Function Study Of The Virlence Genes AcfA And ToxR In Vibrio Alginolyticus

In recent years, more and more evidences showed transcriptional regulator toxR whichwas widely distributed in Vibrio, such as Vibrio cholerae, V. alginolyticus, V.parahaemolyticus,V. mimicus,V. anguillarumand V. vulnificus. The transcriptionalregulatory proteins ToxR encoded by toxR genecould regulate the expression of somevirulence genes and the outer membrane protein in V. cholerae.The acfs (accessorycolonization factors) are the important virulence genes in Vibrio, and the expressionproducts of acfs gene are required for efficient intestinal colonization by Vibrio in somemodel system. The acfs genes cluster are physically linked to toxinco-regulated pilus andare under the regulatory cascade that directs the synthesis of cholera toxin and otherproteins required for colonization.In this study, Based on the sequences of the highly conserved DNA area of the relatedvirulence genes toxR and acfA among vibrios, primers for PCR were designed.The toxRand acfA from V. alginolyticus strain HY9901were cloned by PCR. The results showedthat the complete ORF length of the toxR gene was879bp which encoded a protein with292amino acid residues.The predicted molecular weight (MW) of ToxR was32.9kDawith an estimated pI value of4.68.BLAST analysis showed that the ToxR in V.alginolyticus had a18aa potential transmembrane segments at the site179-196, but signalpeptide couldn t be found. Seven active sites of amino acid residues were found in theToxR, and they could be very important to maintain ToxR three-dimensional structure andfunction. The complete sequence of acfA gene was743bp and the complete ORF lengthwas648bp which encoded a protein with215amino acid residues. A classic-35region,-10region and Shine-Dalgarno sequence couldn t be recognized in upstream in the acfA gene,and the18aa signal peptide could be found in the deduced amino acid sequence of the acfAgene.The predicted molecular weight (MW) of AcfA was22.2kDa with an estimated pIvalue of4.53.The deduced AcfA had5active sitesof amino acid residues.We used the insertion mutation method with the pRE112suicide plasmid as the carrier,and constructed successfully ΔtoxR, Δ acfA mutant strains of V. alginolyticus.Then we usedthe Overlap PCR and homologous recombination technology to construct the contraststrains. Furthermore, we investigated the changes of the physiologyand pathogenicity ofΔtoxR, ΔacfA mutant strains compared with the strain HY9901, and used two-dimensional electrophoresis technologyof proteomics to search the protein expression differences.Theresults showed the ΔtoxR mutant didn t affect the growth level and the flagella synthesis,but could affect the biofilm synthesis and the production of ECPase.The ΔacfA mutantcould promote growth of the late V. alginolyticus, significantly inhibit biofilm synthesis,flagella synthesis and affect the production of ECPase. The LD50results showed the ΔtoxRor ΔacfA mutant could affect the virulence of V. alginolyticus, but there aren t thesignificant statistical difference between the strain HY9901and the mutant strains. The2D-DIGE results showed the ΔtoxR or ΔacfA mutant could affect the expression of someenergy metabolism proteins, transportersand structural proteins, but couldn t significantlyaffect the related virulence factors of V. alginolyticus.The present study can provide aplat form of the valid insertion technology for thestudy of the other virulence genes from V. alginolyticus genome and establish the base tofurther study the pathogenic mechanism of V. alginolyticus.