Spr1875, a Pneumococcal Antigen Figure 4. Protection induced by immunization with recombi- nant R4-GST or by the whole recombinant Spr1875 protein fused to a biotinylated tag (Spr1875-BIO). (A) Groups of CD1 mice were immunized with R4-GST or with GST, used as a negative control, and challenged with the D39 strain (16105 CFU). Results represent cumulative data from 3 experiments; n = 28, total number of animal per each group. (B) Groups of CD1 mice were immunized with Spr1875-BIO or with CAT-BIO, used as a negative control, and challenged with the D39 strain (16105 CFU). Results represent data from one experiment involving 14 animals per group. *, statistically different (p,0.05) from mice challenged with D39 strain, as assessed by Kaplan-Meier estimator of survival. doi:10.1371/journal.pone.0036588.g004 absence of inhibitors. Figure 5 shows that up to 87% of the reactivity of such sera was inhibited by R5-GST, while only 25% was inhibited at saturation by R4-GST. The latter fragment, however, completely inhibited reactivity of anti-Spr1875 antibod- ies in sera from R4-GST-immunized animals (data not shown). These data indicate that the large majority of serum antibodies from animals immunized with the whole Spr1875 protein was directed against the R5, and not the R4, portion of the molecule. Protection induced by other Spr1875 fragments We next hypothesized that antibodies directed against the R5 portion of the molecule were non-protective. To test this hypothesis, animals were immunized with R5-GST, R4-GST or with GST and challenged with the D39 strain as described above. Indeed, in contrast with the results obtained with R4-GST, R5- GST was totally unable to confer immunoprotection (Fig. 6A). Collectively, these data indicated that immunization with the whole Spr1875 protein induced antibodies predominantly directed against immunodominant epitopes located in R5, a portion of the molecule that does not contain protective epitopes. Since these data indicated that Spr1875 has immunogenic properties that may radically differ from those observed by using its fragments, we recombinantly expressed three fragments of Figure 5. The antibody response to recombinant Spr1875 is predominantly directed against epitopes located outside of the R4 fragment. Shown are inhibition ELISA experiments measuring the reactivity against Spr1875-GST of a serum pool (with an antibody titer of 1 16,000) from animals immunized with anti-Spr1875-BIO in the presence of inhibitors. The recombinant Spr1875 fragments R4-GST and R5-GST (see upper diagram) were used as inhibitors. Results are from one experiment representative of three. doi:10.1371/journal.pone.0036588.g005 approximately equal length covering the whole Spr1875 protein. We designated these fragments as R1-GST, R2-GST and R3- GST (Fig. 6, upper panel). The complete sequences of the fragments are provided in Table S3 in the supplemental material. As can be seen in Figure 6B, immunization with R2-GST, but not R1-GST or R3-GST, was protective, and the degree of protection was similar to that of R4-GST immunization. Thus, R4 and R2, but not other portions of the Spr1875 molecule, appeared to contain protective epitopes. Discussion The present study describes the identification and character- ization of a novel protein antigen, Spr1875, and of recombinant fragments encompassing the length of this protein. Spr1875 was found to elicit antibodies in the course of human pneumococcal infection, since it was identified by the ability of its R4 fragment to bind antibodies in a convalescent serum. Moreover, further data indicated that the R4 fragment is recognized by a high proportion of sera from patients recovering from pneumococcal disease. Little is presently know on the biological function of Spr1875. The spr1875 gene was previously found to be strongly upregulated by the VicR component of the VicRK two-component regulatory system together with three other genes encoding one known virulence factors (pspA) and putative membrane (spr0709) and cell wall (spr0096) proteins [9]. Interestingly, Spr0096 and Spr1875 are the only two proteins in S. pneumoniae to contain LysM peptidoglycan-binding motifs. Several LysM proteins are known to be virulence factors and/or protective antigens of human bacterial pathogens and some function as adhesins [10]. For example, Staphylococcus aureus produces five LysM proteins, which are all involved in virulence [10,11]. Similar to Spr1875, protein PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36588