNA levels remained unchanged in the presence of both toxins alone and together (Fig. 5C). These results illustrate that the toxin-mediated decrease in DRA protein was recapitulated in a toxigenic mouse model of CDI and also occurred at the posttranscriptional level. Patients with recurrent CDI exhibited a significant loss in colonic DRA protein. To further identify the role of DRA in CDI, we obtained slides from sections of transverse colonic biopsies from healthy subjects and patients with recurrent CDI. Using immunofluorescent staining of DRA, we found that CDI patients exhibited a drastic reduction in DRA protein levels compared with healthy subjects (Fig. 6). Notably, this decrease in DRA expression appeared to be more robust in the CDI patient biopsies than in our toxigenic mouse model. This marked decrease in CDI patients further confirmed our findings from cell culture and the toxigenic mouse model of CDI. DISCUSSION Clostridium difficile infection is the primary cause of nosocomial diarrhea and hospital acquired infection in the United AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. G48 C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION Fig. 4. TcdA and TcdB had no effect on DRA mRNA in Caco2 cells. Confluent Caco2 cells (14 days postplating) were treated with purified TcdA (0.5–10 ng/ml) (A) or TcdB (0.1–5 ng/ml) (B) for 6 and 24 h. The mRNA levels of DRA were analyzed by RT-PCR and normalized to GAPDH. Values are expressed as relative expression compared with untreated cells. States (5, 39). In recent years, the emergence of hypervirulent strains and higher recurrence rates have made CDI an increasing health concern worldwide. Despite its prevalence and increasing severity, the pathophysiology underlying C. difficile-associated diarrhea remains poorly understood. In these studies, we have examined the key colonic Cl⫺ transporter DRA (SLC26A3) and found that purified C. difficile toxins TcdA and TcdB significantly reduced DRA protein levels in intestinal epithelial cells. Our data also showed that this decrease in DRA was not due to cellular toxicity as we saw no change in LDH release in vitro. These toxin-mediated effects appeared to be specific to DRA as PAT-1 and NHE3 protein levels and mRNA levels were unchanged in the presence of either toxin. Our in vitro studies also showed no change in DRA mRNA levels, indicating that the downregulation of DRA by TcdA and TcdB is likely occurring at the posttranscriptional level. Utilizing an intrarectal mouse model of CDI, we also found that TcdA and TcdA ⫹ TcdB administration, but not TcdB alone, significantly reduced colonic DRA protein expression in mice. Similar to our results in vitro, this downregulation of DRA protein was not seen at the mRNA level in these mice. Last, using colonic biopsies from healthy subjects and recurrent CDI patients, we found that patients with recurrent CDI exhibited a drastic loss of colonic DRA protein compared with healthy controls. These studies, for the first time, illustrate the effect of C. difficile toxins on DRA expression and provide a link between the diarrheal phenotype associated with CDI and intestinal epithelial ion transport. Colonic electroneutral NaCl absorption is primarily mediated through the Na⫹/H⫹ exchanger 3 (NHE3) and the Cl⫺/ HCO⫺ 3 exchanger (DRA) (29). Previous work has shown that TcdB causes internalization of NHE3 from the apical surface of renal and placental cell lines (13). This mislocalization of NHE3 was suggested to have caused an inhibition of NHE3 during CDI. However, when using a human intestinal organoid Table 1. Effect of TcdA and TcdB on mRNA levels of intestinal ion transporters and inflammatory cytokines at 6 and 24 h Gene CFTR MCT1 (SLC16A1) MDR1 (ABCB1) NHE2 (SLC9A2) NHE3 (SLC9A3) PAT1 (SLC26A6) SERT (SLC6A4) IL-8 Cystic fibrosis transmembrane conductance regulator Monocarboxylate transporter 1 Multidrug resistance protein 1 or P-glycoprotein Na⫹/H⫹ exchanger 2 Na⫹/H⫹ exchanger 3 Putative anion exchanger 1 Serotonin transporter Interleukin 8 TcdA 6 h TcdB 6 h TcdA 24 h TcdB 24 h ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ⫹ ns ns ns ns ns ns ns ns ns ns ns ns ⫹ Confluent Caco2 cells (14 days postplating) were treated with purified TcdA (10 ng/ml) or TcdB (5 ng/ml) for 6 and 24 h. The mRNA levels of CFTR, MCT-1, MDR1, NHE2, NHE3, PAT-1, SERT, and IL-8 were analyzed by RT-PCR and normalized to the housekeeping gene ␤-actin. Symbols used indicate changes in relative expression normalized to housekeeping gene: ⫹ (increase), ns (not significant). AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION G49 Fig. 5. TcdA and TcdA ⫹ TcdB decreased DRA protein, but not mRNA, levels in the colon of C57BL/6 mice. Ten- to 12-wk old female C57BL/6 mice were intrarectally administered purified TcdA (10 ␮g), TcdB (10 ␮g), or TcdA/TcdB (5 ␮g each) in 100 ␮l PBS. After 4 h, mice were euthanized and colonic mucosal scrapings harvested. A: relative DRA levels (normalized to ␤-actin) in total protein extracted from colonic mucosa shown by Western blotting (n ⫽ 5). *P ⬍ 0.05. B: representative image of immunostaining of DRA (green) and villin (red) in distal colonic mucosal sections. C: relative mRNA abundance of DRA in total RNA samples from colonic mucosa was shown via RT-PCR using gene-specific primers. Values were normalized to GAPDH as an internal control (n ⫽ 5). (HIO) model system, Engevik et al. (8) found that NHE3 was reduced at both the mRNA and protein levels when HIOs were infected with toxin-producing C. difficile. These findings on NHE3 may indicate that C. difficile causes inhibition of Na⫹/H⫹ exchange via multiple mechanisms during infection. Interestingly, our current studies did not find alterations in NHE3 protein or mRNA levels in Caco2 cells. This may be due to variations in the model system and/or cell type used in addition to the different effects commonly seen in different strains of C. difficile and varying concentrations of toxins (16, 23, 24). DRA, which is functionally coupled to NHE3, also plays an essential role in intestinal chloride absorption with its downregulation being implicated in both infectious and inflammatory models of diarrhea including enteropathogenic E. coli infection (EPEC) (11, 24), but the current study is the first one to investigate DRA in the context of CDI. Additionally, our studies found no changes in PAT-1, another member of the SLC26 family. PAT-1, like DRA, is an intestinal luminal Cl⫺/ HCO⫺ 3 exchanger found predominantly in the duodenum, je- junum, and ileum with mRNA transcripts also seen in the heart, kidneys, and pancreas (17). Although both PAT-1 and DRA facilitate intestinal luminal Cl⫺ absorption, DRA is highly expressed in the colon, the primary site of C. difficile colonization and infection (24, 28, 39). Furthermore, DRA, but not PAT-1, knockout mice exhibit a diarrheal phenotype similar to that seen in congenital chloride diarrhea (CLD) (17, 18). Thus our current studies show that the effects of TcdA and TcdB are specific to DRA, further supporting previous findings that regulation of DRA is implicated in infectious models of diarrhea. Symptoms associated with C. difficile infection (diarrhea, pseudomembranous colitis, disruption of the intestinal barrier) are primarily associated with two major cytotoxins, TcdA and TcdB (5, 35). Both TcdA and TcdB are large glucosylating enterotoxins that irreversibly glucosylate Rho family GTPases (Rac1, Cdc42, Rho) at Thr-35 or Thr-37 (25, 35). This inactivation of Rho GTPases elicits cytoskeletal changes, disrupts tight junctions, induces inflammatory cascades, and causes cell AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. G50 C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION Fig. 6. Patients with recurrent Clostridium difficile infection (CDI) exhibited a significant loss in colonic DRA protein. Immunostaining of DRA (green) in transverse colonic biopsies of healthy and CDI patients and semiquantitative analysis of surface DRA expression compared with healthy colon. Representative images from observations seen in n ⫽ 3 healthy subjects and 3 CDI patients. **P ⬍ 0.01. death by both apoptotic and necrotic mechanisms (2, 5, 16, 21, 40). TcdA and TcdB are also known to alter barrier function in Caco2 cells through the reorganization of actin filaments and tight junction proteins such as ZO-1, occludin, and claudin (27). As expected, our studies also showed a decrease in transepithelial electrical resistance (TEER) in Caco2 cells treated with purified C. difficile toxins, indicating that our toxins are capable of affecting Caco2 cells and disrupting normal barrier function (data not shown). Furthermore, our current findings show a dose-dependent decrease in nonglycosylated Rac1 in the presence of TcdA and TcdB. Thus future studies remain to be done that focus on the mechanisms underlying the toxin-mediated decrease in DRA protein levels and whether these changes are dependent on Rho GTPase inactivation. Previous studies have shown that cytoskeletal components are also implicated in the context of intestinal ion transport. Asghar et al. (1) showed that keratin-8 deficient mice have decreased DRA protein and mRNA levels. Additionally, silencing of K8 in Caco2 cells resulted in decreased DRA protein levels. Therefore, future studies are needed to investigate the relationship between cytoskeletal reorganization and DRA downregulation by C. difficile toxins. Some strains of C. difficile, including recent hypervirulent NAPI/027 strains, also produce a third toxin, binary toxin or C. difficile transferase (CDT). CDT is known to cause microtubule-based protrusions and F-actin depolymerization via ADP-ribosylation of actin at arginine 177 (10, 33). Given its emerging role in CDI, our preliminary studies also investigated the effects of purified CDT on DRA protein levels and found no significant change (data not shown). Thus, although the overall contribution of CDT to CDI-associated pathology remains unclear, our data show that only the major C. difficile toxins, TcdA and TcdB, decrease DRA protein levels in vitro. Modulation of intestinal ion exchangers NHE3 and DRA has been implicated in a variety of inflammatory and infectious models of diarrhea (1, 11, 20, 29). Consistent with these studies, TcdA and TcdB caused a significant decrease in DRA protein levels in vitro. However, our current studies did not indicate alterations in DRA mRNA, an observation frequently seen in other studies (1, 19). These results indicate that toxin- mediated alterations in DRA protein are likely occurring posttranscriptionally. One possible mechanism for this reduction in DRA is increased protein degradation via the ubiquitin/proteasomal pathway. Premature protein degradation of cytosolic proteins by bacterial toxins has been shown in a variety of bacterial infections, including L. monocytogenes and enteropathogenic E. coli (EPEC) (30, 36). These bacterial toxins utilize posttranslational modifications of ubiquitin and ubiquitin-like proteins, such as SUMO and Nedd8, to covalently attach ubiquitin to lysine residues of proteins, thus targeting them for degradation (22). Thus future studies remain to be done that examine the potential interplay between toxin-mediated reduction in DRA protein levels and the ubiquitin/proteasomal pathway of protein degradation. In addition to the many in vitro effects of TcdA and TcdB, clinical hallmarks of CDI are also toxin-mediated including the production of proinflammatory cytokines, disruption of the intestinal epithelial barrier, and activation of the innate immune system (32, 34). One of the most commonly used in vivo models is the small animal ileal loop method, a surgical procedure used in involving ligation of the terminal ileum after injection with C. difficile toxins (7, 14). Although this method is a mainstay of CDI animal models, it is not without its drawbacks. In addition to the risks associated with small animal surgery, the primary target of human CDI is the colon, not the ileum. Therefore, our studies utilized a toxigenic intrarectal mouse model developed by Hirota et al. (15). This mouse model, in contrast to previous methodologies (4, 38), requires no prior antibiotic treatment and allows direct intrarectal instillation of C. difficile toxins into the colon. Using this method, we found that mice given purified TcdA alone and TcdA ⫹ TcdB had significant decreases in DRA protein levels. Interestingly, DRA protein levels remained unaffected in mice given TcdB alone, a finding different from our in vitro studies. In the development of this intrarectal model, Hirota et al. (15) found that only TcdA was capable of inducing colonic tissue damage and intestinal inflammation. Given that the efficacy of TcdA or TcdB in various models of CDI has been heavily debated (3, 23), it is possible that TcdA is more potent in this animal model of CDI. Thus mechanistic studies of TcdA- AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION specific downregulation of DRA protein in the toxigenic mouse model will significantly aid in the understanding of C. difficile infection in vivo. We also investigated the effect of both TcdA and TcdB on DRA mRNA in vivo. Consistent with our results in Caco2 cells, neither TcdA nor TcdB altered expression of DRA mRNA. This finding further indicated that toxin-mediated downregulation of DRA occurs at the posttranscriptional level. Given that C. difficile infection is the primary cause of nosocomial diarrhea and represents a significant burden to human health, we also examined the levels of DRA in colonic biopsies of CDI patients. In the current studies, we found that patients with recurrent CDI had a drastic reduction in colonic DRA expression compared with healthy subjects. This reduction in DRA levels is similar to the findings of Engevik et al. (8), who observed that CDI patients have reduced expression of NHE3, a colonic Na⫹/H⫹ exchanger that is functionally coupled to DRA in the human intestine. Together with our results in vitro and in vivo, this finding indicated that the toxin-mediated decrease in DRA protein is a phenomenon recapitulated in multiple models of CDI. In summary, our current studies demonstrate, for the first time, that C. difficile toxins reduce DRA protein, but not mRNA, levels in intestinal epithelial cells and, in the case of TcdA, in a toxigenic mouse model of CDI. Last, patients with recurrent CDI also showed significantly lower levels of colonic DRA protein than healthy subjects. Given the critical role for DRA in intestinal NaCl absorption and its implications in infectious and inflammatory diarrhea, these findings indicate that a downregulation of DRA may be a critical factor in CDI-associated diarrhea. These findings are in agreement with earlier studies showing the inhibition of NHE3 during C. difficile infection (8, 13). Taken together, these studies show a direct targeting of intestinal ion transporters by C. difficile toxins and highlight a potentially novel target for therapeutic intervention in CDI-associated diarrhea. GRANTS These studies were supported by the National Institute of Digestive and Kidney Diseases Grants DK-71596 (W. A. Alrefai), DK-98170 (R. K. Gill), and DK-54016, DK-81858, and DK-92441 (P. K. Dudeja) and Department of Veterans Affairs (VA) Grants BX 002011 (P. K. Dudeja), BX 000152 (W. A. Alrefai), and BX 002687 (S. Saksena) and VA Research Career Scientist Awards (P. K. Dudeja, W. A. Alrefai). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS H.P.C. and P.K.D. conceived and designed research; H.P.C., S.P., A.N.A., and S.T. performed experiments; H.P.C., S.P., A.N.A., C.S., S.T., S.S., R.K.G., W.A.A., and P.K.D. analyzed data; H.P.C., S.P., A.N.A., C.S., M.A.E., J.V., M.B.Y., B.R.Y., S.T., S.S., R.K.G., W.A.A., and P.K.D. interpreted results of experiments; H.P.C. prepared figures; H.P.C. drafted manuscript; H.P.C., S.P., A.N.A., C.S., M.A.E., J.V., M.B.Y., B.R.Y., S.S., R.K.G., W.A.A., and P.K.D. edited and revised manuscript; H.P.C., S.P., A.N.A., C.S., M.A.E., J.V., M.B.Y., B.R.Y., S.T., S.S., R.K.G., W.A.A., and P.K.D. approved final version of manuscript. REFERENCES 1. Asghar MN, Priyamvada S, Nyström JH, Anbazhagan AN, Dudeja PK, Toivola DM. Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon. Am J Physiol Gastrointest Liver Physiol 310: G1147–G1154, 2016. doi:10.1152/ajpgi.00354.2015. G51 2. Bezerra Lima B, Faria Fonseca B, da Graça Amado N, Moreira Lima D, Albuquerque Ribeiro R, Garcia Abreu J, de Castro Brito GA. Clostridium difficile toxin A attenuates Wnt/␤-catenin signaling in intestinal epithelial cells. Infect Immun 82: 2680 –2687, 2014. doi:10.1128/IAI. 00567-13. 3. Carter GP, Chakravorty A, Pham Nguyen TA, Mileto S, Schreiber F, Li L, Howarth P, Clare S, Cunningham B, Sambol SP, Cheknis A, Figueroa I, Johnson S, Gerding D, Rood JI, Dougan G, Lawley TD, Lyras D. Defining the roles of TcdA and TcdB in localized gastrointestinal disease, systemic organ damage, and the host response during Clostridium difficile infections. MBio 6: e00551–e005515, 2015. doi:10. 1128/mBio.00551-15. 4. Chen X, Katchar K, Goldsmith JD, Nanthakumar N, Cheknis A, Gerding DN, Kelly CP. A mouse model of Clostridium difficile-associated disease. Gastroenterology 135: 1984 –1992, 2008. doi:10.1053/j. gastro.2008.09.002. 5. Chumbler NM, Farrow MA, Lapierre LA, Franklin JL, Lacy DB. Clostridium difficile toxins TcdA and TcdB cause colonic tissue damage by distinct mechanisms. Infect Immun 84: 2871–2877, 2016. doi:10.1128/ IAI.00583-16. 6. Citalán-Madrid AF, García-Ponce A, Vargas-Robles H, Betanzos A, Schnoor M. Small GTPases of the Ras superfamily regulate intestinal epithelial homeostasis and barrier function via common and unique mechanisms. Tissue Barriers 1: e26938, 2013. doi:10.4161/tisb.26938. 7. de Araújo Junqueira AFT, Dias AAM, Vale ML, Spilborghs GMGT, Bossa AS, Lima BB, Carvalho AF, Guerrant RL, Ribeiro RA, Brito GA. Adenosine deaminase inhibition prevents Clostridium difficile toxin A-induced enteritis in mice. Infect Immun 79: 653–662, 2011. doi:10. 1128/IAI.01159-10. 8. Engevik MA, Engevik KA, Yacyshyn MB, Wang J, Hassett DJ, Darien B, Yacyshyn BR, Worrell RT. Human Clostridium difficile infection: inhibition of NHE3 and microbiota profile. Am J Physiol Gastrointest Liver Physiol 308: G497–G509, 2015. doi:10.1152/ajpgi. 00090.2014. 9. Feng Y, Cohen SN. Upregulation of the host SLC11A1 gene by Clostridium difficile toxin B facilitates glucosylation of Rho GTPases and enhances toxin lethality. Infect Immun 81: 2724 –2732, 2013. doi:10.1128/ IAI.01177-12. 10. Gerding DN, Johnson S, Rupnik M, Aktories K. Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes 5: 15–27, 2014. doi:10.4161/gmic.26854. 11. Gill RK, Borthakur A, Hodges K, Turner JR, Clayburgh DR, Saksena S, Zaheer A, Ramaswamy K, Hecht G, Dudeja PK. Mechanism underlying inhibition of intestinal apical Cl/OH exchange following infection with enteropathogenic E. coli. J Clin Invest 117: 428 –437, 2007. doi:10. 1172/JCI29625. 12. Hansen A, Alston L, Tulk SE, Schenck LP, Grassie ME, Alhassan BF, Veermalla AT, Al-Bashir S, Gendron F-P, Altier C, MacDonald JA, Beck PL, Hirota SA. The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction. PLoS One 8: e81491, 2013. doi:10.1371/journal.pone. 0081491. 13. Hayashi H, Szászi K, Coady-Osberg N, Furuya W, Bretscher AP, Orlowski J, Grinstein S. Inhibition and redistribution of NHE3, the apical Na⫹/H⫹ exchanger, by Clostridium difficile toxin B. J Gen Physiol 123: 491–504, 2004. doi:10.1085/jgp.200308979. 14. Hirota SA, Fines K, Ng J, Traboulsi D, Lee J, Ihara E, Li Y, Willmore WG, Chung D, Scully MM, Louie T, Medlicott S, Lejeune M, Chadee K, Armstrong G, Colgan SP, Muruve DA, MacDonald JA, Beck PL. Hypoxia-inducible factor signaling provides protection in Clostridium difficile-induced intestinal injury. Gastroenterology 139: 259 –269.e3, 2010. doi:10.1053/j.gastro.2010.03.045. 15. Hirota SA, Iablokov V, Tulk SE, Schenck LP, Becker H, Nguyen J, Al Bashir S, Dingle TC, Laing A, Liu J, Li Y, Bolstad J, Mulvey GL, Armstrong GD, MacNaughton WK, Muruve DA, MacDonald JA, Beck PL. Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure. Infect Immun 80: 4474 –4484, 2012. doi:10.1128/IAI.00933-12. 16. Johal SS, Solomon K, Dodson S, Borriello SP, Mahida YR. Differential effects of varying concentrations of clostridium difficile toxin A on epithelial barrier function and expression of cytokines. J Infect Dis 189: 2110 –2119, 2004. doi:10.1086/386287. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022. G52 C. DIFFICILE TOXINS DECREASE DRA PROTEIN EXPRESSION 17. Kato A, Romero MF. Regulation of electroneutral NaCl absorption by the small intestine. Annu Rev Physiol 73: 261–281, 2011. doi:10.1146/ annurev-physiol-012110-142244. 18. Kim KH, Shcheynikov N, Wang Y, Muallem S. SLC26A7 is a Cl⫺ channel regulated by intracellular pH. J Biol Chem 280: 6463–6470, 2005. doi:10.1074/jbc.M409162200. 19. Kumar A, Alrefai WA, Borthakur A, Dudeja PK. Lactobacillus acidophilus counteracts enteropathogenic E. coli-induced inhibition of butyrate uptake in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 309: G602–G607, 2015. doi:10.1152/ajpgi.00186.2015. 20. Kumar A, Anbazhagan AN, Coffing H, Chatterjee I, Priyamvada S, Gujral T, Saksena S, Gill RK, Alrefai WA, Borthakur A, Dudeja PK. Lactobacillus acidophilus counteracts inhibition of NHE3 and DRA expression and alleviates diarrheal phenotype in mice infected with Citrobacter rodentium. Am J Physiol Gastrointest Liver Physiol 311: G817– G826, 2016. doi:10.1152/ajpgi.00173.2016. 21. LaFrance ME, Farrow MA, Chandrasekaran R, Sheng J, Rubin DH, Lacy DB. Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity. Proc Natl Acad Sci USA 112: 7073–7078, 2015. doi:10.1073/pnas.1500791112. 22. Lemichez E, Barbieri JT. General aspects and recent advances on bacterial protein toxins. Cold Spring Harb Perspect Med 3: a013573, 2013. doi:10.1101/cshperspect.a013573. 23. Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, Phumoonna T, Poon R, Adams V, Vedantam G, Johnson S, Gerding DN, Rood JI. Toxin B is essential for virulence of Clostridium difficile. Nature 458: 1176 –1179, 2009. doi:10.1038/nature07822. 24. Malakooti J, Saksena S, Gill RK, Dudeja PK. Transcriptional regulation of the intestinal luminal Na⫹ and Cl⫺ transporters. Biochem J 435: 313–325, 2011. doi:10.1042/BJ20102062. 25. May M, Wang T, Müller M, Genth H. Difference in F-actin depolymerization induced by toxin B from the Clostridium difficile strain VPI 10463 and toxin B from the variant Clostridium difficile serotype F strain 1470. Toxins (Basel) 5: 106 –119, 2013. doi:10.3390/toxins5010106. 26. Nicholas A, Jeon H, Selasi GN, Na SH, Kwon HI, Kim YJ, Choi CW, Kim SI, Lee JC. Clostridium difficile-derived membrane vesicles induce the expression of pro-inflammatory cytokine genes and cytotoxicity in colonic epithelial cells in vitro. Microb Pathog 107: 6 –11, 2017. doi:10. 1016/j.micpath.2017.03.006. 27. Popoff MR, Geny B. Rho/Ras-GTPase-dependent and -independent activity of clostridial glucosylating toxins. J Med Microbiol 60: 1057–1069, 2011. doi:10.1099/jmm.0.029314-0. 28. Priyamvada S, Anbazhagan AN, Kumar A, Soni V, Alrefai WA, Gill RK, Dudeja PK, Saksena S. Lactobacillus acidophilus stimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 310: G599 –G608, 2016. doi:10.1152/ajpgi.00210.2015. 29. Priyamvada S, Gomes R, Gill RK, Saksena S, Alrefai WA, Dudeja PK. Mechanisms underlying dysregulation of electrolyte absorption in inflammatory bowel disease-associated diarrhea. Inflamm Bowel Dis 21: 2926 – 2935, 2015. doi:10.1097/MIB.0000000000000504. 30. Ribet D, Hamon M, Gouin E, Nahori M-A, Impens F, Neyret-Kahn H, Gevaert K, Vandekerckhove J, Dejean A, Cossart P. Listeria monocytogenes impairs SUMOylation for efficient infection. Nature 464: 1192– 1195, 2010. doi:10.1038/nature08963. 31. Saksena S, Goyal S, Raheja G, Singh V, Akhtar M, Nazir TM, Alrefai WA, Gill RK, Dudeja PK. Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice. Am J Physiol Gastrointest Liver Physiol 300: G1115– G1123, 2011. doi:10.1152/ajpgi.00027.2011. 32. Savidge TC, Pan W-H, Newman P, O’brien M, Anton PM, Pothoulakis C. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology 125: 413–420, 2003. doi:10.1016/ S0016-5085(03)00902-8. 33. Schwan C, Kruppke AS, Nölke T, Schumacher L, Koch-Nolte F, Kudryashev M, Stahlberg H, Aktories K. Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence. Proc Natl Acad Sci USA 111: 2313–2318, 2014. doi:10.1073/pnas.1311589111. 34. Sun X, Hirota SA. The roles of host and pathogen factors and the innate immune response in the pathogenesis of Clostridium difficile infection. Mol Immunol 63: 193–202, 2015. doi:10.1016/j.molimm.2014.09.005. 35. Sun X, Savidge T, Feng H. The enterotoxicity of Clostridium difficile toxins. Toxins (Basel) 2: 1848 –1880, 2010. doi:10.3390/toxins2071848. 36. Taieb F, Nougayrède J-P, Oswald E. Cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells. Toxins (Basel) 3: 356 –368, 2011. doi:10.3390/toxins3040356. 37. Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. The role of short-chain fatty acids in health and disease. Adv Immunol 121: 91–119, 2014. doi:10.1016/B978-0-12-800100-4.00003-9. 38. Theriot CM, Koumpouras CC, Carlson PE, Bergin II, Aronoff DM, Young VB. Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains. Gut Microbes 2: 326 –334, 2011. doi:10.4161/gmic.19142. 39. Vedantam G, Clark A, Chu M, McQuade R, Mallozzi M, Viswanathan VK. Clostridium difficile infection: toxins and non-toxin virulence factors, and their contributions to disease establishment and host response. Gut Microbes 3: 121–134, 2012. doi:10.4161/gmic.19399. 40. Warny M, Keates AC, Keates S, Castagliuolo I, Zacks JK, Aboudola S, Qamar A, Pothoulakis C, LaMont JT, Kelly CP. p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis. J Clin Invest 105: 1147–1156, 2000. doi:10.1172/JCI7545. 41. Wedenoja S, Höglund P, Holmberg C. Review article: the clinical management of congenital chloride diarrhoea. Aliment Pharmacol Ther 31: 477–485, 2010. doi:10.1111/j.1365-2036.2009.04197.x. 42. Xiao F, Yu Q, Li J, Johansson MEV, Singh AK, Xia W, Riederer B, Engelhardt R, Montrose M, Soleimani M, Tian DA, Xu G, Hansson GC, Seidler U. Slc26a3 deficiency is associated with loss of colonic HCO3⫺ secretion, absence of a firm mucus layer and barrier impairment in mice. Acta Physiol (Oxf) 211: 161–175, 2014. doi:10.1111/apha.12220. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00307.2017 • www.ajpgi.org Downloaded from journals.physiology.org/journal/ajpgi (054.167.240.005) on January 25, 2022.