in virulence. Moreover immunization with the R4 fragment of Spr1875 conferred protection from intravenous challenge with virulent pneumococci. Results Spr1875 is expressed on the bacterial surface A lambda phage displayed library of the pneumococcal genome (strain R6) was previously used to identify several antigenic fragments based on their reactivity with human serum antibodies [6]. By this approach, in the present study, we identified a novel 161 amino acid-long fragment, herein referred to as R4, using serum antibodies from a patient convalescing from invasive pneumococcal disease. The sequence matched ORF spr1875 of the S. pneumoniae R6 strain genome (GenBank accession no. AE007317), encoding a 380 amino acid-long protein with an N-terminal peptidoglycan interaction lysine motif (LysM) domain, which is found in cell wall degrading enzymes and in virulence factors (Fig. 1). The predicted protein sequence of Spr1875 contains a leader peptide with a leader sequence and a cleavage site present in variety of streptococcal surface proteins. We next produced a recombinant R4-GST fusion protein and assessed its ability to bind to serum antibodies from patients recovering from pneumococcal infection. It was found that a high proportion of such serum samples, but not control samples, displayed high anti- R4 antibody titers (Table S1). To assess whether the Spr1875 protein is actually expressed on the bacterial surface, we used R4-GST to immunize mice. Mice were also immunized with recombinant GST and CCR6, a crude pneumococcal surface protein extract, to obtain negative and positive control sera, respectively. Figure 2A (upper panels) shows that sera from mice immunized with R4 fused to GST, but not sera from mice immunized with GST alone, bound to the surface of the rough R6 strain, or to an unencapsulated D39 mutant (D- D39, Fig. 2A). Antibodies from CCR6-immunized mice positively reacted with all strains tested, as expected. In addition, anti-R4 antibodies did not bind to the surface of the parental D39 encapsulated strain (Fig. 2A). These data indicate that Spr1875 is expressed on the bacterial surface, but is largely masked by the polysaccharide capsule. Spr1875, a Pneumococcal Antigen Spr1875 is expressed in S. pneumoniae strains of different serotypes Next, we investigated whether Spr1875 is expressed in strains of different pneumococcal serotypes. To this end, the unencapsulated derivatives of TIGR4, 23F-Spain-1, and 19F-Taiwan-14 were examined by immunofluorescence flow cytometry analysis using anti-R4 mouse sera (Fig. 2A, lower panels). Anti-R4 antibodies bound to the surface of each of these strains, indicating that immunoreactive Spr1875 is expressed on pneumococcal strains of different serotypes and geographical distribution. Next, we compared the spr1875 gene sequences in 27 pneumococcal strains using sequences available in DNA data bases. Figure 2B shows the degree of sequence variability at different positions in the deduced amino acid sequences. These sequences showed a remarkable degree of conservation with no or only few variations at each amino acid position. Such variations are mostly located in the central portion and at the C- and N-terminal regions of the protein sequence. A complete list of the aligned deduced amino acid sequences is reported in Table S2 in supplemental material, which shows that the 27 sequences belonged to one of 14 different sequence types. Spr1875 is required for in vivo pneumococcal growth The pathogenicity of pneumococci has been attributed to various virulence factors, mostly located on its surface. To evaluate whether Spr1875 has an impact on pneumococcal virulence, we constructed Dspr1875, a deletion mutant of the virulent, encapsulated D39 strain. Spr1875 was not detectable, by western blot, in Dspr1875 cell lysate using anti-R4 mouse serum (Fig. S1). As a control, we also constructed DpspA, an isogenic D39 mutant devoid of PspA, a well-characterized virulence factor that prevents complement deposition on the bacterial surface [8]. Figure 3 shows survival plots of mice inoculated intravenously with the wild-type D39 strain and Dspr1875 or DpspA mutants. Using the lower bacterial doses (Fig. 3A and 3B), we observed markedly increased survival in both Dspr1875- and DpspA-challenged mice, as compared with the wild type D39 strain. The highest challenge dose (76106 CFU) killed all mice within 5 days in each of the D39-, Dspr1875- or DpspA-infected groups (Fig. 3C). However, with this dose, Dspr1875-infected mice had a significantly Figure 1. Schematic structure and deduced amino acid sequence of protein Spr1875 of Streptococcus pneumoniae. (A) SP, signal peptide; LysM, LysM peptidoglycan interaction domain. The extension of the fragment isolated from the phage display library (R4) is also indicated. (B) Amino acid sequence of Spr1875, as deduced from ORF spr1875 in the strain R6 genome. The arrow indicates the predicted signal peptidase cleavage site, according to SignalP v3.0 predictions. The LysM domain and the R4 fragment are indicated by the box and by the bold characters, respectively. doi:10.1371/journal.pone.0036588.g001 PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36588