Saxena Sneha, Khan Naeem, Dehinwal Ruchika, Kumar Ajay, Sehgal Devinder
Molecular Immunology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
PLoS One. 2015 Feb 17;10(2):e0118154. doi: 10.1371/journal.pone.0118154. eCollection 2015.
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.
肺炎链球菌是细菌性肺炎、败血症和脑膜炎的主要病因。为克服现有基于多糖的肺炎球菌疫苗的局限性，人们正在探索肺炎链球菌的表面可及蛋白以开发基于蛋白质的疫苗。为鉴定潜在的疫苗候选物，我们使用二维凝胶电泳解析了肺炎链球菌TIGR4菌株的表面相关蛋白，随后用针对全热灭活TIGR4产生的抗血清进行免疫印迹。通过质谱分析鉴定出10个免疫反应性斑点，其中包括一种假定的脂蛋白SP0845。对来自36株肺炎球菌菌株的sp0845同源物的推断氨基酸序列分析表明，SP0845高度保守（>98%同一性），与任何人类蛋白的同一性均低于11%。我们的生物信息学和功能分析表明，SP0845是一种ATP结合盒（ABC）转运蛋白的底物结合蛋白，该转运蛋白参与核苷摄取，以胞苷、尿苷、鸟苷和肌苷作为优选底物。编码SP0845的基因缺失使肺炎球菌无毒，表明它对毒力至关重要。免疫印迹分析表明，SP0845在体外培养的肺炎球菌中以及小鼠感染期间均有表达。免疫荧光显微镜和流式细胞术数据表明，SP0845在包膜菌株中暴露于表面且可被抗体识别。用重组SP0845进行皮下免疫可在小鼠体内诱导产生高滴度抗体。针对SP0845产生的超免疫血清在血液杀菌试验中促进了对包膜肺炎球菌菌株的杀伤。用SP0845免疫可保护小鼠免受异源肺炎球菌血清型的腹腔攻击。基于其表面可及性、在毒力中的作用以及引发保护性免疫的能力，我们认为SP0845可能是基于蛋白质的肺炎球菌疫苗的潜在候选物。
