Construction Of Listeria Monocytogenes ?inlABJ Deletion Strain And Effect Of Inflammatory Factors On Blood-brain Barrier Injury In Mice

Object:In this study,we used Listeria monocytogenes?Lm90?which was isolated from the sheep with encephalitis as the material,and a mutant?Lm90-?inlABJ?was constructed on the basis of lacking inlA and inlB.The biological characteristics and pathogenic effects on mice and sheep were analyzed.Then we constructed a mouse infection model in vivo,we observed the impairment of blood-brain barrier?BBB?and explored the potential mechanisms.In the next research,we constructed in vitro BBB models to further verify the effect of inflammatory factors on BBB injury in mice.The results provide theoretical basis in preventing listeriosis and also provide reference for further study on the bacterial meningitis.Methods:?1?The primers were designed using Primer 5.0 software,the inlJ gene upstream and downstream homologous sequences were amplified by PCR,then were used in splice overlap extension PCR?SOE-PCR?to obtain the fusion fragment of inlJ deletion gene??inlJ?.The recombinant shuttle plasmid pKSV7-?inlJ was constructed and transformed into the Lm90-?inlAB competent cell by electroporation.Homologous recombination was achieved under the dual pressure of temperature and chloramphenicol;the recombinant Lm90-?inlABJ was screened and tested for genetic stability.?2?The growth and culture characteristics of Lm90-?inlABJ?Lm90-?inlAB and Lm90were compared and analyzed by bacteriological method,and the difference of invasion ability of bacteria to cells and pathogenicity of animals were also analyzed.?3?In this study,a mouse model of infectious encephalitis was built on the basis of Lm90,a Listeria strain causing sheep encephalitis.The mouse infection model was evaluated by clinical symptoms observation,histopathological examination,and bacterial recovery tests.At the same time,the changes of BBB permeability and expression of tight junction protein after infection in mice were analyzed using Evan's blue?EB?,Western blot?WB?and immunohistochemistry.Furthermore,the level of inflammatory factors were measured and the possible mechanism of BBB injury in mice was explored.?4?Primary mouse brain microvascular endothelial cells?MBMEC?and astrocyte cells?AC?were isolated from newborn mice,and identified by morphology and immunofluorescence.Then two cells were seeded on transwell cell culture membrane with0.4-?m pores to co-culture to establish an in vitro BBB model.The permeability of the model was evaluated by trans-endothelial electronic resistance?TEER?,leakage tests and horseradish peroxidase permeability?HRP?,and the expression of tight junction protein was detected by western blot.Using qRT-PCR and ELISA to detect the expression of inflammatory factors after Lm90 infected mouse macrophages,and the supernatant of macrophages infected with Lm90 was applied to the BBB model in vitro.The TEER assay,HRP permeability test,and qRT-PCR,Western Blot,and cellular immunofluorescence techniques were used to analyze the regulatory effect of inflammatory factors on BBB tight junction protein to further verify the effect of inflammatory factors on BBB in vivo.Results:Shuttle plasmid pKSV7-?inlJ was successfully constructed and combined with electrotransformation and homologous recombination technology,a inlJ gene deletion strain Lm90-?inlABJ was successfully constructed and had a good genetic stability.The inlJ gene deletion does not affect the growth rate of Lm90,and there was no difference among Lm90-?inlABJ,Lm90-?inlAB and Lm90 in vitro culture.Cell infection test showed that the adhesion and invasion rates of Lm90-?inlABJ to MBMEC were 2.54%and0.22%,respectively.Compared with Lm90,the difference was statistically significant?P<0.05?,compared to Lm90-?inlAB,the adhesion rate and invasion had no significant difference?P>0.05?.In the mouse infection experiment,the LD₅₀ showed that in virulence in Lm90-?inlABJ was 0.3 and 1.6 orders of magnitudes higher than that of Lm90-?inlAB and Lm90,respectively.The tissue load test results showed that the load of Lm90-?inlABJ in liver and spleen decreased by 0.57 and 0.26 logarithmic orders of magnitude compared with that of Lm90-?inlAB after 72 hours of infection,all results indicated deletion of inlJ genes reduces the virulence of bacteria.Sheep infection tests showed that Lm90-?inlABJ is still pathogenic and can cause sheep encephalitis.The colonization of Lm90 in the mouse brain was time-dependent.The EB test showed that the permeability of BBB did not change significantly in the early stage of infection and the permeability increased significantly when the mice showed severe illness and dying.Western blot and immunohistochemical showed that Lm90 infection can down-regulate the expression of Occludin and Claudin-5 and increase BBB permeability.ELISA results showed that the expression of TNF-??IL-6?IL-1?in the supernatant of mouse brain homogenate and serum were up-regulated after Lm90 infection.Combined with the change of BBB permeability,we concluded that that BBB showed damage and increased permeability after infection in mice,which may be related to up-regulation of inflammatory factors and down-regulation of tight junction proteins Occludin and Claudin-5.Primary BMECs and AC were isolated from newborn mice,morphological observation of BMECs showed a typical cobblestone-like structure,AC had slender synapses and shallow cytoplasm,and the cells were correctly identified by immunofluorescence.After co-cultivation of BMECs and AC,TEER value stabilized at 72 h and reached 403?·cm^-2 at96 h;HRP permeability test results were consistent with that of TEER,the test of liquid leakage was positive.WB test showed high expression of tight junction protein between cells.Lm90 infection can induce induce up-regulation of inflammatory factors?IL-1?,IL-6,TNF-??in RAW264.7 cells.Furthermore,the RAW264.7 products infected with L.monocytogenes and IL-1?could down-regulate BMECs tight junction proteins and the in vitro test results were consistent with in vivo.Conclusion:A three-gene deletion strain?Lm90-?inlABJ?was successfully constructed and the mutant had good genetic stability.The results of biological studies showed that the lack of inlA,inlB,and inlJ had no significant effect on their growth.The invasiveness of the deletion strain on cells and pathogenicity in mice had decreased significantly,but the deletion strain is still pathogenic and can still cause sheep encephalitis.After Lm90 infected mice,the BBB permeability did not change significantly in the early stages of infection,but the BBB permeability were increased significantly when mice were severely infected and dying,and BBB showed a damaging effect.The impairment of BBB with increased permeability in mice was probably associated with the phenomenon of up-regulation of inflammatory factors?TNF-?,IL-6.IL-1??and down-regulation of tight junction protein Claudin-5 and Occludin.And the results of in vitro tests have further confirmed.