Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection

Abstract

Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease worldwide. Fragments of the cell wall and the cytolytic toxin pneumolysin have been shown to contribute substantially to inflammatory damage, although the interactions between pneumococcal components and host-cell structures have not been elucidated completely. Results of a previous study indicated that cell-wall components of pneumococci are recognized by Toll-like receptor (TLR)2 but suggested that pneumolysin induces inflammatory events independently of this receptor. In this study we tested the hypothesis that pneumolysin interacts with surface proteins of the TLR family other than TLR2. We found that pneumolysin stimulates tumor necrosis factor-alpha and IL-6 release in wild-type macrophages but not in macrophages from mice with a targeted deletion of the cytoplasmic TLR-adapter molecule myeloid differentiation factor 88, suggesting the involvement of the TLRs in pneumolysin recognition. Purified pneumolysin synergistically activated macrophage responses together with preparations of pneumococcal cell walls or staphylococcal peptidoglycan, which are known to activate TLR2. Furthermore, when compared with wild-type macrophages, macrophages from mice that carry a spontaneous mutation in TLR4 (P712H) were hyporesponsive to both pneumolysin alone and the combination of pneumolysin with pneumococcal cell walls. Finally, these TLR4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with pneumolysin-positive pneumococci than were control mice. We conclude that the interaction of pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus.

Figure 1

Differential induction of IL-8 secretion in transfected HEK cells by ethanol-killed vs. heat-killed S. pneumoniae. HEK 293 cells stably transfected with CD14, TLR2, or TLR4 were stimulated over 18 h with medium (white bars), 10 ng/ml LPS (gray bars), ethanol-killed pneumococcus (black bars), and heat-killed pneumococcus (hatched bars).

Figure 2

Pneumolysin induces NF-κB translocation in CHO cells. Clone 7.19, a CHO/CD14 cell line that contains an ELAM.tac (CD25) reporter plasmid, is LPS-nonresponsive because of a point mutation in MD2 (C95Y). This cell line was stimulated with ethanol-killed D39 (corresponding to 10⁸ cfu/ml) or PLN-A (corresponding to 10⁸ cfu/ml) over 18 h. After stimulation, cells were stained with anti-CD25 mAb and analyzed by flow microfluorometry.

Figure 3

The inflammatory activity of pneumolysin is MyD88-dependent. Peritoneal macrophages from C57BL/6 (WT, black) and MyD88^−/− (white) mice were stimulated with medium or indicated concentrations of pneumolysin (Ply) over 18 h.

Figure 4

The inflammatory activity of pneumolysin is TLR4-dependent, heat-labile, and augmented by costimulation with TLR2 ligands (peptidoglycan or killed whole pneumococcal cells PLN-A). Peritoneal macrophages from C3H/HeOuJ (WT, black) and C3H/HeJ [TLR4 mutants (P712H), white] mice were stimulated with 10 ng/ml LPS, 1 μg/ml pneumolysin (Ply), heated pneumolysin with or without staphylococcal peptidoglycan (PGN, 1 μg/ml) (A), killed pneumococcal cells [PLN-A, concentrations of 10⁷ (E7) cells per ml], pneumolysin, or PdT (a nonhemolytic mutant of pneumolysin, 1 μg/ml) with or without PLN-A E7, heated PdT with PLN-A E7 (B).

Figure 5

Nuclear translocation of NF-κB due to pneumolysin is TLR4-dependent. Peritoneal macrophages from C57BL/6 or TLR4^−/− mice were stimulated for 1 h with medium, 10 ng/ml LPS, 5 μg/ml lipoprotein (Pam₃Cys-Ser-Lys-4), or 10 μg/ml pneumolysin, and then nuclear extracts were prepared and analyzed by EMSA.

Figure 6

Mice lacking functional TLR4 are more susceptible to pneumococcal nasopharyngeal colonization. Emitted photons by luciferase-tagged S. pneumoniae in the nasopharynx from anesthetized C3H/HeJ (Upper) and C3H/HeOuJ (Lower) mice were detected by a charge-coupled camera after intranasal inoculation with 7 × 10⁷ cfu of a type-3 pneumococcus (strain A66.1 Xen 10). Results are from 24 h after inoculation and represent the course of colonization in these animals. The animals with bioluminescence at 24 h (five C3H/HeJ and one C3H/HeOuJ mice in these pictures) subsequently developed bacteremia and died.