The ClpP protease of Streptococcus pneumoniae modulates virulence gene expression and protects against fatal pneumococcal challenge

Abstract

Streptococcus pneumoniae usually colonizes the nasopharynx of humans asymptomatically but occasionally translocates from this niche to the lungs, the brain, and the blood, causing potentially fatal infections. Spread to other host tissues requires a significant morphological change and the expression of virulence factors, such as capsular polysaccharide, and virulence proteins, such as pneumolysin (Ply), PspA, and CbpA. Modulation of the expression of pneumococcal virulence genes by heat shock and by heat shock proteins ClpL and ClpP, as well as the attenuation of virulence of a clpP mutant in a murine intraperitoneal infection model, was demonstrated previously. In this study, we further investigated the underlying mechanism of virulence attenuation by the clpP mutation. The half-lives of the mRNAs of ply and of the first gene of the serotype 2 capsule synthesis locus [cps2A] in the clpP mutant were more than twofold longer than those of the parent after heat shock, suggesting that the mRNA species were regulated posttranscriptionally by ClpP. In addition, the clpP mutant was defective in colonization of the nasopharynx and survival in the lungs of mice after intranasal challenge. The mutant was also killed faster than the parent in the murine macrophage RAW264.7 cell line, indicating that ClpP is required for colonization and intracellular survival in the host. Furthermore, fractionation studies demonstrated that ClpP was translocated into the cell wall after heat shock, and immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with S. pneumoniae D39, making ClpP a potential vaccine candidate for pneumococcal disease.

FIG. 1.

Detection of relative mRNA stabilities of cps(2)A and ply by real-time RT-PCR. (A) To determine the mRNA half-lives at 30°C, S. pneumoniae strains were grown at 30°C, and then rifampin was added. Aliquots for RNA extraction were withdrawn before the addition of rifampin and at 10 and 20 min after rifampin was added (R10m and R20m, respectively). (B) To determine the mRNA half-lives after heat shock, S. pneumoniae strains were grown at 30°C and then heat shocked (HS) at 42°C. After 10 min at 42°C, rifampin was added. Aliquots for RNA extraction were withdrawn before and after heat shock and at 10 and 20 min after the addition of rifampin at 42°C. (C) To determine the effect of heat shock on the mRNA half-lives, S. pneumoniae strains grown at 30°C were treated with rifampin for 10 min and then heat shocked at 42°C. Aliquots for RNA extraction were withdrawn before and at 10 min after the addition of rifampin at 30°C (R-10m) and then at 10 and 20 min after heat shock (R+HS10m and R+HS20m, respectively). Between RNA extracts, levels of individual mRNA species were corrected by reference to that obtained for the internal 16S rRNA control. Data points represent means and standard deviations of quadruplicate samples from each RNA extract.

FIG. 2.

Translocation of ClpP after heat shock. Exponentially growing S. pneumoniae at 30°C was heat shocked at 42°C for 30 min. Cells were collected by centrifugation, and the proteins were fractionated and subjected to SDS-PAGE. Subsequently, ClpP was visualized by immunoblot analysis with polyclonal ClpP-specific antiserum. W, cell wall; M, membrane; C, cytoplasm.

FIG. 3.

Bacterial recovery from the nasopharynx of CD1 mice after intranasal challenge with D39 and its isogenic clpP derivative over a 4-day period. The values are means and standard errors of the means (n = 5) for each time point.

FIG. 4.

Survival of the clpP mutant in macrophage cells. RAW264.7 cell monolayers were infected with ca. 10⁷ CFU of pneumococci (bacterium/cell ratio, 10:1) in RPMI 1640 culture medium. Samples were taken at different times after infection to quantify intracellular pneumococci after gentamicin treatment. Three independent assays were carried out in triplicate for each bacterial strain. Error bars indicate standard deviations. Single and double asterisks indicate P values of <0.05 and <0.01, respectively, for comparisons with parent strain D39.

FIG. 5.

Western immunoblot analysis of whole-cell lysates of S. pneumoniae D39 (A) and purified preparations of PdB (53 kDa), PspA fragment (43 kDa), and ClpP (21 kDa) (B) showing specificities of antibody responses to protein antigens. The proteins were separated by SDS-PAGE and then electroblotted onto nitrocellulose. They were then reacted with sera from groups of mice immunized with the proteins. Nitrocellulose membrane strips were reacted with sera from mice immunized with AlPO₄ adjuvant (lane 1), PdB plus AlPO₄ (lane 2), PspA plus AlPO₄ (lane 3), and ClpP plus AlPO₄ (lane 4).

FIG. 6.

Survival times for mice after intraperitoneal challenge. Groups of 12 CBA/N mice were immunized with the indicated antigens and challenged 2 weeks after the third immunization with approximately 7.5 × 10⁵ CFU of capsular type 2 strain D39. Each datum point represents one mouse. A horizontal line denotes the median survival time for the group.