A Model Host To Study Pathogenicity Of Listeria Monocytogenes And Research On The Role Of Its Membrane Vesicles

Listeria monocytogenes is a gram positive pathogen causing listeriosis.It can penetrate three major barriers of the human and animals:placental barrier,blood brain barrier and intestinal barrier.In human infection,the mortality rate is 20%-30%.In order to deeply analyze the pathogenic mechanism of L.monocytogenes and the interaction between L.monocytogenes and its host,wecarried out the following research:(1)The establishment of an infection insect model of L.monocytogenes;(2)The isolation and functional exploration of L.monocytogenes membrane vesicles.1.Helicoverpa armigera as an infection models of Listeria monocytogenesThis part used H.armigera larvae as an insect model to explore the pathogenicity of L.monocytogenes,in order to lay a foundation for further study of the interaction mechanism between L.monocytogenes and its host.First of all,five strains of L.monocytogenes with different virulence were injected into the 4th instar H.armigera larvae respectively,i.e.The wild strain EGDe(the middle virulent strain),PrfA deletion strain EGDe?prfA(the attenuated strain),PrfA constitutive high expression mutant EGDe?prf?+pERL3-prfA*(the highly virulent strain),the wild strain carrying empty plasmid EGDe+pERL3(the middle virulent strain)and PrfA revertant strain EGDe?prfA+pERL3-prfA(the highly virulent strain).The results showed that L.monocytogenes could not only successfully infect and cause the death of H.armigera larvae,but also the 50%lethal dose of worms against bacteria and their midgut cellular lesion degrees were positively correlated with the virulent levels of L.monocytogenes.Secondly,the immune response of H.armigera to different virulent L.monocytogenes strains was explored by detecting changes of the number and encapsulation function of hemocytes as well as the difference of expression level of immune-related genes after infection with L.monocytogenes.The results showed that EGDe?prfA+pERL3-prfA*had the strongest effect on the immune system of H.armigera larvae,the number of hemocytes in larvae infected with it decreased by 71%,it also had the strongest inhibitory effect on encapsulation of hemocytes.And the hemocytes of H.armigera infected by EGDe?prfA were only reduced by 27%,and there was had almost no inhibitory effect on encapsulation function of H.armigera.In addition,RT-qPCR results showed that the transcriptional levels of HaCTL1 and HaPGRP-A in H.armigera were up-regulated after infection for 24 h,the highest level was found in the worms infected with EGDe?prfA+pERL3-prfA*,and no significant changes in that of the attenuated strain EGDe?prfA.To sum up,the different virulent L.monocytogenes can activate the innate immune system of H.armigera larvae,and the higher the virulence of strains is,the greater the damage to the H.armigera larvae is,and the stronger the immune response will be.In addition,in order to explore the relationship between the pathogenicity of L.monocytogenes and the detoxification of H.armigera,we detected the hemolytic activities of different virulent L.monocytogenes in vitro and in vivo and the corresponding glutathione S-transferase(GSTs)activities of the infected larvae.The results suggested that the different virulent L.monocytogenes have different pathogenic effect on the larvae.The highly virulent strain EGDe?prfA+pERL3-prfA*has the strongest pathogenicity to H.armigera larvae,by inhibition of larval detoxifying enzymes activities,such as GSTs,which may facilitate bacteria dissemination and prolong the larval recovery time.2.Isolation and functional study of Listeria monocytogenes membrane vesiclesIn this part,we planned to establish an effective method for the isolation of L.monocytogenes membrane vesicles,and then analyze the yield,morphology and protein composition of the membrane vesicles secreted by different virulent L.monocytogenes.In addition,we investigated the effect of membrane vesicles secreted by different L.monocytogenes on their biofilm formation,and also the hemolytic activity and the pathogenicity to H.armigera larvae,in order to lay a foundation for the in-depth analysis of the physiological function of Gram-positive bacteria-derived membrane vesicles,and also provide some new ideas for the study of the pathogenic mechanism of L.monocytogenes.Firstly,we isolated membrane vesicles from different virulent L.monocytogenes(EGDe,EGDe?prfA and EGDe?prfA+pERL3-prfA*)by ultrafiltration concentration or Optiprep density gradient centrifugation,we called these membrane vesicles as"EGDe-MVs,AprfA-MVs and prfA*-MVs",respectively.The results showed that the yield of EGDe-MVs was the highest,followed by ?prfA-MVs,and the yield of prfA*-MVs was the lowest.In addition,L.monocytogenes with different virulence could secrete membrane vesicles with a diameter of 20-200 nm,and the morphological structure of them had no obvious relationship with the virulence of strain.Comparing the two methods,it was found that these vesicles extracted by Optiprep density gradient centrifugation had high yield and good observation effect under transmission electron microscope,but it appeared to be more complicated to operate and time-consuming.In contrast,although the extraction effect of the ultrafiltration concentration method was not as good as the former,it was economical and time-saving,and considering the limited laboratory equipment,we mainly used ultrafiltration concentration to isolate membrane vesicles of L.monocytogenes in this study.Secondly,the membrane vesicles from different virulent L.monocytogenes strains isolated by ultrafiltration concentration were used to detect the composition of membrane vesicles proteins by SDS-PAGE and silver staining,the effect on biofilm formation,the hemolytic activity and the pathogenicity to H.armigera larvae.The results showed that:(1)L.monocytogenes-derived membrane vesicles contained variable proteins which molecular mass were mainly concentrated in 50-70 kD.Based on the analysis of the publications,we speculated that the proteins of 58 kD and 71 kD might be virulent factors LLO and internalin InlB,respectively.(2)The membrane vesicles inhibited biofilm formation of L.monocytogenes,harbored a certain hemolytic activity,reduced the pupation and survival rate of the Helicoverpa armigera larvae,and even caused them death.The toxicity of these vesicles to the larvae showed a clear link with the virulence of strains they originated.To sum up,H.armigera larvae(at least 4th instar H.armigera larvae)are suitable to be used as an infection models to study the pathogenesis of L.monocytogenes,and different virulent bacteria can induce different levels of immune responses in worms.In view of the death of H.armigera larvae caused by L.monocytogenes,we consider whether L.monocytogenes-derived membrane vesicles may also play an important role in pathogenic process of bacteria,so we tried to detect the biological activity of the membrane vesicles and tried to explore its biological function,we found all different virulent L.monocytogenes strains can secrete membrane vesicles with 20-200 nm in diameter,and no obvious differences in their shapes and sizes.However,these vesicles inhibited biofilm formation of L.monocytogenes,carried virulent factors,presented hemolytic activities,and led to the death of H.armigera larvae or reduced their survival and pupation rates.Here by,we believe that the membrane vesicles secreted by L.monocytogenes may be directly involved in the pathogenicity of L.monocytogenes,and play an important role in bacteria-host interactions.