Studies On Regulation Of Serine/Threonine Protease In Vibrio Parahaemolyticus On Type Ⅵ Secretion System

Vibrio parahaemolyticus (VP) is a gram-negative halophilic bacterium, which can grow in a wide range of pH and salt concentration. It was first discovered and successfully isolated from a food-borne illness in Japan in 1950. It is widely distributed in seafood products and can infect fish, shrimp and shellfish resulting in shrimp red body disease and fishes'skin ulcers, etc. The outbreaks of food poisoning were almost exclusive with consumption of contaminated or inadequately cooked seafood. The common symptoms are usually presented as gastroenteritis characterized mainly by watery diarrhea and abdominal cramps, sometimes a dysentery-like illness with bloody or mucoid stoos. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) were known as the major virulent factors, and they were correlated firmly with the pathogenecity of vibrio parahaemolyticus.With the development of the aquaculture industry, vibriosis outbreaks are more and more frequently, and spreads faster and faster, which bring a huge economic losses to the world aquaculture. It is the main bacterial which can contaminate seafood, and several times returned and destroyed were happened because of Vibrio parahaemolyticus excessive during China's exports. The EU resolution 368 (97/368/EC) and 587 (97/587/EC) required to test batches of Vibrio parahaemolyticus on the origin of aquatic products in China. Data of food-borne disease showed, since 1998 the outbreaks of Vibrio parahaemolyticus have increased significantly and been to the first rank instead of Salmonella.1. Serine/threonine kinase (STPK) and tyrosine kinase are numbers the two main protein kinase superfamily. STPK makes serine or threonine hydroxy-phosphorylation of special substrate proteins. Through catalyzing a variety of functional proteins (such as enzymes, receptors, transport proteins, regulatory proteins, nuclear proteins, etc.) phosphorylation, STPK can change conformation of functional proteins, leading to functional proteins activity and properties changes to regulate various cell life activities. Reversible phosphorylation of serine/threonine protein kinases and protein phosphatases plays a switch role on signaling processes. Protein kinases catalytic the formation of phosphate ester bonds, cell protein activity occurred significantly change, and the properties of phosphate ester bonds are very stable. The bioinformatics analysis found that Vibrio parahaemolyticus processes two serine/threonine protein kinase, which are located on chromosomesⅠandⅡ, named Vpk1 and Vpk2, respectively, and a protein phosphatase, which is located on chromosomeⅡ, named Vpp2.2. TypeⅥsecretion system (T6SS) is a new secretion system discovered recently. It is widely found in gram-negative bacteria, and consists of structural proteins, translocon proteins, secreted proteins and some auxiliary functions proteins. T6SS can enhance the adaptability of bacteria to the external environment, and mediate bacterial virulence on host cells, etc. T6SS plays an important role for gram-negative bacteria, but plays different roles in different bacteria. Therefore, its structure, function and regulation are all urgent further study. The laboratory analyze by bioinformatics to find that Vibrio parahaemolyticus processes two sets of T6SS, which locates on chromosomeⅠand chromosomeⅡ, respectively. Hemolysin co-regulated protein (Hcp) and valine-glycine repeat protein G (VgrG) can be detected in the supernatant of T6SS gene cluster. They together constitute the pipeline structure of secreted proteins, assisting secreted protein transmembrane transport and eventually reach the host cell. Hcp1 and Hcp2 locates on chromosomeⅠand chromosomeⅡ, respectively.3. To reveal the regulation effect serine/threonine kinase system on T6SS, we designed 6 primers (A, B, C, D, E and F) according to vpk1, vpk2, vpp2, hcp1 and hcp2 genes. First, a common PCR was carried to amplify AB, CD or EB, CF, and then we obtained vpk1-AD, vpk2-AD, vpp2-AD, hcpl-AD, hcp2-AD through the fusion PCR. Next, these fusion fragments were digested and connected pYAK1 vector to construct gene deletion vectors. We successfully obtained mutants HZ-△vpk1, HZ-△vpk2, HZ-△vpp2, HZ-△hcp1,HZ-△hcp2 through conjugation. Primers E and F were used to identify these mutants.4. In present study, we linked the fusion fragment into pYAK1 plasmid by homologous recombination, and transformed into host bacteria CC118λpir. The fusion PCR technique we used overcomes the disadvantage of traditional homologous recombination, and eliminates the digestion process of connecting to compare with the digested ligation. In this study, we utilized plasmid pYAK1 as the donor, the parent strain HZ as a receptor, and pRK2013-HB101 as an auxiliary bacteria in conjugation. Because the recombination rate is relatively low, we used 10%sucrose medium for the second re-screened positive recombinants. To optimize conjugation by Escherichia coli fimbriae, we mixed the donor strain, recipient strain and auxiliary bacteria before dropping in the filter membrane of 0.22μM, and the helper plasmid transfer pRK2013 or 10mM Mg2+was added to increase bacterial cell membrane permeability to increase with the transfer efficiency. The results showed that we successfully transferred the plasmid pYAKl into Vibrio parahaemolyticus strain HZ and obtained mutants HZ-△vpk1, HZ-△vpk2, HZ-△vpp2, HZ-△hcp1, HZ-△hcp2. This lays the foundation for further research the effect of serine/threonine protein kinase and phosphatase on expression of Hcp protein.5. We analyzed different gene deletion mutant effect on Hcp1 and Hcp2 expression by western blotting. In order to decrease the differences on bacterial growth, we used the BHI medium containing high magnesium and low calcium (named BHI'). Meanwhile, BHI'medium can also increase the permeability of bacterial cells to facilitate secretion of protein secretion and translocation. Besides, through a series experiments of temperature and incubation time, the translocation of Hcp2 was optimized at 28℃for 6h and 14h. A relatively high soluble Hcp2 and RpoB were obtained in 15-20℃for 12h in prokaryotic expression E.coli. Western blotting results showed that 1) we failed to detect Hcp1 in the parent HZ strain and△vpk1,△vpk2,△vpp2 deletion mutant in precipitation and supernatant protein, however, Hcp1 protein was detected after complementing hcpl in precipitation, showed that the T6SS1 translocation protein was low expressed under normal conditions.2) Hcp2 was not detected in the supernatant of bacteria with absence of vpkl, vpk1, vpp2, showed that the serine/threonine kinase Vpk1, Vpk2 and phosphatase Vpp2 played important roles on the translocation of Hcp2 protein. But we detected Hcp2 protein in cell precipitation with absence vpk2, vpp2.3) Hcp2 expression was significantly higher than the parent HZ strain in△vpp2, showing that the serine/threonine phosphatase downregulate Hcp2 expression.4) In△vpkl bacteria precipitation, we did still not detect Hcp2 protein, indicating that the serine/threonine kinase Vpkl not only regulates the translocation of Hcp2, but also increases Hcp2 protein. Taken together, two T6SS in Vibrio parahaemolyticus may be cross-regulated by STPKs, and it also showed that serine/threonine kinase syetem regulate T6SS in the diversity ways.