Construction Of The Salmonella Typhimurium ΔsodA Strain And Its Effect On Macrophage Autophagy

As a pathogenic bacteria,Salmonella typhimurium encountered a high level reactive oxygen species(ROS)environment that produced by immune protection of host after it entry into the host.The excess ROS have a produce toxic effects on the body’s DNA,proteins,and lipids.Superoxide dismutase(SOD)as a unique antioxidant enzymes than can eliminate the superoxide anion(·O2-)and constitute a primary source of cellular defenses against ROS damages.Previous studies have shown that SOD involved in the growth of bacteria,biofilm formation,virulence.However,little is known about function and role of SOD genes of Salmonella typhimurium.Thus,this study structure the mutant strain SM52ΔsodA and complementary strain SM52CΔsodA.We study the biological characteristics of mutant strain SM52ΔsodA and complementary strain SM52CΔsod A,and its effects on cell autophagy.We also express the SodA protein and explore the enzymatic property.This study establish a good foundation for further study of the function of sodA genes and its role in autophagy.The main contents include the following: 1.Construction and characteration of SM52Δsod A mutant strainThe sodA gene mutant strain SM52ΔsodA and complementary strain SM52CΔsodA was constructed successfully and determined its role in the biological characterization of Salmonella typhimurium.The biological characterization results showed the reduce of SM52Δsod A on the growth rate,enzymatic activity,biofilm formation ability,antioxidant stress,resistant to environmental stress compared with SM52 and SM52CΔsod A.However,SM52 and SM52CΔsodA have the same results in the biological characterization.It showed that the complementary strains can restore the defects of mutant strain.2.Expression of SodA of Salmonella typhimurium and enzymatic propertyThe sod A was cloned and inserted into the p ET-28 a vector to construct the recombinant expression plasmid pET-28a-sodA and induced to express by IPTG.The SodA protein was purified by nickel-affinity chromatography and study the enzymatic property.Optimum temperature and pH for SodA were 40℃ and 7.0,respectively.The enzymatic activity of Sod A was stimulated by Mn2+,Zn2+ and Cu2+and was inhibited by SDS,chloroform-ethanol,and Fe3+,while it was not affected by H2O2、Mg2+、Ca2+、Ba2+、Na+、K+.The kinetic parameters Vmax and Km in pyrogallol were 2.03U/min and 0.801mmol/L,respectively.3.The influence of sod A on macrophage cells autophagyLaser confocal microscope analysis showed that the expression of LC3-Ⅱ of SM52ΔsodA infected groups was high than SM52 and SM52CΔsod A infected groups at first hour of infection,and it was no significant difference at 2h and 3h of infection.WB detection showed that the expression of LC3-Ⅱ and Beclin-1 of SM52ΔsodA infected groups was high than SM52 and SM52CΔsodA infected groups at first hour of infection,while the expression of p62 was lower than SM52 and SM52CΔsodA infected groups at first hour of infection.The number of active bacteria in SM52ΔsodA infected groups was significant lower than SM52 and SM52CΔsod A infected groups at 2h and 3h of infection.All results showed that sodA could enhance supression of macrophage cells autophagy in the early stage of infection,and increase Salmonella typhimurium survival in cells.