Study On The Mechanism Of STa Induced Apoptosis In Intestinal Epithelial Cells By Mitochondrion

Enterotoxigenic Escherichia coli（ETEC）can not only cause diarrhea in travelers and children in developing countries,but also cause diarrhea and even death in economic livestock such as cattle,sheep,and pigs within a few days of birth and after weaning,resulting in serious economic losses to the global livestock industry.After unclean food and water with ETEC entry in the host through oral,ETEC adheres to intestinal epithelial cells through the adhesin,mainly to small intestinal epithelial cells,then secreting enterotoxins into the intestinal lumen.Enterotoxins bind to receptors on the surface of intestinal epithelial cells or enter the cells,which changes the level of proteins and activates signal transductions in the host cells,such as increasing the cell membrane permeability of the host cells and disrupting the intracellular ion homeostasis,finally leading to secretory diarrhea in the host.The heat-stable enterotoxin（ST）produced by ETEC is one of the main virulence factors of secretory diarrhea in humans and livestock,the mechanism of which induced diarrhea was detailedly describe in the current studies,but the mechanism of how ST leading to intestinal epithelial cell damage and even apoptosis has not yet been clearly explained.STa,a subtype of ST,has a small molecular weight and is a hapten with little or no immunogenicity.Therefore,host infection with ETEC carrying STa enterotoxin cannot stimulate the body’s immune response to produce corresponding antibodies.Researchers build vaccines of live attenuated bacteria or recombinant proteins to prevent and give antibiotics to treat ETEC infection.The recent vaccines for STa are mostly tandem with other immunogenic macromolecular proteins or multiple STa attenuating mutations and have complicated operation.Antibiotic treatment can lead to the emergence of resistant strains.In consequence,this study hopes to discover a new way to prevent and treat ETEC（STa⁺）infection by exploring the specific molecular mechanism of STa-induced intestinal epithelial cell injury,and also to provide new ideas for the new type of drugs.Relevant experimental studies were carried out in vivo（in suckling mice）and in vitro（IPEC-J2 cell line）to explore the injury mechanism of STa on intestinal epithelial cells in this study.First,a two-plasmid CRISPR/Cas9 gene editing system was used to construct ETEC O142ΔestA which was deleted the whole CDS region of STa encoding gene estA from ETEC O142（STa⁺,F5⁺,F41⁺）.Secondly,IPEC-J2 cells were infected with the original strain and the gene deletion strain respectively to explore the optimal time of infection and multiplicity of infection（MOI）in vitro.The effect of STa on host cell damage during ETEC infection was analyzed by comparing the difference of cell damage caused by the two strains.Then,the mechanism of cell damage caused by STa was explored according to RNA sequencing and further analyzed.Finally,the results of the in vitro experimental studies were validated by in vivo experiments.The experimental results are as follows:（1）The gene deletion strain ETEC O142ΔestA was successfully constructed in this study.The results of the growth curve showed that the gene deletion strain and the original strain had the same growth performance.The results of genetic stability showed that the gene deletion strain had no reverse mutation after 50 consecutive passages.The results of blocking ELISA showed that the original strain could secrete STa enterotoxin,but the gene deletion strain could not secrete STa enterotoxin,which proved that the gene encoding STa was deleted.（2）The results of lactate dehydrogenase assay showed that ETEC O142 and ETEC O142ΔestA caused different mortality of IPEC-J2 cells under different MOI and infection time.The optimal in vitro infection conditions were MOI 1:100 and infection time 2 h.The results of flow cytometry and transmission electron microscopy indicated that STa had the ability to induce apoptosis and changes in mitochondrial morphology.（3）Transcriptome sequencing results showed that 58 significantly differentially expressed genes were detected between the ETEC O142 infection group and the ETEC O142ΔestA infection group,including 17 up-regulated genes and 41 down-regulated genes.The results of GO and KEGG enrichment analysis showed that apoptosis induced by STa was mainly related to the down-regulation of oxidative phosphorylation pathway.The differentially expressed genes in the oxidative phosphorylation pathway showed that STa induced up-regulation of NDUFS8 gene and down-regulation of ND2,COXII,CYTB and ATP6 genes in IPEC-J2 cells.（4）Intracellular antioxidant markers,reactive oxygen species（ROS）,8-OHd G,mitochondrial membrane potential,ATP content,and mitochondrial respiratory chain complex I activity detection results showed that,STa inhibited cellular oxidative phosphorylation function by disrupting the antioxidant system and increasing intracellular ROS.Increasing ROS levels led to DNA fragmentation and cellular oxidative stress,which in turn causes the destruction of mitochondrial function,including reducing mitochondrial membrane potential,intracellular ATP content and mitochondrial respiratory chain complex I activity.STa induced apoptosis in the mitochondrial pathway of IPEC-J2 cells.（5）The experimental results of detecting cellular DNA damage and mitochondrial functions after NDUFS8 overexpression and silencing showed that,NDUFS8 was the core subunit of complex I of the mitochondrial respiratory chain and plays an important role in stabilizing mitochondrial functions.Overexpression of NDUFS8 did not cause DNA damage and damage mitochondrial function,but it could not totally protect cells from STa-induced cell damage.NDUFS8 silencing caused DNA damage,impaired mitochondrial function,and further increased cell damage by STa.（6）STa up-regulated NDUFS8 expression by activating PKA/CREB1 in IPEC-J2 cells.STa increased the phosphorylation of CREB1 Ser¹³³ residues by increasing PKA.The transcription of NDUFS8 was regulated by phosphorylated CREB1 Ser¹³³ binding to the proximal end of the NDUFS8 promoter.The PKA/CREB1/NDUFS8 anti-apoptotic system to reduce cell death,but this system did not completely protect cells from STa-induced apoptosis.（7）After oral perfusion of ETEC O142 and ETEC O142ΔestA with different CFUs of 3-day-old suckling mice,the best model of the in vivo infection was screened by comparing the differences of suckling mice in the mental state,body condition and histopathology.The experimental results showed that STa could cause lethargy,cyanosis,watery diarrhea,and death in severe cases.The pathogenicity of STa to suckling mice increased with the increase of ETEC O142 bacterial count.The optimal in vivo infection conditions were bacterial concentration of 3×10⁹ CFU/100μL,infection time of 9 h,and experimental animals of 3-day-old suckling mice.STa could also induce apoptosis in the mitochondrial pathway in suckling mice,which was detrimental to the host and even led to the death.In conclusion,STa infection of the host could cause cellular oxidative stress,thereby activating mitochondria-mediated apoptosis,and at the same time activating PKA/CREB1/NDUFS8 anti-apoptotic system.The damage of STa to small intestinal epithelial cells in vivo and in vitro was enhanced with the increase of the number of infected ETEC bacteria.The discovery of the pathogenic mechanism added new molecular biological information to the in-depth understanding of the damage mechanism of enterotoxins to host cells,and also provided ideas for the development of new drugs.