Functional Definition Of LuxS, An Autoinducer-2(AI-2) Synthase And Its Role In Full Virulence Of Streptococcus Suis Serotype2

Streptococcus suis serotype2(SS2) is a major swine pathogen for causing a widevariety of diseases in pigs including meningitis, septicemia and endocarditic andresponsible for important economic losses to the swine industry worldwide. It can alsoinfect human accidently through skin lesions, respiratory or the oral route to causemeningitis and septicemia etc. The human cases often occurred sporadically and have agood prognosis.Notably two major emerging infectious disease outbreaks of SS2occurredin China (one in Jiangsu Province,1998, and the other in Sichuan Province,2005)characterized by the prevalence of streptococcal toxic shock syndrome (STSS) manifestingitself as acute high fever, multiple organ failures, short course of disease and highlethality.(62.7%~81.3%).Our research group explore a detailed and complete study on theevents and firstly reported the new clinical symptom caused by SS2infection-streptococcaltoxic shock syndrome which indicated virulence variation occurred in the epidemic strains.Our published article received extensive attentions in the academic community home andabroad. Comparative genomics analyses have suggested that virulent Chinese strains of SS2feature a specific,89-kb-long DNA fragment showing preliminary evidence that89-kb-longDNA may function as a pathogenicity island. Our further studies revealed a uniquetwo-component signal transduction system (TCSTS) which is orthologous to the SalK/SalRregulatory system of Streptococcus salivarius locating in the candidate89K PAI is apositive molecular switch on virulence controlling. Furthermore, a GI type IV secretionsystem (T4SS) encoded in89K was verified to involve in not only the pathogenesis ofSTSS, but also in the excision, duplication and transfer of89K. Then we concluded that theepidemic strain obtain the PAI89K through horizontal transfer mediated by a GI-type T4SSand turn to be virulent strain.Since the recent recognition of the revalence of S. suis humandisease with high virulence and mortality in southeast and east Asia, the interest of the scientific community in the pathogenesis of this pathogen has significantly increased.Intensified research efforts have been made to explore the potential contribution of differentdescribed S. suis virulence factors at each step of the pathogenesis of the infection,including the process of SS2colonizing the host, breaching epithelial barriers, reaching andsurviving in the bloodstream, invading different organs, and causing exaggeratedinflammation. Amounts of classical virulence factors and virulence factor candidates havebeen described such as Cps, Suilysin, Muramidase-released protein, Extracellular proteinfactor, Dipeptidyl peptidase IV etc. Some new virulence-associated regulon includingtwo-component signal transduction system (TCSTS) SalK-SalR and CiaRH, an orphanregulator, CovR, Rgg-like regulators etc. have been identified by our group recently tointerpret their roles in genome transcription regulation and virulence of SS2.Quorum sensing is a widespread chemical communication system in response tofluctuation of bacterial population density and has been regarded as a transcription regutorysystem associated with bacterial biofilm formation and virulence etc. AI-2mediatedquorum sensing is one of the important QS in bacteria and has been known to exist in boththe Gram positive and Gram negative bacteria extensively. LuxS, an autoinducer-2(AI-2)synthase have been reported to have homologue in more than80bacteria species with highconservation, is determined to catalyze the last committed step of AI-2biosyntheticpathway, producing AI-2with similar structure which can be recognized by differentbacteria and acts as universal language among intraspecies and interspecies communication.In addition, LuxS is also an important RH cleavage enzyme in activated methyl cycle andacts an important role in metabolic balance. After decades of efforts, LuxS/AI-2systemhave been confirmed to play multiple/varied roles in different bacterial species, such asrelated with growth, biofilm formation, virulence, antibiotics susceptibility and motility.Besides, LuxS/AI-2seemed to affect global transcription response in some microorganism.The pleiotropic roles of luxS and AI-2function in S suis2remained unclear. Here weidentified a functional member of the LuxS protein family from a Chinese SS2isolate,05ZYH33and tried to explore if it would be part of the regulatory network in SS2thatsenses changing surroundings and responds to changed environment for a better survival.The following experiments are conducted and results obtained:1. Bioinformatics analysis, Cloning and prokaryotic expression of the luxS gene in 05ZYH33:A luxS ortholog encoding160aa of polypeptide (05SSU0420) is found to locate on thereverse strand of S. suis05ZYH33. This LuxS homologue exhibits highly similarity tothose known LuxS proteins from other Streptococcus species (>80%aa identity). Not onlyare three highly conserved sites critical for zinc binding (His58, His61&Cys127) observedclearly in this suspected LuxS from05ZYH33, but also a recently-reported amino acid(Gly82) is present that was reported to be required for AI-2production. Structural modelingshowed that LuxS homologue adopts similar folding mode of tetranary structure, i.e., fourβ-sheets plus three α-helixs, indicating its possibility of being a functional member of LuxSfamily protein. In addition, bacterial genome annotation-based analyses showed that thisputative luxS gene is opposite to its two neighboring genes, suggesting that it might betranscribed in an independent operon. In addition, we prokaryoticly expressed genesegment including the luxS OFR plus its upstream putative promoter and downstreamsequence using the pET-32a system. SDS-PAGE results showed that the proteinfunctionally expressed in E.coli BL21with the expected molecular size.2. Knockout of luxS and functional complementation:The luxS gene in strain05ZYH33(WT) was inactivated by allelic replacement with aspectinomycin resistance (SpcR) cassette. The applified upstream and downstream DNAfragments adjacent to luxS (~1kb) were cloned into pUC18vector (Takara), and then theintermediate vector was inserted with the spcRgene, giving the luxS knockout vectordesignated pUC::luxS. The knockout plasmid was electroporated into S. suis competentcells. The expected mutant in which a double-crossover event has been undergone wasconfirmed by series of approaches including clony PCR, Southern blot, RT-PCR as well asdirect DNA sequencing.Reverse transcription-PCR (RT-PCR) detection was carried out toconfirm the successful deletion of the luxS gene in the mutant, designated ΔluxS. Forfunctional complementation, the DNA fragment covering the luxS coding region plus itsputative upstream promoter and downstream sequence was amplified from thechromosomal DNA of05ZYH33and cloned into an E. coli-S. suis shuttle vector, pVA838yielding plasmid pVA838::luxS. The resulting plasmid was introduced into the ΔluxSmutant to make the complementary strain (C-ΔluxS).3. The effects of luxS deletion on the phenotypes and virulence of05ZYH33: Different phenotypic properties of the wild type strain05ZYH33and the ΔluxS mutantwere compared under the same conditions. Firstly, Gram staining analyses showed that theΔluxS mutant tends to aggregate into clusters without apparent formation of chains, andexhibit abnormal morphology relative to the wild type05ZYH33. Transmission electronmicroscopy-based observation revealed that the capsule of ΔluxS is significantly thinnerthan that of its parental strain. These phenotypes can be restored in part by functionalcomplementation (C-ΔluxS). Secondly, we noted growth defect in the ΔluxS mutant of S.suis exhibiting the lagged logarithmic phase relative to the wild type. Thirdly, we foundthat AI-2activity was apparently present in strain05ZYH33while in ΔluxS mutant it ismuch lower than that of wild type. Fourthly, Cellular adhesion assays using two differentcell lines (Hep-2and HUVEC cells) demonstrated that the deletion of luxS significantlyweakened the capability of S. suis adherence on its host cells. In addition, the disruption ofluxS gene from S. suis seemed to improve slightly its resistance to H2O2challenge (18~36mM). Phenotype alteration above except AI-2activity cannot be restored by the addition ofDPD, a precursor for AI-2production, indicating that these phenotypes are related to someother unknown function of LuxS rather than its putative role in quorum sensing ofStreptococcus suis. We also noticed the decreased concentration of sialic acid. Notably, weobserved that ΔluxS had significantly lower lethality than the WT strain in an experimentalpiglet infection model. Pathological examination of the two groups of sacrificed pigletsrevealed that many organs suffered less damage in the mutant-infected group.Co-colonization experiment results showed that the ΔluxS mutant infected the specifictissues less effectively than the wild type strain. Therefore, we can conclude that loss ofluxS in SS2attenuates its pathogenicity in the piglet infection model. In addition, all thesephenotype alterations of SS2were restored after complementation.4. Analysis by microarray on the altered global gene transcription profile ofΔluxS and AI-2regulated genes:By Agilent DNA microarray, we compared the differentially expressed genes betweenΔluxS strain and the WT strain grown to middle-exponential phase and verified the thereliability of microarray data by qPCR assays. Totally,14.5%of all the putative genesencoded in SS2genome are affected by the luxS mutation, of which144genes areup-regulated, and168are down-regulated. These genes were categorized into the following various functions: metabolism, transcription regulators, virulence-related factors etc. Wefocus on the following genes potentially involving in the phenotpe alterations.(i) Someknown virulence-related determinants: CPS biosynthesis locus, sialic acid synthase; MRP;EPF are less abundant in luxS mutant, which is somewhat consistent with the attenuation ofvirulence by luxS mutation.(ii) Three transcription factors down-regulated in the ΔluxSmutant, which have ever been determined to be related with pathogenicity of S. suis andother pathogens including RevS orphan response regulator,a homolog of PadR regulator ofphenolic acid metabolism and may be regulated by quorum sensing plus a catabolite controlprotein A (CcpA);(iii)A conserved cell division related FtsA homologue and a cellshape-determining protein (MreC) both were downregulated by luxS deletion, whichvalidates the morphological changes observed with the ΔluxS mutant;(iv) A collection ofheat shock proteins (HSP)-encoding genes are elevated significantly due to luxS disruption,such as Hsp33, ClpL,and ClpE, which may agree with the increased oxidative tolerance andattenuated virulence of the luxS mutant strain.We also observed that71genes in the ΔluxSmutant are differentially expressed in response to the addition of DPD, among them29genes are thought to be regulated by AI-2associated QS.To sum up, inactivation of luxS gene led to a wide range of phenotypic changesincluding thinner capsular walls, increased tolerance to H2O2, reduced adherence capacityto epithelial cells and attenuated virulence in experimental model of piglets, functionalcomplementation restored these phenotypes nearly to the level of parent strain.Genome-wide transcriptome analyses suggested the global effects of luxS on thetranscription profiles. Simultaneously,29of71genes with differentially expression levelare proposed to be targets candidate regulated by LuxS/AI-2-dependent quorum sensing.Wethen draw a conclusion that LuxS/AI-2plays an important role in both the metabolic andQS function in SS2.