Figure 2. Surface expression of Spr1875 and spr1875 gene polymorphism in different pneumococcal strains. (A) Immuno- fluorescence flow cytometry analysis of different S. pneumoniae strains using anti-R4-GST mouse serum (black lines). Anti-GST (grey peaks) and anti-CCR6 (grey lines) murine sera were used as negative and positive controls, respectively. D-D39, D-TIGR4, D-23F-Spain-1 and D-19F- Taiwan-14 are unencapsulated mutants of D39, TIGR4, 23F-Spain-1 and 19F-Taiwan-14, respectively. (B). Schematic representation of spr1875 deduced amino acid sequence variability within S. pneumoniae. Twenty-seven strains were used for gene variability analysis. Abscissa, amino acid position; ordinate, degree of polymorphism. The baseline represents the sequence of the P1031 S. pneumoniae strain, which was the longest sequence. The height of the line indicates the total number of different amino acids found at that particular position. doi:10.1371/journal.pone.0036588.g002 increased survival time relative to either D39- or DpspA-infected mice. In each experiment, pneumococci were confirmed as the cause of death by organ colony counts in moribund animals. These data show that the spr1875-deficient strain is attenuated in virulence, suggesting that the Spr1875 protein may play a role in pneumococcal sepsis. Protective activity of the R4 peptide fragment To assess whether immunization with the recombinant R4 fragment had protection-eliciting activity against pneumococcal infection, groups of mice were immunized with 50 mg of the R4- GST fusion protein. After three administrations, all mice had R4- specific serum antibody titers ranging from 1 8,000 to 1 64,000 (data not shown). R4-GST-immunized mice were challenged with an approximately 90% lethal dose (16105 CFU) of the D39 strain. Lethality was observed for 14 days and compared with that observed in mice immunized with the GST tag only. Cumulative data from three experiments indicated that immunization with the R4-GST fragment resulted in 57% (16 mice out of 28) survival, while only 18% (5 mice out of 28) of the GST-immunized animals survived (p,0.05; Fig. 4A). Further data indicated that the protection induced by immunization with R4-GST was antibody- mediated, since it could be transferred to unimmunized animals by pooled sera from animals immunized with R4-GST, but not from those immunized with GST alone (Fig. S2). We next recombinantly produced the whole Spr1875 protein fused to a biotin (Spr1875-BIO) or to a GST (Spr1875-GST) tag Spr1875, a Pneumococcal Antigen Figure 3. Lethality induced in mice by a D39 mutant (Dspr1875) lacking Spr1875. Wild type D39 and a mutant lacking PspA (DpspA) were used as controls. CD1 mice were challenged intravenously with 36104 (A), 76104 (B) or 76106 (C) CFU. Results are from one experiment per challenge dose involving 10 mice per group. *, statistically different from D39 strain, as assessed by Kaplan-Meier estimator of survival. doi:10.1371/journal.pone.0036588.g003 and used them for immunizing animals as in the experiments described above. Surprisingly, neither Spr1875-BIO nor Spr1875- GST immunization resulted in significant protection when compared with immunization with the respective negative control protein (Fig. 4B and Fig. S3). Based on these data we hypothesized the induction of a different antibody response to the whole Spr1875 protein as compared to immunization with the R4 fragment. To gain insights into the portion of the Spr1875 protein against which serum antibodies were directed, we expressed a recombinant fragment, designated R5, fused to GST (R5-GST). This fragment encompassed the whole length of the Spr1875 protein excluding the R4 fragment (Fig. 5, upper panel). Next, we conducted inhibition experiments in which R4-GST and R5-GST were used to inhibit the reactivity of anti-Spr1875 serum antibodies from Spr1875-BIO-immunized animals. In these ELISA experiments, Spr1875-GST was used as a coating antigen and antibody titers were measured in the presence and in the PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36588