Mechanism Investigation Of Xenophagy In Temporal Regulation The Propagation Of Intracellular S.Typhimurium

Salmonella enterica serovar Typhimurium(S.Typhimurium)is a pathogenic Gram-negative bacterium to survive in host cells,which is well known to cause gastrointestinal infections and even death in human through food contamination.S.Typhimurium can successfully infect and enter into host cytosol,establishing a replicative niche termed Salmonella-containing vacuole(SCV)based on the formation of ruffles in the host cells membrane,which permits S.Typhimurium survive and reproduce within SCV.Meanwhile,part of S.Typhimurium escape from SCV leading to the robust replication within cytosol,which is called as Hyper-replication bacteria.As one of the host cells innate immunity,xenophagy was initially found to eliminate intracellular Salmonella.With the deepening of research,xenophagy help the reproduction of intracellular Salmonella and maintain the structural integrity of SCV.But,so far,the research on xenophagy has been based on the mixed count of two types of intracellular bacteria,which makes it difficult to elaborate and accurately describe the function of xenophagy during Salmonella infection.In this study,enzyme-linked immunosorbent assay(ELISA)combined with flat colony counting method to quantify the binding amount and binding rate between Salmonella and anti-Salmonella antibody.According to the specific binding of antigen and antibody,it is clear that the anti-salmonella antibody with a dilution ratio of 1:500 can bind about 1700Salmonella,and the binding rate reaches about 70%.Then the biotin-streptavidin-amplified enzyme-linked immunosorbent assay(BA-ELISA)combined with flat colony counting method to quantify the corresponding relationship between Salmonella and the OD450nm value,the result shows that the linear positive correlation between lg^{(number of Salmonella)}and lg^OD450nm,which can quickly and accurately calculate the total number of Salmonella bound by the antibody within ELISA plate,and then improve the counting method for intracellular Salmonella.In order to isolate and quantify the number of cytosolic Salmonella and vacuolarSalmonella,and clarify the proliferation trend of cytosolic Salmonella and vacuolar Salmonella,Using epithelial cells Henle-407 and embryonic fibroblasts MEF as cell models,the homogenizer crushed the cells to obtain intracellular Salmonella,and separated cytosolic Salmonella and vacuolar Salmonella by BA-ELISA during various periods of Salmonella infection.The proportion of vacuolar Salmonella in Henle-407 cells decreased from?70%(2h)to?23%(16h),and the cytosolic Salmonella increased from?22%(2h)to?65%(16h);However,cytosolic Salmonella of MEF cells didn't multiply indefinitely during Salmonella infection,accounting for?20%of the total intracellular Salmonella,but SCV has always been the main lifestyle of Salmonella in MEF cells,which further proved that the method is feasible to separated cytosolic Salmonella and vacuolar Salmonella.In order to explore the effect of xenophagy on the reproduction of intracellular Salmonella,the verification was carried out from two directions at the same time.First,construction of a stable Atg5 gene knockdown cell line in Henle-407 cells by lentivirus infection;Second,using the autophagy inhibitor 3-MA to inhibit xenophagy.untreated Henle-407 cells as control,then we statistical and analysis total intracellular Salmonella,cytosolic Salmonella and vacuolar Salmonella.In the absence of xenophagy,the reproduction trend of total intracellular Salmonella are significantly reduced,and the numbers and proportion of cytosolic Salmonella are remarkably reduced,vacuolar Salmonella is also showing a downward trend,but it has always been the main lifestyle for Salmonella to survive in host cells,which indicated that xenophagy are likely to help cytosolic Salmonella reproduction within host cytoplasm,which further improve our understanding of the pathogenic mechanism of Salmonella,find new targets for the drug treatment of Salmonella infection,and provide more comprehensive insights for the prevention and treatment of salmonellosis.