Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( Isc) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable Isc. Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization. NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host’s cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux ( Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current ( Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host’s cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira’s ability to inhibit NHE3 transcription, resulting in diarrheal disease. NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host’s cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.

Kinetic characterization of electrogenic sodium‐dependent transport in Ussing chambers of d ‐glucose and d ‐galactose demonstrated sigmoidal/Hill kinetics in the porcine jejunum and ileum, with the absence of transport in the distal colon. In the jejunum, a high‐affinity, super‐low‐capacity (Ha/ sL c) kinetic system accounted for glucose transport, and a low‐affinity, low‐capacity (La/Lc) kinetic system accounted for galactose transport. In contrast, the ileum demonstrated a high‐affinity, super‐high‐capacity (Ha/ sH c) glucose transport and a low‐affinity, high‐capacity (La/Hc) galactose transport systems. Jejunal glucose transport was not inhibited by dapagliflozin, but galactose transport was inhibited. Comparatively, ileal glucose and galactose transport were both sensitive to dapagliflozin. Genomic and gene expression analyses identified 10 of the 12 known SLC 5A family members in the porcine jejunum, ileum, and distal colon. Dominant SGLT 1 ( SLC 5A1) and SGLT 3 ( SLC 5A4) expression was associated with the sigmoidal Ha/ sL c glucose and La/Lc galactose transport systems in the jejunum. Comparatively, the dominant expression of SGLT 1 ( SLC 5A1) in the ileum was only associated with Ha glucose and La galactose kinetic systems. However, the sigmoidal kinetics and overall high capacity (Hc) of transport is unlikely accounted for by SGLT 1 ( SLC 5A1) alone. Finally, the absence of transport and lack of pharmacological inhibition in the colon was associated with the poor expression of SLC 5A genes. Altogether, the results demonstrated intestinal segregation of monosaccharide transport fit different sigmoidal kinetic systems. This reveals multiple transporter populations in each system, supported by gene expression profiles and pharmacological inhibition. Overall, this work demonstrates a complexity to transporter involvement in intestinal electrogenic monosaccharide absorption systems not previously defined.

The porcine epidemic diarrhea virus (PEDV), an alphacoronavirus affects the small intestine of suckling piglets causing acute watery diarrhea. Here, the effects on agonist‐induced electrogenic secretory response by PEDV was assessed to determine its role in the diarrheal disease. Jejunal tissues were compared in healthy (n=12) and diseased (n=20) one‐week‐old crossbred Yorkshire x Landrace piglets in Ussing chambers. Diseased jejunal tissues displayed elevated changes in Isc following agonist addition compared to control. Activation of cAMP driven secretion via isoproterenol (adrenergic agonist) and forskolin/3‐isobutyl‐1‐methylxanthine (IBMX) demonstrated a significantly increased change in short‐circuit current (Isc). Cholinergic activation of secretion via carbachol revealed no significant difference in Isc, however, 9/20 diseased jejunal tissues had a negative change in Isc while the remaining 11/20 had a positive change in Isc. Bumentanide inhibitable Isc was not significantly different in diseased tissues compared to controls. Inhibition of cAMP induced secretion via glibenclamide revealed a significant increase in Isc, while niflumic acid, a potent inhibitor of calcium‐activated chloride channels, had no significant effect. Non‐selective potassium channel blocker tetraethylammonium chloride (TEA) revealed a significant decrease in the Isc of diseased animals compared to control. Furthermore, significant decrease in transepithelial electrical resistance (TEER) was noted in jejunal tissues infected with PED virus compared to controls. RT‐qPCR was conducted to determine if elevated chloride and potassium channel gene transcripts were responsible for the increase in cAMP induced secretion. Inflammatory cytokines were also assessed to determine their role in ion channel modulation. mRNA expression of the Cystic Fibrosis transmembrane conductance regulator (CFTR) in the jejunum of PEDV infected piglets was not significantly different than control, while potassium voltage‐gated channel subfamily Q member 1 (KCNQ1) was significantly down‐regulated in diseased animals. However, mRNA expression of potassium calcium‐activated channel subfamily N member 4 (KCNN4) and Transmembrane member protein 16A (TMEM16A) were significantly up‐regulated 3‐fold (P < 0.05) and 5‐fold (P < 0.05) respectively in the jejunum of diseased pigs compared to control. Furthermore, gene expression of proinflammatory cytokines was determined to be significantly elevated in the jejunum of diseased piglets. Collectively, these findings suggest that upregulation of KCNN4 by proinflammatory cytokines is responsible for the increased change in agonist induced Isc by driving chloride secretion via CFTR and TMEM16A.Support or Funding InformationThis research was supported by Genome Alberta GAB‐PEDH ‐ U of S – 344078, the Saskatchewan Agricultural Development Fund (ADF) Project 20140215, and the National Sciences and Engineering Research Council of Canada Discovery Grant 371364‐2010 (M.E.L.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mucosal-to-serosal flux of 14C 3- O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities ( Km “fingerprints”) between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.