Elsevier

Gene

Volume 284, Issues 1–2, 6 February 2002, Pages 63-71
Gene

Allelic variation in the highly polymorphic locus pspC of Streptococcus pneumoniae

https://doi.org/10.1016/S0378-1119(01)00896-4Get rights and content

Abstract

PspC, also called SpsA, CbpA, PbcA, and Hic, is a surface protein of Streptococcus pneumoniae studied for its antigenic properties, its capability to bind secretory IgA, C3 and complement factor H, and its activity as an adhesin. In this work we characterized the pspC locus of 43 pneumococcal strains by DNA sequencing of PCR fragments. Using PCR primers designed on two unrelated open reading frames, flanking the pspC locus, it was possible to amplify the pspC locus of each of the 43 strains of S. pneumoniae. In 37 out of 43 strains there was a single copy of the pspC gene, while two tandem copies of pspC were found in the other six strains. The sequence of the pspC locus was different in each of the 43 strains. Insertion sequences were found in the pspC locus of 11 out of 43 strains. Analysis of the deduced amino acid sequence of the PspC variants showed a common organization of the molecules: (i) a 37 amino acid leader peptide which is conserved in all proteins, (ii) an N-terminal portion which is essentially alpha-helical, and is the result of assembly of eight major sequence blocks, (iii) a proline-rich region, and (iv) a C-terminal anchor responsible for the cell surface attachment. By sequence comparison we identified 11 major groups of PspC proteins. Proteins within one group displayed only minor variations of the amino acid sequence. An unexpected finding was that PspC variants could differ in the anchor sequence. While 32 of the PspC proteins displayed the typical choline binding domain of pneumococcal surface proteins, 17 other PspCs showed the LPXTG motif, which is typical of surface proteins of other gram-positive bacteria. This major difference in the anchor region was also observed in the adjacent proline-rich regions which differed considerably in size and composition.

Introduction

Surface proteins of Streptococcus pneumoniae (pneumococcus) have been investigated for their role in pneumococcal pathogenicity and as candidate antigens for protein-based vaccines. The most extensively studied molecule is pneumococcal surface protein A (PspA), which was shown to be essential for virulence and to elicit protection against pneumococcal infection (McDaniel et al., 1987, Briles et al., 1998, Briles et al., 2000, Ogunniyi et al., 2000). While typical surface proteins of gram-positive bacteria are covalently linked to the cell wall via the LPXTG motif at their C-terminal end (Navarre and Schneewind, 1999), PspA was found to be attached by a novel mechanism by which its C-terminal repeat region binds to the choline residues of the lipoteichoic acid of S. pneumoniae (Yother and White, 1994). By searching the pneumococcal genome data (Tettelin et al., 2001, Dopazo et al., 2001, Hoskins et al., 2001, Oggioni and Pozzi, 2001) it is possible to identify a variety of C-terminally anchored surface proteins, which contain the LPXTG motif or the choline-binding domain.

Different biological functions have been associated with pneumococcal surface protein C (PspC), and different allelic forms of PspC have been referred to by different names, including choline-binding protein A (CbpA) (Rosenow et al., 1997), S. pneumoniae secretory IgA binding protein (SpsA) (Hammerschmidt et al., 1997), C3-binding protein A (PbcA) (Cheng et al., 2000), and factor H-binding inhibitor of complement (Hic) (Janulczyk et al., 2000). PspC plays an important role in pneumococcal pathogenesis by functioning as an adhesin (Rosenow et al., 1997), by promoting invasion of epithelial cells (Zhang et al., 2000), and by binding soluble host factors such as the IgA secretory fragment, C3 and complement factor H (Hammerschmidt et al., 1997, Cheng et al., 2000, Janulczyk et al., 2000, Dave et al., 2001). Used as an immunogen in the mouse model, PspC proved to be a good candidate for anti-pneumococcal vaccines (Brooks-Walter et al., 1999, Briles et al., 2000).

Given this multiplicity of phenotypes, it is important to investigate the variability of the pspC locus among different isolates, to gain a better understanding of PspC as a virulence factor and to facilitate design of PspC-based vaccines. In this work we characterized the pspC locus of 43 clinical isolates of S. pneumoniae.

Section snippets

Bacterial strains, media and growth conditions

The 43 pneumococcal strains characterized in this study include standard strains D39 (Avery et al., 1944, Iannelli et al., 1999) and 8R1 (Bernheimer, 1979), four strains from the American Type Culture Collection, and 37 recent clinical isolates randomly chosen from an Italian collection (including the type 19F strain G54 whose draft genome sequence is annotated) (Pozzi et al., 1996, Dopazo et al., 2001). Bacteria were grown at 37 °C in tryptic soy broth or tryptic soy agar (Difco) supplemented

PspC nomenclature

The term PspC is preferred over other designations, for two reasons: (i) it was used for the first sequence deposited in GenBank (Accession number: U72655), and (ii) it is a generic name referring only to the surface location of the molecule. Based on deduced amino acid sequence analysis we identified 11 major groups of PspC proteins, and used numbers from 1 to 11 to designate proteins belonging to each group (Fig. 1, see below). Single proteins within each group which display minor variations

Discussion

In this work we characterized the highly polymorphic pspC locus of S. pneumoniae. Sequencing of the pspC locus in 43 pneumococcal isolates allowed comparison with already available pspC sequences, and provided insights into the nature of the pspC gene product, a surface protein important for pathogenesis and vaccine development. Results indicate that: (i) each pneumococcal strain contains a pspC gene at the same chromosomal location, (ii) each pneumococcal strain has a unique DNA sequence at

Acknowledgements

We thank Pat Cleary for critically reading the manuscript. The authors also thank The Institute for Genomic Research (TIGR) for preliminary sequence data of the type 4 S. pneumoniae TIGR4. This work was supported in part by grants from MURST (Cofinanziamento 1999), CNR (P. F. Biotecnologie), and the Commission of the European Union (QLK2-2000-00543).

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