Mechanisms Of Autophagy Induced By Group A Streptococcus

Objective: Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. During this process, the double-membrane vesicle detached from the no-ribosome attachment region of rough surfaced endoplasmic reticulum wrapped some cytoplasmic, protein and organelles that need to be degraded. Then it forms an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled. Studies have shown that a variety of factors such as oxidative stress, hunger, heat, intracellular damage, bacterial infection will induce autophagy. Autophagy is very important for normal homeostasis of the body. In starvation response process, the lack of amino in different organs such as liver or the culturing cells could induce autophagy. Then macromolecules are decomposed and generated into intermediate metabolite that is necessary in the catabolic and anabolic processes. In normal cell activities such as developmental by metamorphosis, aging and differentiation of animal cells, autophagy is responsible for the degradation of proteins to rebuild cells.Group A streptococcus is a kind of Gram-positive bacterium, about 90% of human streptococcal infections are caused by group A streptococcus. Group A streptococcus infection can cause purulent diseases like local skin infection and subcutaneous tissue infection, toxic diseases like scarlet fever and allergic diseases like rheumatic fever and acute glomerulonephritis. Group A streptococcus has strong invasive ability mainly because it can produce a variety of extracellular enzymes and toxins, such as lipoteichoic acid(LTA), M protein, pyrogenic exotoxin and hemolysins. These extracellular enzymes and toxins have their respective functions. LTA can enhance the adhesion of bacteria to host cells. M protein is a major virulence factor of group A streptococcus. Pyrogenic exotoxin can change the permeability of the blood brain barrier. Hemolysin is divided into streptolysin O and streptolysin S and plays an important role in the process of hypersensitivity caused by streptococcus. Whether autophagy occurs when the body is invaded by group A streptococcus and if it occurs, the mechanism of group A streptococcus-induced autophagy is not yet known. In this study, western blot and immunofluorescence techniques are used to monitor the trends of LC3 and P62 following stimulation of Hep2 cell by GAS, GAS and GAS deficient strain. Cellular localization of LC3 proteins is detected by immunofluorescence techniques to study whether autophagy is induced by GAS, GAS or GAS deficient strain. To verify our conclusions, autophagy-related protein is detected by western blot after the cells is stimulated by purified M protein and Fba A protein. Subsequently, protein fragments of Fba A are used to stimulate cells to further evaluate the Fba A fragment related to the occurrence of group A streptococcus induced autophagy.Methods:1 GAS strain is used to stimulate the Hep2 cells, A549 as control group. After the invasion, intracellular bacteria is gathered according to the principle of osmotic pressure. Then the intracellular bacteria is swabbing inoculated on blood agar medium after dilution of different multiples to detect the invasion efficiency of cells.2 Hep2 cells is separately stimulated by GAS,GAS and GAS deficient strain, then the changes of autophagy-related protein LC3, P62 at different time points are detected by western blot.3 Hep2 cells is separately stimulated by GAS,GAS and GAS deficient strain, then the distribution of LC3 in these cells is detected by immunofluorescence.4 Affinity chromatography is used to purify Fba A protein and M protein.5 Hep2 cells is separately stimulated by Fba A protein and M protein, then the changes of autophagy-related protein LC3, P62 of different concentrations at different time points are detected by western blot.6. Protein fragment of Fba A are used to stimulate Hep2 cells, then the changes of autophagy-related protein LC3, P62 of different concentrations at different time points are detected by western blot. The protein fragments used are 37-104, 104-277, 68-164 and 164-320.Results:1 Ihe average invasive rate of GAS is 33% to Hep2 cells, and 25.6% to A549.2 The changes of LC3 and P62 in the cells stimulated by different strains show that P62 and LC3-1is gradually reduced, while LC3-2 is gradually increased with the stimulation time when the Hep2 cells is stimulated byGAS and GAS deficient strain of MOI 1:100. P62 and LC3 have no obvious characteristic changes when the Hep2 cells is stimulated by GAS deficient strain of MOI 1:100.3 The distribution of LC3 detected by immunofluorescence shows that: LC3 gradually changed from dispersive distribution to aggregated distribution when the Hep2 cells are stimulated by GAS and GAS deficient strain of MOI 1:100 for 6 hours. LC3 is still in dispersive distribution when stimulated by GAS deficient strain of MOI 1:100.4 The changes of LC3 and P62 in the cells stimulated by purified Fba A or M proteins show that P62 and LC3-1 are all gradually reduced, and LC3-2 is gradually increased when the Hep2 cells is stimulated by Fba A. P62 and LC3 have no obvious characteristic changes when the Hep2 cells is stimulated by M protein.5 The changes of LC3 and P62 in the cells stimulated by different protein fragments of Fba A show that P62 and LC3-1 is gradually reduced, while LC3-2 is gradually increased when Hep2 cells are stimulated by 68-164 protein fragment. No indicative trend of autophagy is appear by the stimulation of the other fragments.Conclusions:1 The invasion rate of GAS to Hep2 cells is higher than that to A549 cells. And the invasion rate to Hep2 cells achieves to study level.2 GAS and GAS deficient strain can induce the autophagy of Hep2 cells, while GAS deficient strain cannot.3 GAS-induced autophagy of Hep2 cells is related to Fba A protein of GAS.4 Major functional areas of Fba A induced autophagy is the 68-164 protein fragment.