Transcriptional Regulation Of Target Genes Of Biofilm By The Master Regulator AphA In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a gram-negative halophilous pathogenic bacterium which causes acute gastroenteritis in humans when people eat raw or undercooked seafood. V. parahaemolyticus is widely found in brackish saltwater. V. Parahaemolyticus can adherent to a surface to form biofilm in life cycle which is the one key factor for environmental survival and transmission. Biofilm formation regulated by Quorum sensing (QS) systems and c-di-GMP synthesis. Vibrio parahaemolyticus AphA and OpaR are the two master quorum sensing (QS) regulators that are abundantly expressed at low cell density (LCD) and high cell density (HCD), respectively, with a feature of reciprocally gradient production of them with transition between LCD and HCD regulated by the Qrr sRNAs.The phenotypic experiments disclosed that AphA is an activator of c-di-GMP synthesis and biofilm formation in V. parahaemolyticus but the molecule mechanism is still poorly understood in V. parahaemolyticus.Objective:Using the phenotypic and molecular biochemical experiments to study the regulation mechanism of biofilm formation by AphA in Vibrio parahaemolyticus.Methods:Colony morphology and crystal violet staining assays were carried out to analyze the phenotypic changes between the aphA null mutant (AaphA) and the wide-type (WT) strains. The intracellular levels of c-di-GMP in the AaphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method. The base sequence of N-terminal domain facing the cytoplasm of AphA was amplified by PCR from V. parahaemolyticus strain RIMD2210633, and then cloned into the BamHI and Hind III sites of the vector pET-28a-c(+). The recombinant plasmid pET-28a-c(+) was transformed into BL21(λDE3). Over-expression of His-AphA in the LB medium was induced by addition of lmM IPTG (isopropyl-b-D-thiogalactoside). The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Then the electrophoretic mobility shift assay (EMSA) was carried out to analyze the DNA-binding activity of His-AphA to the promoter-proximal DNA regions of target genes. The promoter-proximal regions of these target genes were cloned into the pHRP309containing a promoterless lacZ gene, respectively. Then, these recombinant LacZ reporter plasmids were transformed into the wide-type strain (WT) and the aphA null mutant strain(△aphA), respectively, to measure the promoter activity (the P-Galactosidase activity) of the target genes in WT and△aphA by using the β-Galactosidase Enzyme Assay System. Total RNAs were extracted from the△aphA and WT. And then, quantitative RT-PCR was applied to calculate the transcriptional variation of scrABC and scrG between the△aphA and WT.Results:The phenotypic experiments disclosed that AphA is an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus. We successfully expressed His-AphA recombinant proteins which had the ability to bind to the upstream DNA regions of target genes. As showed by EMSA, the His-AphA was able to bind to the promoter regions of VP0376,VP0485and VP1469. But it can not bind to the promoter regions of PA1513(scrABC),VP1377(scrG),VP0117,VPA0198,VP1403and VP1476. The LacZ fusion results showed that the transcription of VP1469and VP0485were positive regulated by AphA, but VPA1513,VP1377and VP0376were negative regulated by AphA in V. Parahaemolyticus. The quantitative RT-PCR and LacZ fusion results showed that the transcription of scrABC and scrG are under negative control of AphA.Conclusion:The recombinant His-AphA protein could be used for studying the transcriptional regulation mechanism in V. parahaemolyticus. One of the accumulation mechanisms of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus was that AphA represses the transcription of scrABC, scrG notwithstanding indirectly and directly represses the transcriptional expression of VP0376. AphA can directly actives the expression of VP0485,VP1469to regulate biofilm formation, and thus the expression of the biofilm formation and exopolysaccharides genes occurs which promotes the bacterial colonization and infection.