Ovomucin Hydrolysates Reduce Bacterial Adhesion and Inflammation in Enterotoxigenic Escherichia coli (ETEC) K88-Challenged Intestinal Epithelial Cells

Ovomucin Hydrolysates Reduce Bacterial Adhesion and Inflammation in Enterotoxigenic Escherichia coli (ETEC) K88-Challenged Intestinal Epithelial Cells. X. Bao, M. G. Gänzle, and J. Wu. Journal of Agricultural and Food Chemistry 2024 72 (13), 7219-7229

About

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of diarrhea in children and travelers. It is also the most common cause of diarrhea in neonatal and postweaning pigs, which leads to economic losses in the swine industry. In the swine industry, over 80% of antimicrobial substances are administered between birth and 10 weeks of age for disease management, including severe diarrhea in neonatal and weaned piglets that is mainly caused by ETEC. With the increasing concern about antimicrobial resistance, developing effective antimicrobial alternatives are crucial. Given that bacterial adhesion to the host cell surface is the initial step of pathogenesis, antiadhesive therapy provides a promising strategy to interfere with the interactions of microbes and toxins with host cells, inhibiting pathogenic colonization and infection. Thus, this study aimed to investigate the effects of these two ovomucin hydrolysates on ETEC K88-induced epithelial cell integrity damage and inflammation. Ovomucinhydrolysates treated with sialidase were used to determine mechanisms in addition to inhibiting pathogenic adhesion, in porcine intestinal epithelial (IPEC-J2) and human Caco-2 cells.

Approach

The porcine K88acETEC strain ECL13795 (O149; virotype STb:LT:EAST1:F4) was kindly provided by the Escherichia coli Laboratory at the University of Montreal. Ovomucin (OVM), OP, and OPP were prepared from egg whites according to the established protocols. The adhesion test was done using procedures adapted from the methods described by González-Ortiz et al. To test the effects of ovomucin hydrolysate samples against LPS stress, IPEC-J2 cells were preincubated with OP or OPP (1%) for 24 h before being exposed to LPS.

Analysis of Results

Ovomucin Hydrolysates inhibited ETEC K88 adhesion to intestinal epithelial cells. Intact ovomucin (OVM) coated onto the bottom of 96-well plate exhibited the highest adhesion to ETEC K88, and removing sialic acids significantly decreased its interaction with the bacterial cells. Removal of the terminal sialic acid residues significantly reduced ETEC K88 interaction with OVM and OP, but did not significantly interfere with bacterial adhesion to OPP. At higher concentrations, OP (2.5% and 5.0%) and OPP (5%) significantly inhibited bacterial adhesion to IPEC-J2 cell surfaces. Ovomucin Hydrolysates restored tight junction integrity in Caco-2 Cells. Further research needs to determine whether ovomucin hydrolysates functioned through mediating the migration and diffusion of claudin-3 to maintain functional tight junction permeability. Taken together, these data suggested that ovomucin hydrolysates could preserve epithelial cell integrity in Caco-2 cells, supported by ovomucin hydrolysates’ ability to maintain the abundance and distribution of tight junction proteins against ETEC K88 challenge.

Application

Ovomucin from chicken egg white and its hydrolysates have been demonstrated to interact with diverse microorganisms, which indicates their potential to interfere with pathogenic adhesion, colonization, and resulting infectious diseases. In conclusion, this study demonstrated that ovomucin hydrolysates OP and OPP inhibited bacterial adhesion. OP partially restored epithelial cell integrity against ETEC K88 challenge in both cells, but the abundance and networks of tight junction proteins were maintained only in Caco-2 cells, but not in IPEC-J2 cells. Both hydrolysates could effectively alleviate ETEC-induced inflammatory response and oxidative stress, whereas the interactions between ovomucin hydrolysates and the virulence factors especially toxins are to be established. Overall, our results indicate possible applications of ovomucin hydrolysates in preventing ETEC K88 infection, thus alleviating antimicrobial resistance issues arising from agricultural sectors. Further in vivostudies using ETEC K88-challenged piglet models and on-farm studies are needed to validate their efficacies in practice.

Abstract

Enterotoxigenic Escherichia coli (ETEC) K88 is the most common cause of diarrhea in neonatal and postweaning pigs. After adhering to small intestinal epithelial cells via glycoprotein receptor recognition, the pathogen can produce enterotoxins, impair intestinal integrity, trigger watery diarrhea, and induce inflammation via nuclear factor κB (NF-κB) and mitogen-activated protein kinase phosphatase (MAPK) pathways. Inhibiting ETEC K88 adhesion to cell surfaces by interfering with the receptor-fimbriae recognition provides a promising strategy to prevent the initiation and progression of infection. Ovomucin is a highly glycosylated protein in chicken egg white with diverse bioactivities. Ovomucin hydrolysates prepared by the enzymes Protex 26L (OP) and pepsin/pancreatin (OPP) were previously revealed to prevent adhesion of ETEC K88 to IPEC-J2 cells. Herein, we investigated the protective effects of ovomucin hydrolysates on ETEC K88-induced barrier integrity damage and inflammation in IPEC-J2 and Caco-2 cells. Both hydrolysates inhibited ETEC K88 adhesion to cells and protected epithelial cell integrity by restoring transepithelial electronic resistance (TEER) values. Removing sialic acids in the hydrolysates reduced their antiadhesive capacities. Ovomucin hydrolysates suppressed ETEC-induced activation of NF-κB and MAPK signaling pathways in both cell lines. The ability of ETEC K88 in activating calcium/calmodulin-dependent protein kinase 2 (CaMK II), elevating intracellular Ca2+ concentration, and inducing oxidative stress was attenuated by both hydrolysates. In conclusion, this study demonstrated the potential of ovomucin hydrolysates to prevent ETEC K88 adhesion and alleviate inflammation and oxidative stress in intestinal epithelial cells.