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. 2002 Sep;45(5):1389-406.

Large-scale Identification of Serotype 4 Streptococcus Pneumoniae Virulence Factors

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Free PMC article

Large-scale Identification of Serotype 4 Streptococcus Pneumoniae Virulence Factors

David L Hava et al. Mol Microbiol. .
Free PMC article

Abstract

Streptococcus pneumoniae (the pneumococcus) is carried in the nasopharynx of healthy individuals, but can spread to other host sites and lead to pneumonia, bacteraemia, otitis media and meningitis. Although it is logical to think a priori that differential gene expression would contribute to the ability of this pathogen to colonize different sites, in fact very few genes have been demonstrated to play tissue-specific roles in virulence or carriage. Using signature-tagged mutagenesis to screen 6149 mariner-transposon insertion strains, we identified 387 mutants attenuated for infection in a murine model of pneumonia. Among these mutants are ones with disruptions in a number of putative tissue-specific transcriptional regulators, surface proteins, metabolic proteins and proteins of unknown function, most of which had not previously been associated with virulence. A subset of these, including most of those with insertions in putative transcriptional regulators,was examined for phenotypes in murine models of bacteraemia and nasopharyngeal carriage. Four classes of mutants defective in infection models of the: (I) lung, (II) lung and blood, (III) lung and nasopharynx,and (IV) all three tissues were identified, thus demonstrating the existence of tissue-specific pneumococcal virulence factors. Included in these strains were two with disruptions in a genetic locus that putatively codes for a transcriptional regulator, three surface proteins and three sortase homologues. Mutation analysis revealed that three of the seven genes in this locus are virulence factors that are specific to mucosal surfaces.

Figures

Fig. 1
Fig. 1
A. Schematic representation of the S. pneumoniae rlrA locus. rlrA is divergently transcribed from at least six different genes indicated by black arrows, and the entire locus is flanked by two IS1167 elements. The left element contains a frameshift mutation and is therefore predicted to be inactive. The sites of magellan2 insertions identified by STM in rlrA and srtD is shown as open triangles and the magellan5 insertions generated by in vitro transposition and used in additional animal experiments are show as black triangles. B. The predicted C-terminal sorting signals of RrgA, RrgB, and RrgC are listed. The LPXTG motif of each of the proteins is conserved, with the exception of the first amino acid. All three proteins have a stretch of hydrophobic residues (underlined) and a charged tail, characteristic of proteins that are anchored to the cell wall by sortases.
Fig. 2
Fig. 2
Analysis of rlrA locus mutants in animal models of lung infection (A), nasopharyngeal carriage and bacteraemia (B). The in vivo competitive index (CI) was calculated as described in the text; each circle represents the CI for a single mouse in each set of competitions. A CI of less than one indicates a virulence defect. Open circles indicate that no mutant bacteria were recovered from that animal and therefore 1 was substituted in the numerator when calculating the CI. The geometric mean of the CIs for all mice in a set of competitions is shown as a solid line and statistically significant data are indicated with a symbol (* p < 0.05, # p < 0.07). The in vitro competition results for each of the tested strains are as follows: rrgA – 1.06, rrgB – 0.50, rrgC – 0.75, srtB – 0.94, srtC – 0.69, and srtD – 0.93.

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