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ABSTRACT
Background & Aim
Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects of C. difficile toxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection.
Methods
We studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation.
Results
Intestinal permeability was increased through the size-selective leak pathway in vivo during acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However, Sglt1, Nhe3, and Dra were drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI.
Conclusions
CDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.
GRAPHICAL ABSTRACT
Abbreviations
| CDI | = | Clostridioides difficile infection |
| GI | = | gastrointestinal |
| NHE3 | = | Na+/H+ Exchanger 3 |
| DRA | = | Downregulated in Adenoma |
| BHIS | = | brain-heart infusion-supplemented medium |
| TA | = | taurocholic acid |
| TCCFA | = | taurocholic acid, D-cycloserine, cefoxitin and fructose agar |
| FITC | = | fluorescein isothiocyanate |
| FD4 | = | 4 kDa FITC dextran |
| RD70 | = | 70 kDa rhodamine B dextran |
| KRB | = | Krebs-Ringer Buffer |
| Isc | = | short-circuit current |
| Rt | = | transmucosal resistance |
| Gt | = | tissue conductance |
| SGLT1 | = | sodium-dependent glucose cotransporter 1 |
| ENaC | = | epithelial sodium channel |
| CFTR | = | cystic fibrosis transmembrane conductance regulator |
| PFA | = | paraformaldehyde |
| NBF | = | neutral buffered formalin |
| FFPE | = | formalin-fixed paraffin embedded |
| OCT | = | optimal cutting temperature embedding medium |
| ERM | = | Ezrin/Radixin/Moesin |
| NHERF | = | Na+/H+ exchanger regulatory factor |
| BI/NAP1/PCR-RT | = | restriction endonuclease analysis type B1, North American pulse-field gel electrophoresis type 1, polymerase chain reaction ribotype |
| qRT-PCR | = | quantitative real time-polymerase chain reaction |
Acknowledgments
Thank you to the members of the Lacy lab for stimulating discussions and feedback on this manuscript. Thank you to Miguel Beristain, Greg Gomez, and Lee Brackman for technical assistance with qRT-PCR. Thank you to Kari Seedle and the Vanderbilt Cell Imaging Shared Resource Core for support on imaging and analyses; Vanderbilt Technologies for Advanced Genomics for support with RNA-sequencing; TPSR for histology support; and to Colin Kruse for advice on RNA-sequencing data analyses. The graphical abstract and cartoons in figures were made using BioRender.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Author contributions
Conceptualization, F.C.P.G., I.K., and D.B.L.; Methodology, F.C.P.G., I.K., A.S., J.A.S., and H.M.S.A.; Investigation, F.C.P.G., I.K., A.S., L.M.W., J.A.S., and M.K.W.; Writing – Original Draft, F.C.P.G.; Writing – Reviewing & Editing: I.K., A.S., H.M.S.A, P.K.D., J.R.G., and D.B.L.; Funding Acquisition, F.C.P.G., I.K., H.M.S.A, and D.B.L.; Resources, I.K., L.M.W., H.M.S.A., P.K.D, J.R.G., and D.B.L.; Supervision, I.K. and D.B.L.
BioRxiv preprint
Data availability statement
Large datasets (RNA-seq) are available online on Gene Expression Omnibus with identifier GSE216919. Reagents used in this study are either commercially available or available upon request.
Synopsis
Clostridioides difficile infection (CDI) causes increased intestinal fluid leakage and decreases the colon’s ability to absorb water and electrolytes. Using a mouse model of CDI, we found that intestinal permeability is increased through a size-selective pathway, and key absorptive ion transporters Sglt1, Nhe3, and Dra are downregulated. These factors likely cause diarrhea associated with CDI.
Transcript profiling
RNA-seq data were uploaded into GEO database (GSE216919).
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2225841