This study examines the impact of Brachyspira hyodysenteriae and Brachyspira hampsonii infection on chloride (36Cl−) transport within the porcine colon. Utilizing 3H-mannitol and 36Cl−, we evaluated the bidirectional flux across colonic tissues from both healthy and infected pigs. We observed no change in 3H-mannitol flux, indicating unaffected paracellular movement. However, we observed a significant decrease in mucosal-serosal 36Cl− flux (Jm-s) in the apical segment of the spiral colon of infected pigs, suggesting a compromised chloride absorption due to the downregulation of DRA (SLC26A3), confirmed by reduced mRNA and protein levels. To dissect the molecular dynamics in vitro, we exposed polarized Caco-2 cells to B. hampsonii lysate. This model recapitulated the in vivo responses, including the downregulation of DRA and the concomitant IL-1α upregulation. This in vitro model demonstrated that B. hampsonii lysate upregulates IL-1α expression through p38 MAPK phosphorylation. More importantly, in vitro studies with B. hampsonii lysate, recombinant IL-1α, SB 203580 (p38 MAPK inhibitor), and blocking IL-1α signalling with IL-1 receptor antagonist (IL-1RA) revealed that IL-1α has a dose-dependent negative effect on DRA expression and a positive effect on IL-1α expression. Furthermore, inhibiting IL-1α expression with SB 203580 and blocking IL-1α signalling with IL-1RA not only prevented this downregulation of DRA but also promoted its upregulation, highlighting the regulatory role of IL-1α in chloride absorption. In summary, this study presents an in-depth understanding by which Brachyspira infections can impede chloride absorption in the colon and offers potential intervention targets for conditions involving impaired DRA-mediated Cl− transport. This research was supported by Alberta Livestock Meat Association (ALMA 2013R054R) and Natural Sciences and Engineering Research Council of Canada Discovery Grant 02743-2021 (to M. E. Loewen). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In this study, we uncovered the novel mechanism of IL-1α-mediated downregulated in adenoma (DRA) (SLC26A3) downregulation in the context of Brachyspira spp.-induced malabsorptive diarrhea. Experimentally infected pigs with Brachyspira spp. had significantly reduced DRA expression in the colon accompanied by IL-1α upregulation. This response was recapitulated in vitro by exposing Caco-2 cells to either Brachyspira lysate or IL-1α. Both p38 and MAPK-activated protein kinase 2 (MAPKAPK-2 also referred as MK-2) showed an increased phosphorylation after exposure to either. SB203580 application, a p38 inhibitor blocked the MK-2 phosphorylation and attenuated the DRA and IL-1α response to both lysate and IL-1α. Exposure to IL-1 receptor antagonist (IL-1RA) produced a similar response. In addition, exposure of cells to either of these blockers without IL-1α or lysate results in increased DRA and decreased IL-1α expression, revealing that DRA needs IL-1α signaling for basal physiological expression. Dual inhibition with both blockers completely inhibited the effect from IL-1α while significantly attenuating the response from Brachyspira lysate, suggesting a minor contribution from another pathway. Together this demonstrates that Brachyspira activates p38 MAPK signaling driving IL-1α expression, which activates IL-1R1 causing DRA downregulation while also driving upregulation of IL-1α through p38 in a positive feedback mechanism. In conclusion, we elucidated a major pathway involved in DRA downregulation and its role in Brachyspira-induced diarrhea. In addition, these observations will aid in our understanding of other inflammatory and infectious diarrhea conditions.NEW & NOTEWORTHY The diarrheal disease caused by the two infectious spirochete spp. B. hyodysenteriae and B. hampsonii reduced the expression of DRA (SLC26A3), a major Cl-/HCO-3 exchanger involved in Cl- absorption. This is attributed to the upregulation of IL-1α driven by p38 MAPK. This work also describes a potential new mechanism in inflammatory diseases while showing the importance of IL-1α in maintaining DRA levels.

BACKGROUND:TlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis. METHODS:TlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis. RESULTS:Purified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments. CONCLUSIONS:Oligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence. GENERAL SIGNIFICANCE:Our results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.

Impairment of electroneutral Na (cid:2) transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge with Brachyspira spp. Am Physiol Gastrointest Liver Physiol effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na (cid:2) transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na (cid:2) absorption was assessed in Ussing chambers by radiolabeled 22 Na flux and pharmacological inhibitory studies. Basal radiolabeled 22 Na flux experiments revealed that mu-cosal-to-serosal flux ( J ms ) was significantly impaired in B. hyodysen- teriae and B. hampsonii -diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current ( I sc ) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na (cid:2) absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na (cid:2) /H (cid:2) 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-1 (cid:3) 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 (cid:2) absorption in the porcine colon due to Brachyspira’s ability to inhibit NHE3 transcription, resulting in diarrheal disease. (cid:2) (cid:2) (NHE3) in findings decrease in NHE3 mRNA and protein cytokine Biosci-ences), verified by supplier to react with pig and anti- (cid:5) -actin (C-4; sc-47778; Santa Cruz Biotechnology) in a 10% RapidBlock solution. The membranes then incubated for 1 h at room temperature with secondary antibodies Alexa Fluor 488 Goat anti-Rabbit IgG antibody (A-11008) and ECL Plex Goat anti-Mouse IgG-Cy5 antibody (PA45009; Amersham Biosciences) in 10% RapidBlock solution. Proteins were detected and analyzed using Typhoon Trio and ImageQuant TL System (63005583; GE Healthcare Life Sciences). The sizes of detected proteins were compared with a prestained rec protein ladder loaded on each blot (BP3603500; Fisher BioReagents). Cell culture. The Caco-2 cell line derived from human colorectal adenocarcinoma (HTB-37; ATCC, Manassas, VA) was cultured in DMEM (10-0130CM; Corning, Manassas, VA) containing 10% heat-inactivated fetal bovine serum (Gibco, Burlington, ON, Canada), 1% penicillin-streptomycin (15140-122; Life Technologies), and 1% MEM nonessential amino acids (Gibco, Grand Island, NY) at 37°C in a humidified atmosphere with 5% CO 2 . Cells were plated on polyester Transwell permeable supports (0.4- (cid:9) m pores, 24-mm diameter; Corn-ing) and cultured under standard conditions until confluency. Cells were maintained for 10 days after confluency was achieved before being used in downstream experiments. was analyzed by Kruskal-Wallis one-way ANOVA. Changes in rate of 22 Na transport was analyzed by Kruskal-Wallis one-way ANOVA and Dunn’s test. Fold changes in mRNA expression of diseased colonic segments compared with control obtained by RT-qPCR were log 10 transformed and analyzed by one-way ANOVA and Tukey post hoc. Western blot analysis of NHE3 protein and cell signaling pathways were analyzed by Student’s t test (control vs. Brachyspira species in 2 separate analyses for NHE3 protein, control vs Brachyspira lysate treated for cell signaling pathways). Fold changes obtained by RT- qPCR for in vitro data were analyzed by Student’s t test. Significance was set a priori at P (cid:8) 0.05.

The effects of Brachyspira hyodysenteriae and Brachyspira hampsoniispirochetosis 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 assessedin Ussing chambers by radiolabelled 22Na flux and pharmacological inhibitory studies. Basal radiolabelled 22Na flux experiments revealed that Jmswas significantly impairedin B. hyodysenteriaeand B. hampsonii diseased pigs. Inhibition of ENaC 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 supportedby significant down-regulation of Na+/H+exchanger (NHE1, NHE2, andNHE3) 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 Brachyspira hampsoniilysate significantly down-regulated 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'sability to inhibit NHE3 transcription, resulting in diarrheal disease.

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 radiolabeled22Na flux and pharmacological inhibitory studies. Basal radiolabeled22Na 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.

The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.

Cytolysins produced by Brachyspira hyodysenteriae and “Brachyspira hampsonii” strain 30446 are believed to be important virulence factors in the pathology of swine dysentery. To date four putative cytolysin genes termed tlyA, tlyB, tlyC, and hlyA have been identified, all of which are present in both Brachyspira hyodysenteriae and “Brachyspira hampsonii”. While the contributions of tlyB, tlyC, and hlyA to the pathogenesis of swine dysentery are not yet known, tlyA knockout strains of Brachyspira hyodysenteriae have shown decreased virulence in mouse models. In addition, homologues of tlyA are believed to be important virulence factors for human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, functioning as both cytolysins and contributing to antibiotic resistance through ribosomal RNA methylation. While active hlyA protein has been purified from Brachyspira hyodysenteriae broth cultures, studies of tlyA, tlyB, and tlyC to date have focused on their activity when introduced into Escherichia coli via plasmid. These results were cast into doubt several years later upon the discovery that many lab strains of Escherichia coli exhibit a hemolytic phenotype when foreign genes were introduced. To examine the effects of these putative cytolysins the tlyA and tlyC genes were amplified from “Brachyspira hampsonii” genomic DNA and ligated into expression vectors. The resulting protein products were overexpressed in Escherichia coli and purified by Ni2+ and amylose affinity chromatography. Cytolytic activity of purified recombinant tlyA and tlyC on a sheep erythrocyte suspension was examined, and no cytolysis greater than a phosphate buffered saline control was observed from tlyA at concentrations ranging from 0 to 88 μg/mL or from tlyC at concentrations ranging from 0–34 μg/mL. At the time of writing, attempts are being made to express and purify the α‐toxin from Staphylococcus aureus utilizing the same methodology in order to rule out inactivation of tlyA and tlyC by the purification process. While it is also possible that higher concentrations of tlyA and tlyC are required for hemolysis or that full activity of these proteins require unidentified cofactors or post‐translational modifications not possible in our current Escherichia coli based expression system, the possibility that tlyA and tlyC have been misannotated as cytolysins cannot be ruled out.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) 371364 /2010 to MEL & Alberta Livestock and Meat Association project 2013R054R to JCH, MEL & JEH