Contribution of a response regulator to the virulence of Streptococcus pneumoniae is strain dependent

Abstract

Bacterial two-component signal transduction systems (TCS) enable bacteria to respond to environmental changes and regulate a range of genes accordingly. They have a crucial role in regulating many cellular responses and have excellent potential as antibacterial-drug targets. We have constructed mutations in a TCS response regulator gene for two different strains of the human pathogen Streptococcus pneumoniae. These mutants have been analyzed in our murine model of infection. Data suggest that in a D39 background the response regulator gene is essential for virulence; an isogenic mutant is avirulent via intraperitoneal, intranasal, and intravenous routes of infection. This mutant, which does not show impaired growth in vitro, is unable to grow in the lung tissue or in blood. Mutation of the response regulator in a 0100993 background results in a strain that is fully virulent intraperitoneally and intravenously but shows decreased levels of bacteremia and increased murine survival following intranasal infection. The ability to grow in the lung tissue is not impaired in this mutant, suggesting that it has an impaired ability to disseminate from the lungs to the systemic circulation. Our data highlight the importance of assessing the contribution of putative virulence factors to the infection process at different sites of infection and provide evidence that virulence determinants can behave very differently based on the genetic background of the bacterial strain. These important findings may be relevant to other bacterial pathogens.

FIG. 1.

Gene organization around the TCS09 locus. The rr and hk genes make up S. pneumoniae tcs09. An erythromycin resistance cassette was inserted to replace the rr09 gene. R9F and R9R, primers used to create and confirm mutations within the rr09 gene. Downstream of the tcs09 locus is a conserved hypothetical protein (open arrow) and a gene, zmpB, encoding a putative zinc metalloprotease (3, 23). Immediately upstream lies msrA, encoding a methionine sulfoxide reductase, also important in virulence (35). Primer pairs P1-P2, P3-P4, and P1-P4 were used to confirm that the organization of genes surrounding tcs09 was conserved in the two strains used in this study. The diagram is not drawn to scale.

FIG. 2.

Survival of mice following intraperitoneal infection with 10⁵ CFU of wild-type or mutant bacteria. (a) Strains on a D39 background. ▪, wild-type strain D39; □, the Δrr09 mutant. P < 0.05 for survival time with the Δrr09 mutant compared to that with the D39 parental strain. (b) Strains on a 0100993 background. •, wild-type strain 0100993; ○, the Δ488 mutant. No difference in survival was seen between animals challenged with the Δ488 mutant and the 0100993 parental strain.

FIG. 3.

Intranasal challenge with 10⁷ CFU on a D39 background. (a) Survival; (b) bacteremia. ▪, wild-type strain D39; □, the Δrr09 mutant. P < 0.05 for survival and for bacteremia at all time points after 0 h. In panel b, error bars represent SEMs; dashed line represents the limit of detection (log₁₀ 1.92 CFU ml⁻¹).

FIG. 4.

Intranasal challenge with 10⁷ CFU on a 0100993 background. (a) Survival; (b) bacteremia. •, wild-type 0100993; ○, the Δ488 mutant. P < 0.05 for survival and for bacteremia at all time points after 24 h. In panel b, error bars represent SEMs; dashed line represents the limit of detection (log₁₀ 1.92 CFU ml⁻¹).

FIG. 5.

Bacterial loads in the lung tissue 24 and 48 h following intranasal infection with 10⁷ CFU. (a) Strains on a D39 background. Filled bars, wild-type D39; hatched bars, the Δrr09 mutant. P < 0.05 for wild-type versus mutant lung counts at both time points. (b) Strains on a 0100993 background. Filled bars, wild-type 0100993; hatched bars, the Δ488 mutant. No differences were found between wild-type and mutant lung counts at either time point studied. Values are geometric means ± SEMs. The dashed line represents the limit of detection.

FIG. 6.

Intravenous challenge with 10⁶ CFU on a D39 background. (a) Survival; (b) bacteremia. ▪, wild-type D39; □, the Δrr09 mutant. P < 0.05 for survival and for bacteremia at all time points after 0 h. In panel b, error bars represent SEMs; dashed line represents the limit of detection (log₁₀ 1.92 CFU ml⁻¹).

FIG. 7.

Intravenous challenge with 10⁶ CFU on a 0100993 background. (a) Survival; (b) bacteremia. •, wild-type 0100993; ○, the Δ488 mutant. No differences were seen in survival or level of bacteremia between mice infected with 0100993 or the Δ488 mutant at any of the time points studied. In panel b, error bars represent SEMs; dashed line represents the limit of detection (log₁₀ 1.92 CFU ml⁻¹).