Streptococcus Suis Serotype2:Screening Virulence Related Factors And Identification And Functional Analysis Of The Cell Wall Hydrolase

Streptococcus suis serotype2(SS2) is an important swine and human pathogen responsible for a diverse group of diseases including meningitis,arthritis, and septicemia.It is also recognized as the zoonotic pathogen for human who especially exposed to swine byproducts.Based on capsular antigen,33serotypes have been described,and they are designated from1to31,33and1/2.Among these33serotypes,SS2is considered the most virulent and prevalent type in diseased pigs. However, the molecular pathogenesis of this pathogen is still poorly understood.Recently, SS2has been reported to become increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. In this study,we constructed the transposon mutant library of HA9801,screened the virulence related factors through zebrafish; characterized and functional analyzed the major autolysin Atl in Streptococcus suis,identified protein LytA that performs bactericidal activity by comparative genomics and zymography, BALB/c mice was used to evaluate the anti-infection against SS2.1. Construction of SS2transposon mutant library and screening the virulence related factors through zebrafish.The objectives of this study were to construct transposon pSET4S-miniTn4001for SS2and to identify genes required for infection in zebrafish. Screening500mutants individually through zebrafish infection model,allowing identification of4attenuated strains, which were selected for the analyzation of the insertion sites.The disrupted ORF was identified by plasmid rescue and each identified ORF was deleted by homologous recombination Four genes were identified, including putative glycosyltransferase (Cps2F), transcriptional regulator(tr66), TetR family regulatory protein (TetP), conserved hypothetical protein(chp). Cps2F is involved in capsule biosynthesis, tr66is a regulator which is associated with the sugar metabolism, TetP could regulate the transcription and the virulence expression and chp is a conserved hypothetical gene. Identification of such candidate genes may lead to a better understanding of the pathogenesis of SS2and provide substrate for the discovery of new vaccines.2. Characterizaiton and functional analysis of the major autolysin Atl in Streptococcus suisA novel gene, designated atl and encoding a major autolysin of S. suis2virulent strain HA9801, was identified and characterized in this study. The Atl protein contains1,025amino acids with a predicted molecular mass of113kDa and has a conserved N-acetylmuramoyl-L-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801decreased to20%of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced30%compared to the parental strain. In the zebrafish infection model, the50%lethal dose of the mutant strain was increased up to5-fold. Furthermore, the adherence to HEp-2cells of the atl mutant was50%less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.3. Identification of cell wall hydrolase LytA in Streptococcus suis and characterization its bactericidal activityWhen compared with SS2virulent strain HA9801, there are more cell wall hydrolase in SDS extracts of T15, which is the SS2avirulent strain.The molecular weight25kDa lytic band showed siginificant cell wall lytic activity comparedd with other lytic bands.We sequenced genome of T15,then compared with genome of SS2virulent strain to screen the ORFs that exist in T15while absent in HA9801.The ORFs were selected to perform the Blastn program.One720bps ORF which showed some similarity to the phage-associated cell wall hydrolase in streptococcus pyogenes was chosen for the next analyzation and designated as LytA.Zymography confirmed the cell wall lytic activity of the recombinant LytA and the mutant of LytA in T15resulted in the disappearance of the25kDa lytic band. Furthermore,the hyperimmune serum against LytA can react with the25kDa lytic band while there is no signal in the LytA mutant strain.When tested the bactericidal activity in vitro, the recombinant LytA can reduce sharply the OD600value of HA9801suspension from1.0to0.003and the SS2counts decreased from5.0×108cfu/ml to4.1×106cfu/ml, besides, the plat lyse assay also showed the significant lyse area.All these data indicated that LytA is the gene which encodes the25kDa lytic protein of T15in zymography and shows significant bactericidal activity.The bactericidal characteristics of LytA makes it the potential powerful antimicrobials in the SS2therapy.4. Cell wall hydrolase LytA as a Novel Antimicrobial for Streptococcus BacteremiaWe reported the use of LytA, cell wall hydrolase of SS2, as an intravenous therapy for SS2bacteremia in BALB/c mice. A2,000μg dose of LytA reduced SS2titers from a median of Log107.48to6.07within15min. This dose given1h after intravenous infection led to90%survival, compared to the100%lethal of buffer-treated controls. In advanced bacteremia, treatment with two doses at5and10h still resulted in50%survival.The protein LytA is immunogenic, but the treatment efficacy was not significantly reduced after previous intravenous exposure of mice (p>0.05) and hyperimmune rabbit serum did not neutralize the bactericidal activity. In vitro, the enzyme is active against many serotypes of S. suis and especially effective for SS2. Cell wall hydrolase LytA is unusual but extremely effective antimicrobials and represents a new weapon against infections with SS2.