A locus contained within a variable region of pneumococcal pathogenicity island 1 contributes to virulence in mice

Abstract

We have previously described a 27-kb pathogenicity island of Streptococcus pneumoniae, termed pneumococcal pathogenicity island 1 (PPI1), which contains iron uptake locus piaABCD, required for full virulence in mice, and a further 28 previously uncharacterized genes. We have investigated one of these, Sp1051, which encodes a protein of unknown function. Disruption of Sp1051 does not affect growth in laboratory broth, serum, or blood but impairs virulence in mouse models of infection. When S. pneumoniae capsular serotypes were analyzed by PCR and Southern hybridization, it was found that 33% did not contain Sp1051. Analysis of other genes within PPI1 demonstrated that, compared to the serotype 4 genome published by The Institute for Genome Research (TIGR), the genomes of many strains contain deletions of a variable number of genes between Sp1046 and Sp1064, conforming to one of six different patterns. Amplification by PCR of this PPI1 variable region from a capsular serotype 17 strain and comparison of the sequence to TIGR serotype 4 strain sequence showed that Sp1051 is contained within an 11.3-kb segment of DNA flanked by 7-bp direct repeats within the serotype 4 strain which is not present in the serotype 17 strain. Further comparison of the sequences of this region between the three published S. pneumoniae genomes demonstrated that serotype 19F and strain R6 contain novel complements of genes not present in the serotype 4 strain. These data indicate that there is striking variation in gene content and structure of the 3' region of PPI1 among strains and that this region includes at least one virulence determinant. Gene variation within horizontally acquired DNA such as that of PPI1 may be one factor modulating differences in virulence among strains.

FIG. 1.

(A) Map of PPI1, showing the positions of the iron uptake ABC transporter genes piaABCD and of Sp1050 to Sp1053. Genes within PPI1 are in grey, and those flanking the ends of PPI1 are in black. (B) Genetic organization of Sp1050 to Sp1053. The derived numbers of amino acid residues (aa) for each protein are given below the corresponding gene. Arrows, insertion points for the mutant strains.

FIG. 2.

(A) Growth curves of the PPC34 (circles) and wild-type (diamonds) strains in THY as determined by measuring OD. (B) Survival of groups of 12 mice inoculated i.n. with 5 × 10⁵ CFU of the PPC34 (circles) or wild-type strains (diamonds). (C) Survival of groups of 18 mice inoculated i.n. with 3 × 10⁶ CFU of the PPC34 (circles) or wild-type (diamonds) strains.

FIG. 3.

(A) Results of PCR analysis for genes within PPI1. Each strip is an ethidium bromide-stained agarose gel containing products of PCRs designed to amplify internal fragments of genes within or adjacent to PPI1 from one S. pneumoniae strain. Representative strains are presented for each of the six different patterns of PPI1 identified. The patterns for the 16 strains (capsular serotypes) investigated were as follows: (i) strains 85 (4), M313 (4), and M127 (4); (ii) strains D39 (2), 100993 (3), 154 (3), 152 (9N), 144 (12), 138 (14), 233 (14), and 140 (16); (iii) strain 159 (19F); (iv) strain 142 (17); (v) strains 20 (35F) and 40 (1); (vi) strain M158 (6B). The gene names for each product are listed above the corresponding PCR product. (B) Southern analysis of the distribution of genes within PPI1 from strain KNR.7/87 among other S. pneumoniae strains. Each panel represents a Southern membrane containing HindIII-digested genomic DNA from five different strains representing varied capsular serotypes (listed above) probed with a PCR-amplified internal portion of a gene from within PPI1 (listed to the left).

FIG. 4.

(A) Genetic organization of the right-hand part of PPI1 in strains KNR.7/87 (ii) and 142 (iii). Boxes, open reading frames, with the gene name marked for the larger genes. (i and iv) Nucleotide structure at the junction of the insertion of the PPI1 variable region in strain KNR.7/87 (i) and strain 142 (iv). The sequence in boldface is the direct repeat flanking the 11.3 kb of DNA present in the KNR.7/87 strain but not present in strain 142; the underlined sequences are the complementary nucleotide sequences flanking the insertion point in both strains. (B) Genetic organization of the right-hand part of PPI1 in strain R6. (C) Genetic organization of the right-hand part of PPI1 in strain G54. Light grey, genes present within the KNR.7/87 genome and PPI1; black, first gene flanking the 3′ end of PPI1; dark grey, genes present in strains R6 and G54 but not strain KNR.7/87; white, genes unique to strain G54; speckled, genes unique to strain R6.