Clostridium perfringens is a major cause of human and livestock diseases that originate in the intestines and involve enteritis and/or enterotoxemia, where C. perfringens grows in the intestines and produces toxins that are absorbed into the circulation and then damage organs such as the brain. Intestinal adherence and growth play critical roles in C. perfringens intestinal infections, particularly when these diseases can be chronic, e.g., antibiotic-associated diarrhea. Our published studies established that the C. perfringens type A and C strains causing chronic human intestinal infections produce NanI sialidase. These NanI+ intestinal disease strains are also more adherent to Caco-2 human enterocyte-like cells than are the NanI- C. perfringens strains causing acute intestinal disease. Using nanI null mutants and complementing strains of two NanI+ intestinal disease strains, we showed that NanI production is critical for those NanI+ strains to adhere to Caco-2 human enterocyte-like cells. Using the same strains, additional data was obtained suggesting that NanI+ intestinal dis- ease strains can grow by using NanI to obtain sialic acid from a host source, as may be important in the intest- ines. Last, we showed that the sialidase inhibitor siastatin B reduces NanI+ strain adherence to Caco-2 cells. Given our strong preliminary data, we hypothesize that, i) NanI is an important contributor to intestinal in- fections caused by NanI+ C. perfringens strains and ii) inhibitors affecting NanI represent a potentially novel therapeutic approach against these intestinal infections. The current proposal will now directly test these hypo- theses. Specifically, based upon our in vitro Caco-2 cell studies, Aim 1 will evaluate whether NanI is important for the in vivo (intestinal) attachment and virulence of NanI+ C. perfringens intestinal disease strains. This work will employ NanI+ C. perfringens intestinal disease strains, their isogenic nanI null mutants and complementing strains to address whether NanI enhances the intestinal adherence of NanI+ C. perfringens intestinal disease strains in a newly-developed mouse oral challenge model.
Aim 2 will explore if NanI can support the in vitro and in vivo growth of NanI+ C. perfringens intestinal disease strains. These studies will also use wild-type NanI+ intestinal disease strains, their isogenic nanI null mutants and complementing strains to test if NanI pro- motes, i) the in vitro growth of NanI+ intestinal disease strains using sialic acid removed from Caco-2 cells or mucus protein Muc-2, as intestinally-relevant sialic acid sources, or ii) the in vivo growth/survival of these strains in a mouse intestinal loop model. Last, based upon our in vitro Caco-2 studies, Aim 3 will test if a NanI sialidase inhibitor can reduce the adhesion, growth/survival and virulence of NanI+ C. perfringens intestinal dis- ease strains in the mouse models used in Aims 1 and 2. The proposed studies, with their inclusion of in vivo work, represent the logical next step to build upon strong previous work and will explore the innovative poten- tial use of sialidase inhibitors as a novel therapeutic approach against several important intestinal infections.
Clostridium perfringens is a major cause of human and livestock infections that originate in the intestines and then manifest as enteritis and/or enterotoxemia (where toxins produced in the intestines are absorbed into the circulation and affect extraintestinal organs such as the brain). This project will use cell culture and mouse models to test our hypothesis that NanI sialidase contributes to C. perfringens attachment and growth, both in vitro and in the intestines, and to virulence. We will also explore if a sialidase inhibitor can reduce C. perfringens adherence, growth and/or virulence as a potentially novel therapeutic approach against C. perfringens intestinal infections.
