Virulence Regulation Mechanism Of Type ? Secretion Systems In Vibrio Alginolyticus

Vibrio alginolyticus is a widespread marine pathogen.It can occupy the dominant niche in the environment duo to its fast growth rate.It can infect various marine animals including various fishes,crustaceans and coral insects and cause serious losses to the global aquaculture industry.Bacteria,including Vibrio alginolyticus,depend on some specialized secretion systems to interact with their environmental niches.Type VI secretion system(T6SS)is widespread in Gram-negative bacteria.This multi-component secretion system can mediate contact-dependent killing of eukaryotic cells as well as prokaryotic.Vibrio alginolyticus EPGS encodes two T6SS gene clusters(T6SS1 and T6SS2),each of whom encodes the homologs of 13 core T6SS proteins.However the function of these two T6SSs has not been studied further.In this study,we found the best conditions of the killing ability of V.alginolyticus EPGS on agar plate and the toxicity of the bacterium was not only to E.coli,but also toward some other marine bacteria.We found by confocal microscopy and killing ability assay that this organism principally relies on T6SS2 but not T6SS1 for interbacterial competition on agar plate.The data in qRT-PCR and Western blot experiments showed that the killing activity of T6SS2 is regualted by quorum sensing(QS)system and T6SS2 phosphokinase PpkA2.Furthermore we carried out a phosphoproteomic screen to identify the substrates of PpkA2.Similar to other bacteria,PpkA2 autophosphorylation was critical for T6SS2 functions.Another T6SS2 protein we identified in phosphoproteomic screen is DotU2.It can be phosphorylated by PpkA2 at two threonine residues,and these two phosphorylation sites were redundant for phosphrylation of DotU2.Meanwhile,p-DotU2 can bind to Fha2 to turn on T6SS2 secretion.Interestingly,phosphorylation of a small protein encoded outside of T6SS2 cluster,VtsR,was important for T6SS2 expression and function.It can promote T6SS2 cluster gene expression by augmenting expression of luxR,the key regulator of QS.Thus,kinase PpkA2 can control a phosphorylation cascade that mediates a positive regulatory loop between T6SS and QS to coordinate these pathways to enhance the competitive ability of V.alginolyticus.Except PpkA2,V.alginolyticus EPGS T6SS2 gene cluster also encodes a PP2C-family protein phosphatase,PppA.The primary function of these phosphatase family proteins is about regulating stress signalling pathways.PpkA-PppA pair can also regulate some non-T6SS phenotypes.However,the role of PppA on T6SS activation is now not clear.We found by phosphoproteomic screen that PppA could be the phosphorylation substrate of PpkA2 at threonine 253 and its phosphatase activity can be modulated by PpkA2.Moreover,PppA phosphorylation mediates T6SS2 expression and killing activity on agar plate.Meanwhile,the data indicated that the phosphorylation of PppA can regulated T6SS2 towards LuxR.In our previous work,PppA can positively regulate the expression of LuxR in liquid medium and ? factor RpoE can directly bind on the promoter of LuxR.Using qRT-PCR,it was found that PppA can positively regualte luxR and rpoE on transcriptional level.Then we carried out a Tn-seq screen to identify possible genes related to the regualtion.We found some transcriptional factors and some quorum sensing system genes,which might be related to the regualtion.And some other genes might be only the regulator of LuxR or RpoE,but not be related to PppA.