The Structure And Function Study Of The Lysozyme-Like Putative Protein SP0987 From Streptococcus Pneumoniae

Streptococcus pneumoniae is a serious challenge for the prevention and therapy because of the increasing antibiotic resistance and the defects of the current vaccine. So further research should be done on the pathogenic mechanism of Streptococcus pneumoniae, which can supply the theory background of more effective antibiotic and vaccine development. In our previous study, we have found that a putative protein SP0987 from Streptococcus pneumoniae coded by an in vivo induction gene sp0987, may be a new virulence factor. Till now, the structure and function of SP0987 was unknown, so we decided to do further research on the protein.Objectives The aim of this study was to resole the structure of the protein SP0987 for the first time, to study the function based on the structure and the effect of the sp0987 gene on the pathogenesis of Streptococcus pneumoniae.Methods The structure of sp0987 was analysed by clone, expression, purification, protein crystal growth, diffraction data collection and protein structure resolution. Based on the structure, the enzyme activity and the active sites were analysed through the analysis of enzyme activity and site-directed mutagenesis. The cellular localization of SP0987 was analysed using novel fluorescent reporter system.The virulence study was carried out by the construction of the sp0987 mutant strain, cell adherence and invasion experiment in vitro, colonization experiment in vivo.Results The putative protein SP0987 , coded by the gene sp0987, 266 amino acid, was predicted by bioinformatics to be aβ-1,4-N-acetylmuramidase belonging to glycosylhydrolase family 25, which cleaved theβ-1,4-glycosidic bond between N-acetylmuramic acid(NAM) and N-acetylglucosamine(NAG) in the carbohydrate backbone of bacterial peptidoglycan. The structure of SP0987 was resolved successfully for the first time by clone, expression, purification, protein crystal growth, diffraction data collection and protein structure resolution. By sequence and structural comparison of SP0987 with other lysozymes, we found that SP0987 was a lysozyme-like protein, and the active sites were probably D33, D131, E133 and D222. The protein SP0987 was proved to be a monomer state in solution by size exclusion chromatography; A GFP fusion to SP0987 showed clear membrane localization, as predicted from its primary sequence. The sp0987 gene mutant stain was constructed by LFH-PCR, and the reduced virulence of the mutant suggested the SP0987 may be a virulence factor in Streptococcus pneumoniae. In vitro, the invasion rate of the mutant strain to the host cell was lower than the wild strain, while there was no difference between the mutant stain and the wild strain in adherence. In vivo, when mice were challenged intranazal, the wild strain appeared in blood earlier than the mutant strain, and the number of bacteria in the nasal and lung tissue of the mutant was much smaller than the wild strain.Conclusions The structure of the putative protein SP0987 from Streptococcus pneumoniae was resolved for the first time by X-ray crystallography. The protein SP0987 was a new lysozyme-like membrane protein, and was a monomer state in solution. The sp0987 gene mutant strain showed reduced virulence in the intranasal mouse model , and affected invasion and colonization of Streptococcus pneumoniae. So, the protein SP0987 is a new virulence factor of Streptococcus pneumoniae.