Study On The Quorum Sensing Of LuxS/AI-2 Of Streptococcus Suis Serotype2

Streptococcus suis is an important pathogen associated with a wide range of diseases in pigs, including meningitis, septicaemia, pneumonia, endocarditis, and arthritis. On the basis of the capsular polysaccharides,35 serotypes; have been identified (types 1-34 and 1/2). Serotype 2 is the most prevalent type in assoc ation with diseases in most countries.lt is also an important zoonotic agent for humans in contact with diseased pigs or their products, causing life threatening diseases.Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules,called autoinducers (AI).The only QS system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). It has been proposed that AI-2 is a universal signaling molecule that functions in interspecies cell-to-cell communication. Recent reports have shown that,depending on the bacterium, AI-2 plays a role in the regulation of virulence-related genes,pathogenicity and biofilm formation.The worldwide spread of antibiotic resistance among emerging and re-emerging bacterial pathogens has underlined the need to develop new approaches in control the diseases of SS2.In this context, the disruption of bacterial quorum-sensing, especially luxSIA1-2 QS system in SS2, will be the aim of control SS2 infectivity and pathogenicity. 1 Dectation of AI-2 of Streptococcus suis serotype 2The only quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). AI-2 has been reported to be a key player in the regulation of survival and virulence-related genes or processes.Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen. However,there have been no reports concerning the AI-2 in SS2. A homologue of luxS,the gene required for AI-2 synthesis in Vibrio harveyi (V.harveyi),was isolated from the SS2 genome. The amino acid identity to luxS in V. harveyi was 36% and>80% amino acid identity with various streptococcal luxS sequences. V.harveyi BB170 bioassay demonstrated luxS functionality in SS2 and its ability to complement the luxS-negative phenotype of Escherichia coli DH5a.Further studies showed that AI-2 activity peaked in the late exponential phase. Analysis of luxS/AI-2 QS will provid a solid basis for future research concerning the role of luxS/AI-2 QS in SS2.2 The expression,purification and preparation of multiantibody of LuxS and Pfs from Streptococcus suis Serotype 2LuxS and Pfs catalyze synthesis of the quorum-sensing signaling molecule autoinducer 2 (AI-2), which has been shown to control a variety of cellular processes.The expression and purification LuxS and Pfs from SS2 strain HA9801,two enzymes gave an apparent single protein band,the molecular mass of 21.74 and 28.44kDa on an SDS-PAGE, respectively.Two New Zealand White rabbits were immunized by subcutaneously injection with recombinant LuxS and Pfs proteins for preparation multiantibody.The results of western blotting showed that the LuxS and Pfs have immunogenicity.Moreover,44 strains SS2 have luxS and pfs,which demonstrating clearly that luxS and pfs are conserved in SS2.Such analyses will be the aim of future studies to elucidate the biosynthesis of AI-2 in SS2.3 Pathway of Biosynthesis Autoinducer-2 in vitro of Streptococcus suis Serotype 2Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules,called autoinducers. LuxS and Pfs catalyze the synthesis of the quorum-sensing signaling molecule autoinducer 2(AI-2),which has been shown to control a variety of cellular processes. Expressed and purified LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH),the reaction products were able to induce the luminescence of Vibrio, harveyi BB170, demonstrating clearly that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. The optimum pH and temperature for biosynthesis AI-2 in vitro were 8.0 and 37℃, respectively. The biosynthesis AI-2 in vitro was stimulated by Cr3+,A13+ and Ba2+ and was inhibited by Fe2+, Ni2+, respectively. It was strongly inhibited by Hg2+, Cu2+and Mn2+,while enzyme activity was not affected by Li+, Mg2+and Zn2+.In this study,we identified the pathway of AI-2 synthesis in SS2,and analyzed the impact factor of biosynthesis AI-2 in vitro, which provided a solid basis for future research concerning the importance of AI-2 in SS2.4 Analysis of profile of luxS and pfs transcription in Streptococcus suis serotype 2In this study,we investigated AI-2 production and analyzed the relationship between the transcription level of luxS and pfs and AI-2 production in SS2.The results showed that the maximum level of AI-2 occurred during the late exponential phase, which is consistent with the maximum transcription profile of pfs; however, the maximum transcription profile of luxS occurred in the stationary phase. Further studies showed that sodium chloride or glucose increased the production of AI-2, the results of real time PCR assays showed that NaCl, glucose and sucrose increased the amount of luxS-mRNA, while only NaCl and glucose increased the amount of pfs-mRNA; sucrose had no significant on the amount of pfs-mRNA.Furthermore, although glucose and sucrose increased luxS-mRNA by the same amount, sucrose did not raise the level of AI-2 or transcription of pfs-mRNA.The results presented here demonstrate that the level of transcription of pfs is tightly correlated to the level of AI-2 production, while the level of transcription of luxS does not correlate with the level of AI-2 production.5 Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2Phage display is a powerful technology that allows selection of short peptide ligands with high binding affinities to proteins of interest from a large pool of random peptide permutations. AI-2 is produced in bacteria that express the gene luxS.In the present study,expressed and purified LuxS from Streptococcus suis serotype 2 was used to catalyze the substrate SRH in a reaction that leads to the production of AI-2.The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay.Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display.One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the enzyme’s activity.Furthermore,14 peptides containing the consensus sequence HSIR showed high affinity with LuxS.The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets,thereby opening up new approaches in the development of therapeutic drugs.6 Effects of AI-2 on adherence and gene transcription of SS2AI-2 has been shown to control a variety of cellular processes.In this study, to investigate whether AI-2 affect the capability of SS2 to adhere HEp-2 epithelial cells,we tested the adherence of SS2 by adding different concentration AI-2. The results showed that the maximal adherence of SS2 is 144% at the concentration of 4 umol AI-2. While,it lost 42% of the adherence at the concentration of 16 umol AI-2.Further study by Real-time PCR showed that AI-2 increased the transcription of cps, mrp,sly, gdh,fbps,orf2,MEP,hsp and AbpB, while decreased the transcription of luxS, ef and DAP. These findings will be of benefit to future studies of the role of AI-2 in SS2.