Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection In Vivo

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

肺炎链球菌（Streptococcus pneumoniae）是引发细菌性肺炎、败血症与脑膜炎的主要致病菌。为克服现有多糖基肺炎球菌疫苗的局限性，研究人员正探索以该菌表面可及蛋白为靶点开发蛋白亚单位疫苗。为筛选潜在疫苗候选靶点，本研究采用二维凝胶电泳（two-dimensional gel electrophoresis）分离肺炎链球菌TIGR4菌株的表面相关蛋白，随后以针对全热灭活TIGR4菌株制备的抗血清进行免疫印迹（immunoblotting）分析。经质谱分析（mass spectrometric analysis）鉴定，共获得10个免疫反应阳性斑点，其中包含推定脂蛋白SP0845。对36株肺炎球菌的sp0845同源物的推导氨基酸序列进行分析后发现，SP0845具有高度保守性（同源性＞98%），且与人类任何蛋白的同源性均低于11%。本研究的生物信息学与功能分析结果表明，SP0845是ATP结合盒转运体（ATP-binding cassette transporter，ABC）的底物结合蛋白，该转运体参与核苷摄取过程，优选底物为胞苷、尿苷、鸟苷与肌苷。编码SP0845的基因缺失可使肺炎球菌丧失致病力，提示该蛋白对致病菌的毒力至关重要。免疫印迹分析显示，SP0845可在体外培养的肺炎球菌及小鼠感染模型中表达。免疫荧光显微镜（immunofluorescence microscopy）与流式细胞术（flow cytometry）检测结果表明，SP0845在荚膜菌株中可定位于菌体表面，且可与抗体结合。小鼠经皮下免疫重组SP0845后可产生高滴度抗体；以SP0845制备的高免血清可在血液杀菌试验（blood bactericidal assay）中促进荚膜肺炎球菌菌株的杀伤。经SP0845免疫的小鼠可抵御异源肺炎球菌血清型的腹腔攻毒（intraperitoneal challenge）。综上，结合SP0845的表面可及性、毒力相关功能及诱导保护性免疫的能力，本研究提出SP0845可作为蛋白基肺炎球菌疫苗的潜在候选靶点。