Murine intrarectal instillation of purified recombinant C. difficile toxins enables mechanistic studies of pathogenesis

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota, et al . first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin (H&E) for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo . Furthermore, TcdB mutants lacking either endosomal pore-formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared with slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis. * CDI : Clostridioides difficile infection DxD : D286/288N mutant lacking glucosyltransferase activity H&E : hematoxylin and eosin HBSS : Hank’s Balanced Salt Solution IHC : immunohistochemistry L1106K : mutant lacking pore-forming function Ly6G : lymphocyte antigen 6G (murine myeloid cell marker) MPO : myeloperoxidase Na+/K+-ATP : sodium-potassium ATPase NKCC : sodium-potassium-chloride channel PBS : phosphate buffered saline SEM : standard error of the mean TcdA : C. difficile toxin A TcdB : C. difficile toxin B WT : wild-type VPI : VPI 10463 reference strain (ATCC 43255)