Construction Of Salmonella Enterica Serovar Pullorum AvrA Mutants And Study On Molecular Mechanism Of Anti-Inflammatory Effect Perforemd By AvrA

Salmonella enterica serovar Pullorum(S.Pullorum)is a host-adaptive pathogen that mostly infects chickens within 20 days old,resulting in high morbidity and mortality.The infected adult chickens showed declined egg production without obvious clinical symptoms.S.Pullorum can be transmitted horizontally and vertically,causing huge economic losses in the poultry industry around the world,especially in developing countries.The type ? secretion system(T3SS)is a needle-like or syringe-like device by which many Gram-negative bacteria are used to invade host cells.S.Pullorum can use T3SS to directly secrete multiple effector proteins into eukaryotic host cells.The effector AvrA encoded by T3SS1 is prevalent in the Salmonella enterica serotypes.Unlike other common effector proteins secreted into the host by the pathogen,AvrA can stabilize the host cell tight junction,reduce inflammation and inhibit cell apoptosis.Until now,the study on AvrA mainly has been focused on S.Typhimurium,there are few reports about its function in S.Pullorum.Sequence analysis showed that the AvrA from S.Pullorum lose ten amino acids at the C-end compared to the protein in other Salmonella enteirca serotypes.This study used ?-Red homologous recombination technology to construct the avrA mutant of S.Pullorum,and revealed the molecular mechanism of AvrA in anti-inflammatory response.This study lays a foundation for further study on the molecular mechanism of AvrA in the process of SP infection.1 Construction of the mutant strain C79-13?avrA and S06004?avrAIn this study,we used the ?-Red homologous recombination system to knock out the avrA gene in S.Pullorum and successfully constructed the avrA mutants of S.Pullorum S06004 and C79-13.The avrA gene from S.Pullorum and S.Enteritidis was cloned into plasmid pBR322,respectively.And the recombined plasmids then were electroporated into the mutant strains,respectively.Here we constructed the complementary strains of S06004 and C79-13 with native or S.Enteritidis avrA genes.The basic biological characteristics of the wild,mutant and complementary strains demonstrated that the deletion of avrA do not affect the physiological and biochemical characteristics of S06004 and C79-13,and the mutant strain have good genetic stability.In this study,recombinant eukaryotic expression plasmids of S.Pullorum avrA were constructed using the vectors pcDNA3.1(+)and pCMV-HA.We transferred the recombinant eukaryotic expression plasmids into E.coli DH5?,and identified the expression of AvrA in HeLa and other cells by indirect immunofluorescence technology.The construction of S.Pullorum S06004 and C79-13 avrA mutants and their eukaryotic expression vectors provided the corresponding biomaterials for the father study on the role of AvrA in pathogenicity of S.Pullorum.2 Molecular mechanism of anti-inflammatory response performed by AvrA in S.PullorumThe eukaryotic expression plasmids of avrA were transiently transfected into HEK293T,HeLa and LMH cells,and the NF-?B transcriptional activation activity was analyzed by luciferase reporter assay system.The results showed that the activity of NF-?B signaling pathway was down-regulated when the recombinant eukaryotic expression vector was transiently transfected into HEK293T,HeLa and LMH cells.This indicated that S.Pullorum effector AvrA could inhibit the activation of NF-?B pathway.In addition,the expressed AvrA from S.Pullorum displayed weaker inhibitory effect on TNF-a-induced NF-?B pathway activity than S.Enteritidis AvrA.This result indicates that the deleted fragment of AvrA from S.Pullorum reduced its ability to inhibit the activation of the NF-?B pathway in the level of eukaryotic transfection.On the other hand,HeLa and Caco-2 BBE cells were infected with wild-type,mutant and complementary strains of S.Pullorum,the proinflammatory cytokines secreted were monitored in the cell culture supernatant.The results showed that the proinflammatory cytokines IL-6 and IL-8 of the mutant strain infected group were significantly higher expressed than those in the wild type strain infected group,indicating that S.Pullorum effector AvrA played an anti-inflammatory role in the Salmonella-host interaction.The expression level of proinflammatory cytokines in the culture supernatant of the cells infected with the complementary strain with the native avrA was lower than that of the complementary strain with the extraneous avrA.This indicates that AvrA can exert better anti-inflammatory effects in the native serotype Salmonella.Western-blot analysis showed that S.Pullorum AvrA could inhibit the expression of p-JNK and Beclin-1 proteins during the infected HeLa or Caco-2 BBE cells by S.Pullorum.The deletion fragment of AvrA do not affect its anti-inflammatory function.AvrA exerts better inhibition role on p-JNK and Beclin-1 in its native Salmonella serotype than in other serotypes.Laser confocal microscopy experiment technology results showed that S.Pullorum AvrA can inhibit TNF-a-induced nuclear import of p65.This study tentatively clarified that S.Pullorum efforter AvrA plays an anti-inflammatory role in the host immune response infected by S.Pullorum.