| Listeria monocytogenes is a gram-positive,food-borne pathogenic bacteria.It is widely spread in soil,dairy and ready-to-eat foods.Elderly,children,pregnant women and people with low immunity are susceptible to listeriosis and the mortality rate after infection is up to 30%.L.monocytogenes can accommodate easily in human body and food environment.It is highly adaptable and resistant to unsuitable environment and expresses an array of proteins and virulence factors to survive under these harsh conditions such as low temperature,low pH,high osmotic and oxidative stress.It has become the main pathogens that major threaten to public health security in recent years.Sodium hypochlorite(Na Cl O)is a strong reactive oxygen species(ROS)which widely used as a disinfectant and can effectively kill bacteria,fungi,and viruses.It can react with a wide variety of biomolecules(DNA,RNA,fatty acid groups,cholesterol and proteins).The defense of oxidative stress by bacteria is a complex process,and usually controlled by the oxidative stress regulatory factors.The major function of oxidative defense in bacteria including the detoxification and the repair effect.In addition,a large number of low molecular weight thiol proteins distributed in the cytoplasm can not only neutralize the oxidants in the cells,but also protect the important proteins in the cells from being oxidized.In L.monocytogenes,SOD,KAT and Lmo1604 are function as detoxification for reactive oxygen species(ROS).The function of regulation of oxidative stress in the general stress stress regulator sigB is controversial,and the oxidative stress regulator PerR has a significant effect in oxidative defense under hydrogen peroxide stress.Moreover,the important low molecular weight thiol protein glutathione,NADPH,glutathione reductase and Grx together constitute the Glutathione Redox System and Thioredoxin Redox System(thioredoxin reductase and MetA,Met B)also play an important role in the process of oxidative defense in Listeria monocytogenes.To explore the antioxidant mechanism of L.monocytogenes during the stress of sodium hypochlorite,we studied the physiological and biochemical changes,the reaction of antioxidant stress enzymes and important redox-related genes in L.monocytogenes WaX12 and sigB-deficient strain WaX12-?sigB under medial lethal concentration of sodium hypochlorite(3 mM)stress.The results showed that L.monocytogenes had a strong tolerance to sodium hypochlorite which can survive for more than 1 hour under a medial lethal concentration of 3 mM,and the survival of bacteria was significantly decreased under the content over 3 mM.NaClO can cause oxidative damage by destroying the cell membrane.The ROS levels in the bacteria will rise in NaClO stress.The results showed that the level of ROS increase in bacteria was higher after 30 min and later.The activity of SOD in the bacteria was higher at 30 min and 45 min,and the activity of KAT was highest at 45 min.Then,under the same lethal dose,we compared the changes of bacterial hemolytic activity under NaClO stress and H2O2 stress.Results showed the hemolytic activity of bacteria under NaCl O stress was lower.Suggesting that NaClO has a greater effect on the virulence of bacteria,which may be related to the ability of NaClO to specifically target thiol-containing proteins.Further studies have shown that gene perR,trxA,sigB are most active in the half-lethal concentration of NaClO stress.And with the change of time,more genes in the late time of stress have higher expression.TrxA is an important gene for the repair of Met protein,and the active expression of trxA indicates that Met plays an important role in sodium hypochlorite stress.We observed the effect of oxidative stress by adding Met.The results showed that the survival rate of bacteria increased significantly after adding Met,and the content of GSH in bacteria was also significantly higher than that of bacteria without Met.It is noteworthy that this phenomenon is not obvious in the H2O2 stress.Finally,we investigated the regulation of L.monocytogenes sigB gene on sodium hypochlorite by comparing the increase level of reactive oxygen species(ROS)of LM-WaX12 and its dull sigB mutant at median lethal concentration NaClO.Then we observe the transcription level changes of lmo1433,lmo0906,GRXs,ohrR,gshA during the different infection time.Our results showed that the wild-type had a better ability in defense of NaClO stress than the mutant-type.This phenomenon was much obvious in stationary phase.And the increase level of ROS was 19.97% higher in wild-type strain on the infection time at 30 min.RT-PCR results indicated that the relative expression of lmo1433 gene in wild-type strain is much higher than that in mutant-type(P<0.01).And this consequence also showed in the relative expression of grx and ohrR but not in lmo0906,gshA.sigB gene have an important role during the NaClO infection.And the regulation effect of sigB may work by directly or indirectly activating some anti-oxidation relate genes. |
PDF Full Text Request