The Molecular Mechanisms Of Two-component Systems HssSR Mediating Oxidative Stress Resistance And Pathogenicity Of Listeria Monocytogenes

Listeria monocytogenes(LM)is an important food-borne zoonotic pathogen,which exists widely in nature and can resist the influence of various external factors.Such as acid,alkali,osmotic pressure and oxidizing environment.Food contaminated with listeria monocytogenes can cause listeriosis in animals or humans,which can lead to septicemia,meningitis,gastroenteritis and abortion in pregnant women.The elderly,young children,pregnant women and people with low immunity are susceptible to listeriosis.Although the incidence rate is not high,the mortality rate can reach 30%.After entering the body,Listeria monocytogenes can proliferate in host cells,escape phagosomes through the action of various virulence factors,and then infect neighboring cells.In the body,Listeria monocytogenes will encounter the threat of oxidative environment in the blood and phagosomes,so the ability to resist oxidative stress is necessary for the infection and intracellular proliferation of Listeria monocytogenes.Two-component systems HssSR(Hem E-Sensor System)mainly regulates the Hrt AB(Heme Regulated Transporter)to discharge excess Heme to avoid damage caused by oxidation environment.However,the specific regulatory mechanism against oxidative stress remains unclear.Therefore,we will study the molecular mechanism of the antioxidative stress and pathogenicity of the twocomponent system HssSR in Listeria monocytogenes.1.hssS /hssR gene deletion strains were constructed to compare the effects of hssS/hssR gene deletion on the anti-stress ability and virulence of Listeria monocytogenes.2.Analyze the response of HssSR under oxidation conditions.3.Target genes regulated by HssSR were screened to explore the molecular mechanism of HssSR-mediated antioxidant stress in Listeria monocytogenes.1.hssS /hssR gene deletion strains were constructed to compare the effects of hssS/hssR gene deletion on the anti-stress ability and virulence of Listeria monocytogenesThe hssS/hssR gene deletion strain was constructed by homologous recombination,and then the effect of hssS/hssR gene deletion strain on the anti-oxidative stress ability of Listeria monocytogenes was compared by growth curve measurement and stress survival experiment.The results showed that after the deletion of hssS/hssR gene,the growth ability in oxidative environment was significantly decreased,and the ability to resist oxidative stress was reduced,indicating that hssS/hssR may play an antioxidant role in the process of anti-oxidative stress in Listeria monocytogenes;Cell invasion assay showed that hssS/hssR gene deletion significantly enhanced the invasion ability of Listeria monocytogenes on intestinal epithelial cells Coca-2;also significantly enhanced the ability of the bacteria to resist phagocytosis of macrophage RAW264.7 and intracellular proliferation ability.Animal infection experiments showed that hssS/hssR gene deletion significantly increased the bacterial load of L.monocytogenes in mouse and chicken embryo livers,indicating that hssS/hssR gene deletion could enhance the virulence of L.monocytogenes.2.Analyze the response of HssSR under oxidation conditions.The hssS/hssR gene recombinant expression vector was constructed using the prokaryotic expression vector p ET-28a;the expression was successfully induced and purified recombinant proteins HssS and HssR were obtained;the purified recombinant protein was used to immunize mice to prepare polyclonal antibodies.The complementation strain CΔhssS/hssR was constructed using the complementation plasmid p ERL3;Western blot results showed that the hssS/hssR gene was successfully complemented;Stress survival assays showed that complementation of hssS/hssR genes restored the oxidative stress survival ability of L.monocytogenes.Western blot showed that the expression level of HssR in L.monocytogenes was significantly downregulated under oxidative conditions.This indicates that hssS/hssR may be involved in the anti-oxidative stress of Listeria monocytogenes.3.Target genes regulated by HssSR were screened to explore the molecular mechanism of HssSR-mediated antioxidant stress in Listeria monocytogenes.In order to explore the molecular mechanism of HssSR mediating the anti-oxidative stress and pathogenicity of Listeria monocytogenes,the regulatory effects of HssSR on genes involved in oxidative stress such as kat,sod and fri were screened.First,the GFP reporter plasmids of the kat,sod and fri promoters were constructed using the expression plasmids respectively;the reporter plasmids were transferred into the parental strain 10403 S,the deletion strain ΔhssS/hssR and the complementing strain CΔhssS/hssR,and the report was detected by a microplate reader.Gene expression,the results show that under oxidative stress,hssS/hssR deletion can up-regulate the expression of sod and fri reporter genes;further use gel migration arrest assay(EMSA)to verify the binding of HssR to kat,sod and fri promoters The results showed that HssR could not effectively bind to the sod and fri promoters,suggesting that HssR has a weak regulatory effect on sod and fri.