Vibrio Parahaemolyticus T3SS2 Effector VPA1324 Manipulates Host Immune Signaling Pathways

Vibrio parahaemolyticus(VP)is a gram-negative Vibrio halophilus.It is not only the main pathogen of marine animals,but also the leading cause of gastroenteritis even hematosepsis caused by the marine products worldwide.The pathogenicity of the bacterium is related to a variety of virulence factors,among which the type III secretion systems(T3SSs)have been revealed as an important virulence factor of VP.It mainly secretes virulence proteins(called effectors)into host cells to destroy the normal function of the host cell.T3SS2 is encoded by the VPaI-7 virulence island gene cluster on chromosome 2 and is associated with intestinal toxicity.T3SS2 effectors have been shown to induce intestinal toxicity primarily,through disrupting of the host actin cytoskeleton and interfering with intracellular natural immune signaling pathways.However,only a few effectors have been studied.It is necessary to identify new effectors and reveal the mechanism of their action on host cells.Bis-(3'-5')cyclic diguanylic acid(c-di-GMP or cdG for short)is a signal molecule that is widely present in bacteria.In recent researches,pathogenic bacteria in the process of infecting host cells released the cdG,which can specifically bind to the STING protein,an important signaling molecule in immune and inflammatory reactions.The binding can activate the natural immune signaling pathway and stimulate the immune defense function of the body.Previous researches have shown that VPA1324 is a secreted protein which encoded by T3SS2.In this paper,the bioinformatics analysis found that VPA1324 is a diguanylate phosphodiesterase that contains the EAL domain,and 29 phosphodiesterases that contain the EAL domain in VP.VPA1324 is the only protein encoded by the pathogenic island gene of VPaI-7.It is speculated that VPA1324 can influence the cdG-activated innate immune signaling pathway in the host cell,and then carried out a series of related studies.First,the recombinant expression vector and gene knockout vector related to vpa1324 gene were constructed.Secondly,VPA1324 was expressed and purified,and its product after cdG was detected by LC-MS/MS.It was found that VPA1324 can hydrolyze cdG into pGpG and GMP,confirming that VPA1324 has diguanylate phosphodiesterase activity of EAL.Again,the expression vector pBBR1MCS2-vpa1324-cyaA(adenylate cyclase)was transformed into the T3SS1-deficient strain POR2,and the T3SS2 was activated with bile salts.The coupling protein(VPA1324-CyaA)was detected in the secreted protein fraction of the strain,and it confirms thatVPA1324 is a secretable protein;after the strain infected the host cell,the cyclic adenosine monophosphate(cAMP)in the host cell was detected.The content was significantly increased,confirming that VPA1324 is translocated into eukaryotic cells in a T3SS2-dependent manneris.Then,the eukaryotic expression vector pEGFP-C1-vpa1324 was transfected into the host cells,and then the cdG was transfected.As a result,it was found that the host intracellular transcription factor IRF3 was decreased.The above results preliminarily clarified that VPA1324,as a diguanylate phosphodiesterase containing EAL,inhibits the innate immune pathway of host cells activated by cdG,in the way of degrading the cdG released into the host cell when infected.This discovery reveals a new mechanism of action of the effector against the immune system.It not only helps to understand the virulence of pathogenic bacteria and the mechanism of infection,but also provides new strategies and models for host signal transduction and immune defense research.It has important theoretical and application values for pathogenic microbiology and immunology.