Study On A Novel Virulent Factor Streptococcus Pneumonia Hypothetical Protein Spd0414and The Protective Effect As A Candidate Protein Vaccine

Background As an opportunistic human pathogen, Streptococcuspneumonia is potential to cause serious invasive diseases. With the absurdantibiotics treatment, there are increasing drug resistant strains found in allover the world. However, the prevalent vaccines including23-valentcapsular polysaccharide and7-13-valent capsular polysaccharide conjugate,have more or less limitation, which result in a remaining imminent problemon Prevention and treatment of Sreptococcus pneumonia. So moreresearches of pathopoiesis mechanism and new virulent factors explorationare still necessary to develop more effective antibiotics or vaccines.Our previous study suggested that spd0414is one of the in vivo induciblegenes screened by IVET and DFI, further research indicated that its codedprotein SPD0414may be of great importance in the bacterial infection. Thecurrent study intends to investigate the mechanism underlying the influenceof spd0414in the pathopoiesis of S.pn and elucidate its subcellular location,besides the value of SPD0414used as a protein vaccine is also estimated inour experiment.Methods The pathogenic abilities were investigated by infecting Balb/c nasally or peritoneally with D39，203， D39Δ spd0414and203Δspd0414.The adhere capacity were compared between spd0414deficit strain andtheir wild type parent in vivo and in vitro，and experiments of purifiedSPD0414（27aa-245aa）competitive inhibition and anti-SPD0414serumblock were employed to elucidate whether the SPD0414is directlyinvolved in the adhesion of S.pn to A549. We also estimated the effect oflack of spd0414on the expression of other virulent proteins in S.pn.Thesubcellular location of SPD0414was analysed by Using two vector thatallowed C-and N-terminal GFP fusions for SPD0414to constructrecombinant proteins of SPD0414-GFP and GFP-SPD0414in R6.Finally，we evaluated the protection of SPD0414（27aa-245aa）as a protein vaccineagainst infectious S.pn of three different serotype.Results There was no significant difference in the mice of peritonealinfection no matter whether spd0414was deficient.Besides,D39Δspd0414and D39had a comparable virulence when mice were instilled nasally. Incontrast，nasal administration of203△spd0414showed a obviously lessability in pathopoiesis compared with treatment with203(p<0.05).Furtherdata in vivo suggested both203△spd0414and D39△spd0414had adecreased colony activity in nasopharynx and lung in contrast with that oftheir wild type parents（P<0.05）.This result were confirmed in vitro by the203and203△spd0414adhesion and invasion test towards A549and CNE（P<0.05）.Subsequencely,the disability of purified SPD0414（27aa-245aa） competitive inhibition and anti-SPD0414serum block reflected theSPD0414indirectly mediated the adhesion of S.pn to A549. The mRNAtest showed D39and203in deficiency of spd0414lead to an increasedexpression of Ply and LytA, and a less level in mRNA of PsaA. Unexpected,NanA was overexpressed in203△spd0414while down-regulated in D39△spd0414when compared with their wild type parents respectively. Thewhole cytoplasma displayed an obvious fluorescence illustrated SPD0414was a plasma protein. By mucosal immunization of SPD0414,50%、33.3%、41.7%mice infected nasally with D39（Serotype2）、CMCC31436（Serotype3）、CMCC31614（Serotype14）could survival while all themice dead in control group.Conclusions The lack of spd0414in203caused a less virulence whenadministrated nasally，while this influence was not found in D39. To someextent，SPD0414mediated indirectly the disable adhesion of D39and203on account of the regulation to expression of an important S.pn adherefactor PsaA. Meanwhile, the mRNA levels of LytA, Ply and NanA werealso changed due to the spd0414lost. Our results also suggested SPD0414located in cytoplsama, but it is still unclear whether it could be secretedoutside bacteria. Additionally, mucosal immunization with SPD0414couldprovide some protection to mice against multi-serotype S.pn including D39（Serotype2）、CMCC31436（Serotype3）、CMCC31614（Serotype14）,which indicated SPD0414may be a potential candidate protein for vaccine of S.pn.