Streptococcus Pneumoniae Gts, The Potd And Srta Protein Joint Immune Protection Of Their Infection And Streptococcus Pneumoniae Outer Membrane Protein Spd1741 And Spd0280 Preliminary Crystallographic Studies

Objective: Infections caused by Streptococcus pneumoniae are amajor cause of human diseases such as otitis media, sepsis, meningitis, andfatal pneumonia worldwide. Each year S.pneumoniae causes approximately1.6million deaths globally. Although antibiotics are effective at controllingsome cases of pneumococcal infections, their treatment does not preventmortality within the first48h of presentation. The effectiveness ofantibiotics is further constrained by the widespread occurrence of antibioticresistance in many pneumococcal strains. These factors have led toincreased emphasis on the prevention of pneumococcal infections byvaccination.Therefore, protein-based vaccines are considered to be thenext-generation of pneumococcal vaccines.In this study, we compared theabilities of Gts, PotD and SrtA to elicit protection against pneumococcalinfections and examined the possibility that an immunization of acombination of two or three these proteins could result in superiorprotection over any of the antigens alone using different pneumococcal strains.Methods: We got purified Gts, PotD and SrtA from prokaryoticexpression system. To study antigen-specific responses generated byintranasal immunization with recombinant antigens, both humoral and cellmediated responses were investigated.In order to evaluate protectionagainst death afforded by mucosal immunization with recombinant Gts,PotD and SrtA protein antigens, the invasive pneumococcal infectionmodel by intranasal inoculation of S.pneumoniae D39(1.5x107) wasestablished in mice, which mimic the natural route of pneumococcalinfection. To further evaluate the protection against different strains ofS.pneumoniae elicited by immunization with recombinant Gts, PotD, andSrtA proteins either singly or in combinations, groups of mice wereimmunized intraperitoneally with these protein antigens and challengedwith D39, CMCC31207(serotype6B), or CMCC31614(serotype14)after strong antibody titers being induced.To investigate that the protectionelicited by recombinant protein antigens may be antibody mediated,anti-Gts, anti-PotD and anti-SrtA sera were administered singly or incombinations, and mice were then intraperitoneally challenged by D39. Tofurther determine whether recombinant Gts, PotD and SrtA proteins or theirrespective anti-sera have a functional significance, we tested their ability tointerfere with adhesion of D39to A549cells.Furthermore,to detect naturalantibody responses against these three protein antigens in children or adults, 34sera obtained from healthy children or adults and24sera from pediatricpatients with acute pneumococcal pneumonia were analyzed for anti-Gts,anti-PotD and anti-SrtA IgG levels.Results: In the present study, we cloned and expressed three newpneumococcal protein antigens including Gts, PotD and SrtA. Westernblot analyses using polyclonal antibodies against these three antigensdemonstrated different pneumococcal strains could express Gts, PotD andSrtA without heterogeneity at protein level. Using human lung A549cellmodels, we demonstrated that all of three protein antigens could inhibitadhesion of pneumococcus to A549cells, and combination of two or threeantigens had an additive effect. Besides, anti-sera against these threeantigens had a functional significance, and each of them had the ability tointerfere with adhesion of S. pneumoniae to A549cells. Also, combinationof two or three anti-sera resulted in an additive effect. In mucosalimmunization studies, we found that intranasal delivery of recombinant Gts,PotD, or SrtA could induce mucosal and systemic immunity to S.pneumoniae, and there was a significant increase of Th1cytokine IFN-γ,Th2cytokine IL-4, Treg cytokine IL-10, or Th17cytokine IL-17A in miceintranasally immunized with Gts, PotD or SrtA. All these cytokines havebeen suggested to regulate protective immunity to pneumococcal infections.In the pneumococcal pneumonia model established with serotype19F, wedemonstrated that intranasal immunization with recombinant Gts could effectively reduce pneumococcal nasopharyngeal as well as lungcolonization, and recombinant PotD or SrtA was as effective as Gts inreducing pneumococcal colonization in the nasopharynx and lung. Besides,combinations of two or three protein antigens could significantly decreasepneumococcal nasopharyngeal colonization, while Gts plus SrtA, or Gtsplus PotD plus SrtA could enhance the protection against pneumococcallung colonization when compared single antigens. Furthermore, protectiveefficacy against death afforded by mucosal immunization with recombinantGts, PotD and SrtA protein antigens was also evaluated using a morevirulent D39isolate. The results of the intraperitoneal challengeexperiments indicate that nearly all combinations of the protein antigensprovided higher degrees of protection than any of single antigens alone,corroborating our results in mucosal immunization studies. A combinationof Gts and PotD consistently elicited enhanced protection against challenge,followed by a combination of Gts and SrtA and a combination of PotD andSrtA. However, a triple combination of Gts, PotD and SrtA could notfurther augment the protection when compared to the combinations of twoprotein antigens. passive immunization studies showed that combinationsof anti-Gts, anti-PotD, or anti-SrtA sera could also confer increasedprotection, indicating that protection effects elicited by activeimmunizations are at least antibody-mediated. Conclutions: In summary, this work reports that the formulation ofconserved and multicomponent pneumococcal protein antigens includingGts, PotD or SrtA could enhance the protection against differentpneumococcal strains in a mucosal or systemic delivery way. Since thesethree antigens are also immunogenic in humans, our findings support thedevelopment of them as a human pneumococcal vaccine. Objective:To obtain the crystals of outer-membrane proteinsSPD1741and SPD0280of streptococcus pneumoniae for X-ray crystalstructure and function analysis. Methods:The spd1741and spd0280genesof D39strains from Streptococcus pneumoniae was respectively clonedinto the prokaryotic expression vector pET32a(+), then overexpressionwas obtained in the E.coli BL21(DE3) through transformation of therecombinant plasmid that had been verified by colony PCR and sequencing.Soluble fusion protein with His-tag expressed highly by the induction ofIPTG and was purified by a three step procedures, which includedHis-Bind Column affinity chromatography (Ni-NTA), ion chromatography(DEAE), and gel filtration chromatography. Preliminary screening ofcrystallization conditions was performed using the hanging-dropvapour-diffusing method with Hampton crystal screen kit and PEG crystalscreen kit. The protein crystals X-ray diffraction data were collected from asingle crystal using a MAR CCD detector. Results:The purity of the purified SPD1741and SPD0280recombinant proteins are over90%andmore crystals whose X-ray diffraction respectively reached4.0and3.5were obtained. Conclusions:We successfully prepared SPD1741andSPD0280recombinant proteins with high purity and obtained crystals forX-ray diffraction. These work laid the foundation for the further researchon the3D structure of SPD1741and SPD0280proteins and their biologicalresearch.