Identification of Clostridium innocuum hypothetical protein that is cross-reactive with C. difficile anti-toxin antibodies

Objectives: Previously considered solely an opportunistic pathogen, Clostridium innocuum (CI) was recently reported in Taiwan to be an emerging cause of antibiotic-associated diarrhea and clinically indistinguishable from Clostridioides difficile (CD) infection. We previously identified CI culture supernatant being cross-reactive with commercial CD toxin enzyme immunoassays. We aimed to identify and characterize the cross-reacting protein and determine whether it functioned as a human toxin. Methods: We performed western blots using CI culture supernatants and CD anti-toxin antibodies and identified interacting bands. We identified protein(s) using tandem mass spectrometry and evaluated them by cytotoxicity assays. Results: CI, but not CD, was isolated from stool of 12 children and adults with diarrhea. Culture supernatant from 6/12 CI isolates, and an ATCC reference strain, tested positive for CD toxins (total 7/13 isolates) by commercial EIA. Using two of these isolates, we identified two ∼40 kDa hypothetical proteins, CI_01447 and CI_01448, and confirmed cross-reactivity with CD anti-toxin antibodies by enzyme immunoassay and Western blot. Whole-genome sequencing confirmed all 13 isolates contained both genes, which were highly conserved. We observed no cytopathic or cytotoxic effects to HeLa cells when treated with these proteins. We identified amino acid sequence similarity to the NlpC/P60 family of proteins. Conclusions: Our findings do not suggest CI proteins CI_01448 and CI_01447, which cross-react with antibodies against CD toxins A and B, are toxic to HeLa cells. Further studies are needed to determine the function of these cross-reacting proteins and the potential virulence factors that could be responsible for CI diarrheal disease.