Mechanisms Of The Glutathione Peroxidase In Oxidative Stress Resistance In Listeria Monocytogenes

Listeria monocytogenes（L.monocytogenes,LM）is a gram-positive,facultative anaerobic and non-spore-forming bacteria.As a food-borne zoonotic pathogen,a prerequisite for L.monocytogenes to infect the host is the ability to resist various adverse stresses in the environment and host,such as acid stress,osmotic stress,oxidative stress,etc.Infection of the host with L.monocytogenes will cause listeriosis,which is mainly for the elderly,pregnant women,infants and other immunocompromised people,with a low incidence but high lethality characteristics.The main symptoms of listeriosis are miscarriage,sepsis,meningitis,and even death.The viability of pathogenic bacteria in the host depends on the ability of the bacteria to resist reactive substances such as reactive oxygen species,reactive nitrogen species,reactive chlorine species,and other redox stressors.The key to counteracting oxidative damage is to maintain a dynamic equilibrium of intracellular pro-oxidant agents and antioxidant stressors and repair and remove damaged DNA and proteins.In order to prevent damages caused by excessive oxidative stress,L.monocytogenes has evolved numerous non-enzymatic and enzymatic antioxidant defense systems to protect cells from oxidative damage.The classical enzymatic antioxidant defense systems include superoxide dismutase（SOD）,catalase（CAT）,thioredoxin（Trx）,glutathione reductase（GR）and glutathione S-transferase（GST）.Glutathione peroxidase（GSH-Px）is a class of enzymes with antioxidant functions in eukaryotes,whose main role is to counteract oxidative stress caused by hydrogen peroxide and organic peroxides,and has been less studied in prokaryotes.It has been shown that GSH-Px is widely distributed in different bacteria,suggesting that GSH-Px also plays an important role in prokaryote resistance and bacterial virulence.The main objectives of this study are first to construct gsh-px gene deletion and supplementation strains to investigate the role of gsh-px gene in the resistance to oxidative stress in L.monocytogenes.Secondly,to investigate the role of gsh-px gene in the pathogenicity of L.monocytogenes.The final goal is to screen the regulatory factors of gsh-px and explore the related molecular mechanisms.1.Exploring the role of gsh-px in the resistance to oxidative stress of L.monocytogenes.Construction of deletion strain after successful knockout of gsh-px gene（Δgsh-px）from clinical isolate strain Lm850658 using homologous recombination technique.The deletion strain was supplemented with a supplementation plasmid to construct supplementation strain（CΔgsh-px）.By comparing the growth curves of the parental strain and the deletion strain under different H₂O₂ concentrations,it was found that the growth trend of the deletion strain was consistent with the parental strain under normal conditions（0 m M）,and at low（10 m M）or high（40 m M）concentration of H₂O₂.This indicated that knockout of gsh-px did not affect the growth of L.monocytogenes,and L.monocytogenes also did not respond to oxidative stress generated by H₂O₂.The oxidative stress survival assays showed that the survival rate of gsh-px deficient strain was not significantly different from the parental strain under 10 m M H₂O₂.While under the conditions of 10m M Fe³⁺and 5 m M Cu²⁺,the survival rates of the gsh-px deficient strain were significantly higher than that of the parental strain.The glutathione（GSH）measurement tests revealed that there was no difference in the GSH contents of L.monocytogenes between the H₂O₂ group and the control group,but the GSH of L.monocytogenes treated with metal ions were significantly higher than that the control group.In addition,the gsh-px deletion strain had significantly higher GSH levels than the parental strain,while the complementary strain had GSH levels comparable to the parental strain.These results suggested that GSH-Px might be involved in the anti-oxidative stress effect of L.monocytogenes by affecting the concentrations of GSH.2.Exploring the role of gsh-px gene in the pathogenicity of L.monocytogenesExploring the role of gsh-px in the pathogenicity of L.monocytogenes using cell assays and animal assays.In the cell adhesion and invasion assays,deletion of gsh-px significantly enhanced the adhesion and invasion ability of L.monocytogenes to Caco-2cells.In addition to this,gsh-px deletion enhanced the resistance of L.monocytogenes to macrophage phagocytosis in the RAW264.7 cell assays.In the mice virulence tests,it was found that the bacterial load of the liver and spleen infected with the deletion strain was significantly higher than that of the parental strain 48 h after intraperitoneal injection of the bacteria.The mice survival tests also showed that more mice died in the deletion strain infected group than in the parental strain infected group.The results of the tests with chicken embryos were consistent with the mice tests.Western blot showed that the expression of virulence factors such as Inl A,Inl B,and LLO in the deletion strain was significantly higher than the parental strain,indicating that the deletion of gsh-px had an effect on the virulence of L.monocytogenes,and it was by increasing the expression of virulence factors.3.Screening for regulatory factors of gsh-px and exploring the related molecular mechanismTo explore the related molecular mechanism of gsh-px on the antioxidant stress and pathogenicity of L.monocytogenes,the GFP reporter plasmid of gsh-px was constructed in this study(P_gsh-px-gfp).This reporter plasmid could be successfully expressed in Lm850658 and 10403S.The reporter plasmids were electroporated into 14 pairs of two-component system deletion strains,and the fluorescence intensity was found to be significantly higher in the kdpE/kdpD deletion strain than in the parental strain.The results indicated that kdp E/kdp D is involved in the negative regulation of gsh-px expression.The prokaryotic expression of Kdp E was further obtained and the purified Kdp E was used for EMSA,which revealed that Kdp E did not bind to the promoter of gsh-px directly.It is suggested that Kdp E does not directly regulate gsh-px-mediated anti-oxidative stress and pathogenicity of L.monocytogenes.In summary,in the present study we found that GSH-Px in L.monocytogenes affected antioxidant stress by influencing the concentrations of GSH,and enhanced pathogenicity by increasing the expression of virulence factors such as Inl A,Inl B,and LLO.The two-component system kdp E/kdp D is involved in the negative regulation of gsh-px expression,but not in direct binding to the gsh-px promoter.The findings provided a basis for an in-depth exploration of the role of GSH-Px in L.monocytogenes.