Characterization Of The Hypothetical Protein Spd-0873 As A Novel Lysozyme And Virulence In Streptococcus Pneumoniae D39

Background the increasing problem of drug resistant Streptococcus pneumoniae, coupled with the limitation of vaccine coverage, is a grim problem confronting us. We should further understand its molecular pathogenic mechanism to provide new theory and experimental data for more effective drug and vaccine development.In our previous study, we have established a in vivo-induced gene (ivi) database selected by IVET (in vivo expression technology), which were related to the virulence of Streptococcus pneumoniae. After selected by bioinformatics analysis, we found that a gene, spd-0873, encoding a hypothetical protein SPD-0873,have a similar function domain with lytC in S.pn. And its mutant reduced the formation of the biofilm. So we suppose that SPD-0873 is a new Lysozyme-like virulence in Streptococcus pneumoniae.Objectives The purpose of this study was to confirm the lysozyme activity of the hypothetical protein SPD-0873 and investigate the effect of spd-0873 on the pathogenesis of Streptococcus pneumoniae.Methods The lysozyme activity of the SPD-0873 was identified with a substrate PNP-(GlcNAc)5. The difference on virulence between the mutant and wild strain was evaluated in a mouse intraperitoneal challenge model. Growth in C+Y media, capsule synthesis in vitro or in vivo, the adherence to A549 and HUVEC and the expression of some major virulences at mRNA level between the mutant and wild strain were investigated to the role spd-0873 in the pathogenesis of S.pn.Results SPD-0873 was predicted by bioinformatics to be aβ- 1,4-N-acetylmuramidase belong to glycosyl hydrolase family 25, which cleaves theβ-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the carbohydrate backbone of bacterial peptidoglycan. A classical lysozyme assay was used to identify activity of the SPD-0873 with a substrate PNP-(GlcNAc)5. Both Lysozyme and SPD-0873 could liberate p-nitrophenol from the substrate PNP-(GlcNAc)5 , and the absorbance of OD405nm were 1.166,0.792 respectively. The virulence of the mutant reduced sharply than the wild strain and the growth of mutant was slower than that of the wild strain. Compared with the wild strain had the capsule both in vivo and in vitro, there was capsule around the mutant in vivo, but nothing in vitro. In addition, the rate of adherence of mutant to the host cell was higher than the wild strain. The results of FQ-PCR displayed that the expression of pneumolysin (ply), neuraminidase (nanA) decreased in the mutant, whereas, pneumococcal surface adhesion A (psaA) expressed higher than in the wild strain.Conclusion These results indicate that hypothetical protein SPD-0873 was proved to be a new Lysozyme with low activity. And spd-0873 may be a new major virulence factor, which could effect the virulence by regulated some gene expression at mRNA level.