The streptococcal Blr and Slr proteins define a family of surface proteins with leucine-rich repeats: camouflaging by other surface structures - PubMed (original) (raw)
The streptococcal Blr and Slr proteins define a family of surface proteins with leucine-rich repeats: camouflaging by other surface structures
Johan Waldemarsson et al. J Bacteriol. 2006 Jan.
Abstract
Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Slr protein of Streptococcus pyogenes, which is related to internalin A of Listeria monocytogenes. Here, we show that strains of the human pathogen Streptococcus agalactiae express a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible on S. agalactiae grown in vitro, but surface exposure was increased approximately 100-fold on mutants lacking polysaccharide capsule. In S. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components.
Figures
FIG. 1.
Analysis of the Blr protein of S. agalactiae. (A) The Blr protein exhibits extensive residue identity to Slr of S. pyogenes (46), and both of these predicted lipoproteins are related to L. monocytogenes InlA, which is attached to the wall via an LPXTG motif. Amino acid residue identities of different regions are indicated in percents between the proteins. In all three proteins, the regions with LRRs are probably located distally to the bacterial surface. There are 12.5 LRRs in Blr, 10.5 LRRs in Slr, and 15 LRRs in InlA. The positions of histidine triad motifs (HXXHXH) in the N-terminal part of Blr and Slr are indicated. R, repeat; P, partial repeat. (B) Amino acid sequence of the C-terminal part of the Blr protein, including the LRR domain. The consensus sequence of LRRs in L. monocytogenes InlA is indicated in bold at the top, and the corresponding residues in the LRRs of Blr are highlighted in gray. In Blr, each LRR has a length of 22 residues, with the exception of the seventh repeat, which is one residue shorter. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant Blr and Slr. Molecular weight markers (in thousands) are noted at the left of the gel.
FIG. 2.
Characterization of mutants of S. agalactiae strain BM110 that lack polysaccharide capsule and/or protein Rib. (A) Binding of anticapsular and anti-Rib antibodies to the BM110 wild type (wt). A series of identical bacterial samples were mixed with antisera and diluted as indicated, and bound antibodies were detected by the addition of a standard amount of radiolabeled protein A, as described in Materials and Methods. Binding is presented as percentage of protein A added, explaining why binding may reach a maximal level at lower antibody dilutions and decreases at higher dilutions. (B, C, and D) Binding of anticapsular and anti-Rib antibodies to the BM110 mutants indicated. The capsule-negative mutant (Cap−) is strain BM110-22, the Rib-negative mutant is strain Rm69, and the double mutant is strain Rm69-16. All experiments were performed three times with triplicate samples, and each panel shows representative data from one experiment. Binding observed with preimmune serum (<8%) has been subtracted.
FIG. 3.
The capsule of S. agalactiae inhibits binding of antibodies to Blr but not to protein Rib. (A) Binding of anti-Blr antibodies to S. agalactiae strain BM110 wild type (wt) and mutants of this strain. For each bacterial strain, a series of identical samples were mixed with antisera and diluted as indicated, and bound antibodies were detected as described in Materials and Methods. The mutants used were those described in the legend to Fig. 2. (B) Binding of anti-Blr antibodies to S. agalactiae strain COH1 and its acapsular mutant, COH1-13 (Cap−). (C) Binding of anti-Blr antibodies to S. agalactiae wt strain BM110 and its acapsular mutant, BM110-22 (Cap−), analyzed by immunofluorescence. A control with preimmune (preimm) serum is shown for BM110-22. Bound antibodies were detected as described in Materials and Methods, and representative images from one experiment are presented. For each analysis, the same field is shown in the phase-contrast panel (Phase) and in the immunofluorescence panel (IF). (D) Western blot analysis of bacterial extracts, using anti-Blr as the probe. The extracts were prepared from S. agalactiae BM110 wild type, its Rib- and capsule-negative double mutant (Rm69-16), and the Blr-negative BM110 mutant, Δ_blr_-36. Molecular weight markers (in thousands) are noted at the left of blots. (E) Lack of capsule does not affect surface exposure of protein Rib. Anti-Rib antibodies, diluted as indicated, were incubated with BM110 wild type and its acapsular mutant, BM110-22 (Cap−). This panel was derived from data presented in Fig. 2A and B. The binding experiments shown in panels A, B, and E were performed three times with triplicate samples, and each panel represents data from one experiment. Binding observed with preimmune serum (<8%) has been subtracted.
FIG. 4.
Two surface proteins of S. pyogenes, M protein and protein F, inhibit binding of antibodies to Slr. (A) Mutants of S. pyogenes strain JRS4 (of serotype M6) were analyzed for ability to bind anti-Slr. For each strain, a series of identical samples were mixed with antisera and diluted as indicated, and bound antibodies were detected as described in Materials and Methods. M− refers to the M6-negative strain JRS145, F− refers to the protein F-negative strain SAM1, and M−F− refers to the double mutant SAM2. (B) Binding of anti-Slr to S. pyogenes strain M5 Manfredo and its M-negative mutant, ΔM5 (M−). Each experiment was performed three times with triplicate samples, and representative data from one experiment are shown. Binding observed with preimmune serum (<9%) has been subtracted. wt, wild type.
FIG. 5.
Analysis of immunological cross-reactivity between Blr and Slr. (A and B) Cross-reactivity of purified proteins. Microtiter wells were coated with Blr (coat Blr) or Slr (coat Slr), and binding of anti-Blr and anti-Slr, diluted as indicated, was analyzed by ELISA. The two antisera were adjusted to have the same titer against the homologous antigen. Preimmune serum was used as a control. (C and D) Cross-reactivity between Blr and Slr expressed on the surface of bacteria. The analysis in panel C employed the S. agalactiae strain Rm69-16, which lacks both capsule and Rib, and the analysis in panel D employed S. pyogenes strain SAM2, which lacks both M protein and protein F. For each bacterial strain, a series of identical samples were mixed with antisera and diluted as indicated, and bound antibodies were detected as described in Materials and Methods. Preimmune serum was used as a control. Each experiment was performed three times with triplicate samples, and each panel shows representative data from one experiment.
FIG. 6.
Blr does not affect virulence in an i.p. infection model but is immunogenic in vivo. (A) Mice in one group were infected i.p. with a 90% lethal dose of S. agalactiae strain BM110, and mice in another group were infected with the same number of bacteria of the nonpolar Blr-negative (Blr−) BM110 mutant, Δ_blr_-36. Each group comprised 15 mice. Deaths were recorded regularly for 96 h. (B) Immune response to Blr in mice infected with a sublethal dose of S. agalactiae. An immune response was detected in mice infected with the Blr-expressing strains BM110 (serotype III) and 1954/92 (serotype II) but not in control mice infected with the Blr-negative BM110 mutant, Δ_blr_-36.
References
- Adamou, J. E., J. H. Heinrichs, A. L. Erwin, W. Walsh, T. Gayle, M. Dormitzer, R. Dagan, Y. A. Brewah, P. Barren, R. Lathigra, S. Langermann, S. Koenig, and S. Johnson. 2001. Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis. Infect. Immun. 69**:**949-958. -PMC -PubMed
- Berggård, K., E. Johnsson, E. Morfeldt, J. Persson, M. Stålhammar-Carlemalm, and G. Lindahl. 2001. Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes. Mol. Microbiol. 42**:**539-551. -PubMed
- Bierne, H., S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart. 2002. Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol. Microbiol. 43**:**869-881. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources