Study On The Functions Of The Transcriptional Regulator CalR In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that naturally occurs in estuarine and marine habitats, that causes seafood-related diseases in humans. Infection is acquired by the consumption of contaminated raw or undercooked seafood. Vibrio parahaemolyticus causes three major clinical illness, i.e., gastroenteritis, wound infections and septicemia. Gastroenteritis, the most common illness include symptoms such as diarrhea with abdominal cramps, nausea, vomiting, headache, and low grade fever. The major virulence-associated determinants of V. parahaemolyticus include thermostable direct hemolysin(TDH), TDH-related hemolysin(TRH), type III secretion systems(T3SS), type VI secretion systems(T6SS), capsular polysaccharide. TDH positive strains of Vibrio parahaemolyticus are detected on a special agar known as Wagatsuma blood agar, the identification method referred to as Kanagawa phenomenon. Food-borne disease caused by V.parahaemolyticus remarkly increase in food poisioning case at coastal area of china, so that it is necessary to investigate the pathogen mechanism proudly.LeuO belongs to the lys R familiy transcriptional regulator present in members of Enterobacteriaceae. It was first found in Salmonella, which activate the transcription of the Leucine biosynthesis(leu ABCD) operon. Further work revealed Leu O as important master regulator, which has been shown to be involved in competition with H-NS. The gene cal R of V. parahaemolyticus located on chromosome I, comprising 960 bp content of G+C 47.29%,encode the protein of 319 amino acid residues with calculated molecular weight of 36192.8.Cal R is annotated as a Leu O homologue, which is regulated by the concentration of solidum and calcium ions. The recent research only shows that Cal R inhibits T3SS1 gene expression,cytotoxicity and swarming motility. There is no information and work done on Cal R molecular mechanisms.Objective: To construct the cal R null mutant, the complemented mutant strain and protein expression strain of Cal R in Vibrio parahaemolyticus, which promote analysis of the function of CalR.Methods: Utilizing the homologous recombination characteristics of the suicide plasmid vector pDS132, the recombination plasmid was introduced into Vibrio parahaemolyticus RIMD2210633(WT) by conjugation. The cal R null mutant(Δcal R) was screened and verified by PCR. The complemented mutant strain(Δcal R/cal R::p BAD33, C-Δcal R) and Lac Z strains containing target gene were constructed on the basis of Δcal R. To measure the promoter activity(the β-Galactosidase activity) of the vop N gene in these three strains(WT/p BAD33, Δcal R/p BAD33 and C-Δcal R) the β-Galactosidase Enzyme Assay System used was to determine the regulation of vop N by Cal R. The WT strain was compared withΔcal R to determine the adaptive abilities of some stress conditions, colony transparency,motility, biofilm formation, and cytotoxicity. Stress conditions corresponding to environmental stress in vitro, including cold treatment, low salt and acid stress. The promoter activity(the β-Galactosidase activity) of the target gene was measured using the Lac Z strains,which determine the regulation of target gene by Cal R. Using the recombinant cloning method, Cal Rhis6 was induced and purified by nickel column, which lay the foudation for the research of the activity of CalRhis6 binding to the promoter region of target gene.Results: The Lac Z fusion result showed negative regulation of vop N by Cal R. The mutation and complementation of cal R in V. parahaemolyticus was successful. Significant difference existed between WT and Δcal R at cold stress condition(4℃) in the second day. The Δcal R strain showed more ability to form biofilm than that of WT, Δcal R had different colony morpholoy compared with WT; The Swarming motility was significantly decreased in Δcal R relative to WT, however, there was no significant difference between the two strains in swimming motility. The cytotoxicity against the cells infected with Δcal R and the relative hemolytic activity of Δcal R markedly increased more than that from WT. The Lac Z fusion results showed that the transcription of cps A was positively regulated by Cal R, but exs A, exs B,vop N, syp G, tdh A were negatively regulated by Cal R, while syp A was not regulated by Cal R.Cal Rhis6 recombinant proteins were expressed and purified successfully.Conclusion: Cal R in Vibrio parahaemolyticus was required for adaptive abilities of some stress condition, biofilm formation, motility, transparency, cytotoxicity and hemolytic activity.Cal R inhibited the transcription of T3SS1 and Vp-PAI.