The Enterotoxicity Mechanism Of Vibrio Parahaemolyticus Effector VPA1324 Regulating Host C-di-GMP/NLRP3 Immune Signaling Pathway

Vibrio parahaemolyticus is the main pathogen causing foodborne gastroenteritis.Its type ? secretion system 2(T3SS2)destroy the normal function of the cells by injecting effector proteins into the host cell,which is the key cause of its enterotoxicity.VPA1324 is a T3SS2 effector protein identified by our research group recently,and it also has been confirmed as a c-di-GMP phosphodiesterase.In addition,as a unique second messenger of bacteria,c-di-GMP has become a signal molecule for host recognition of pathogen invasion and can activate innate immunity such as NLRP3 inflammasome.Therefore,whether the degradation of c-di-GMP by VPA1324 is an effective strategy for Vibrio parahaemolyticus to resist host immunity and an important reason for its enterotoxicity remains to be further revealed.In order to confirm the hypothesis that VPA1324 degrades c-di-GMP from bacteria in host cells to prevent the activation of NLRP3 inflammasome and thus escape immunity,a method was first established to effectively transfer VPA1324 protein into cells by cell penetrating membrane peptide PEP-1.The PEP-1-VPA1324 fusion protein can effectively cross the cell membrane and enter the cytoplasm without affecting cell viability.Then,LPS and c-di-GMP were used to establish the activation model of NLRP3 inflammasome in vitro,and the activation of NLRP3 inflammasome was evaluated by comparing the secretion of IL-1? and cell pyrotosis of cells containing and without VPA1324 protein.Collectively,the date demonstrated that the c-di-GMP /NLRP3 immune signaling pathway can be inhibited by VPA1324 significantly.In order to further explore the role of VPA1324 in the toxicity of Vibrio parahaemolyticus,the vpa1324gene-deficient strain KO-T3SS1/?vpa1324 was constructed on the basis of KO-T3SS1,of which the gene tdh A/S and the key structural gene vcr D1 of T3SS1 were deleted.Then the virulence of KO-T3SS1,KO-T3SS1/T3SS2 and KO-T3SS1/?vpa1324 bacterial strain was compared through the rabbit ileum ligation experiment and the mouse infection model.The results indicate that VPA1324 could significantly enhance the enterotoxicity of Vibrio parahaemolyticus and increase the mortality of mice.In conclusion,the completion of this study initially revealed the unique strategy and enterotoxicity mechanism of Vibrio parahaemolyticus to escape innate immunity by secreting VPA1324 into host cells to degrade c-di-GMP,which helps to further clarify the pathogenic mechanism of Vibrio parahaemolyticus,as well as the antagonism between the pathogen and the innate immunity of the host.Meanwhile,it also provides new insights for the research and development of antimicrobial drugs targeting T3SS2.