TGEV Infection Enhances ETEC K88 Adhesion By Promoting Epithelial Mesenchymal Transition In Intestinal Epithelial Cells

Of all the diseases in pigs,diarrhea is the most common and probably the most important.There are many different pathogens and factors that can cause diarrhea,and diarrhea has caused large economic losses to the swine industry.Epidemiological investigations show that pig diarrhea is often caused by mixed infections.Although it is known that coinfection aggravates the severity of diarrhea,the pathogenesis of coinfection has not yet been fully elucidated.Transmissible gastroenteritis virus(TGEV)and enterotoxigenic Escherichia coli K88(ETEC K88)are important contributors to piglet diarrhea,and TGEV and ETEC K88 coinfection have been reported in China recently.In this study,TGEV and ETEC K88 coinfection experiments are conducted using intestinal porcine epithelial cells(IPEC-J2)as a model system.In order to evaluate pig diarrhea caused by TGEV and ETEC K88,the effects of coinfection in IPEC-J2 cells were examined.LC-MS/MS coupled to isobaric tags for relative and absolute quantification(iTRAQ)is used to profile expressed proteins in IPEC-J2 cells infected by TGEV alone,ETEC K88 alone,and by both agents together.Secondly,we explored how persistent TGEV infection stimulates the epithelial-mesenchymal transition(EMT),thereby generating cells that more easily adhere to ETEC K88.Finally,we evaluated the impact of TGEV infection on the small intestine of recovered piglets,and explored EMT of the small intestine,the histopathological changes and immune function.In a word,our results help us better understand the pathogenicity of TGEV and bacteria coinfection,and provide potential targets for the development of new strategies to prevent and treat piglet diarrhea.The research is divided into the following three parts in detail:1 Proteomic analysis of IPEC-J2 cells in response to coinfection by TGEV and ETEC K88In this study,TGEV and ETEC K88 coinfection experiments are conducted using IPEC-J2,and examined the effects of coinfection.In TGEV pre-infected IPEC-J2 cells,ETEC K88 adhesion was enhanced over uninfected cells.ETEC K88 was also found to inhibit the proliferation of TGEV.Additionally,cytokine levels(IL-1?,IL-6,IL-8,and TNF-?)in coinfected cells were lower than cells infected by TGEV alone,and higher than cells infected by ETEC K88 alone.LCMS/MS coupled to isobaric tags for relative and absolute quantification(iTRAQ)is used to profile expressed proteins in IPEC-J2 cells infected by TGEV alone,ETEC K88 alone,and by both agents together.We identified 77,89,and 136 differentially expressed proteins in TGEV infected,ETEC K88 infected,and coinfected cells,respectively.Based on these data,we suspected that integrin a5 might enable TGEV to promote ETEC K88 adhesion.TGEV and ETEC K88 coinfected cells expressed higher levels of integrin ?5,and the inhibitor and agonist of integrin a5 demonstrated that integrin a5 is involved in ETEC K88 adhesion.The results suggest that TGEV infection activates the expression of integrin a5 in IPEC-J2 cells,which may increase ETEC K88 loading in the small intestine and make piglet diarrhea more serious.2 Persistent TGEV infection enhances ETEC K88 adhesion by promoting EMT in IPEC-J2 cellsTGEV persists in the gut for a long time after pigs recover,and recovered pigs can excrete and spread virus continuously.TGEV infected IPEC-J2 cells enhances adhesion of secondary pathogen ETEC K88,which aggravates the severity of diarrhea,but the mechanisms remain unknown.Here,we hypothesized that persistent TGEV infection stimulates the EMT,thereby generating cells that more easily adhere to ETEC K88.First,we detected whether TGEV could develop a persistent infection in IPEC-J2 cells.The viral protein and nucleic acid could be detected in IPEC-J2 cells,and supernatants from the persistent infection could produce virus plaques.Then,we found TGEV cause EMT in IPEC-J2 cells,consistent with multiple changes in key cell characteristics.Infected cells display fibroblast-like shapes,exhibit increases in mesenchymal markers with a corresponding loss of epithelial markers,demonstrate increases in migratory and invasive behaviors,and cellular uptake is also modified in cells that have undergone EMT.Persistent TGEV infection have enhanced expression of IL-1?,IL-6,IL-8,TGF-?,and TNF-? mRNAs.Additional experiments showed that activation of the PI3K/AKT and ERK signaling pathways via TGF-? are critical for the TGEV-mediated EMT process.TGEV-infected cells have higher levels of integrin ?5 and fibronectin and exhibit enhanced adhesion of ETEC K88.Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin a5 and fibronectin.Overall,these results suggest that TGEV infection stimulates IPEC-J2 cells to undergo EMT,enhances the expression of integrin a5 and fibronectin,favors ETEC K88 adhesion,and facilitates dual infection.3 Impact of TGEV infection on the small intestine of recovered pigletsIntestinal epithelial cells are the primary targets of TGEV infection.TGEV infection caused cell exfoliation and intestinal villous atrophy.No investigation has yet been made of the small intestine of piglets that survived infection by TGEV.In this study,we evaluated the impact of TGEV infection on the small intestine of recovered piglets.Western blot exhibit increases in vimentin,integrin a5 and fibronectin,with a corresponding loss of E-cadherin,which indicated TGEV infection induce EMT in vivo.Histological analyses showed that TGEV infection lead to villi atrophy,and reduced villous height and crypt depth.The number of SIgA positive cells,CD3+T cells,and dendritic cells(DCs)in jejunum decreased after TGEV infection in vivo.In contrast,microfold cells(M cells)and cell proliferation increased in infected pigs.TGEV infection also significantly enhanced the mRNA expression levels of cytokine IL-1?,1L-6,TNF-?,IL-10,and TGF-p.In conclusion,TGEV infection induces EMT in vivo,damages the small intestine,and impairs immune functions,which may facilitate secondary infection by other pathogens.