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. 2015 Dec 3;5:17668.
doi: 10.1038/srep17668.

β-Sitosterol Interacts With Pneumolysin to Prevent Streptococcus Pneumoniae Infection

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Free PMC article

β-Sitosterol Interacts With Pneumolysin to Prevent Streptococcus Pneumoniae Infection

Hongen Li et al. Sci Rep. .
Free PMC article

Abstract

Pneumolysin is one of the major virulence factors elaborated by Streptococcus pneumoniae; this toxin is a member of the cholesterol-dependent cytolysins. Engagement of cholesterol induces the formation of a multi-subunit complex by pneumolysin that lyses host cells by forming pores on the membrane. Because pneumolysin released by bacteria which have been killed by conventional antibiotics is still active, agents capable of directly attacking the toxin are considered advantageous against antimicrobials in the treatment of S. pneumoniae infections. Here we found that the phytosterol, β-sitosterol, effectively protects against cell lysis caused by pneumolysin. This compound interacts with the toxin at Thr459 and Leu460, two sites important for being recognized by its natural ligand, cholesterol. Similar to cholesterol, β-sitosterol induces pneumolysin oligomerization. This compound also protects cells from damage by other cholesterol-dependent toxins. Finally, this compound protects mice against S. pneumoniae infection. Thus, β-sitosterol is a candidate for the development of anti-virulence agents against pathogens that rely on cholesterol-dependent toxins for successful infections.

Figures

Figure 1
Figure 1. Chemical structures of four sterols and the inhibitory mechanism of β-sitosterol against PLY.
(A) Chemical structures of four sterols. The red circle indicates the structural differences among cholesterol, β-sitosterol and stigmasterol, which are the critical chemical bonds responsible for the binding to pneumolysin. (B) β-sitosterol did not affect the oligoerization of pneumolysin. Purified pneumolysin at a concentration capable of self-assembly (>10 mg/ml) was incubated with β-sitosterol and the mixture was analyzed by high performance liquid chromatograph (HPLC). The toxin eluted in a profile identical to the control which did not receive β-sitosterol. (C,D) The interactions of pneumolysin with cholesterol or β-sitosterol. Pneumolysin was immobilized on an SPR assay chip and liposomes containing β-sitosterol or cholesterol at the indicated concentrations were used to determine the binding. (E) β-sitosterol protects human alveolar epithelial cells from cells injury caused by PLY. A549 cells were treated with the toxin in medium supplemented with different concentrations of β-sitosterol. Cell injury was measured by the release of LDH. The values in the bars represent the means±SD of three independent experiments. *p < 0.05 and **p < 0.01.
Figure 2
Figure 2. The binding and distance modes of PLY-CHO and PLY-BSS.
(A) The binding modes of PLY-CHO and PLY-BSS. The binding sites of BSS (molecule in green) with pneumolysin are identical to the binding sites (Val372, Ala370, Leu460 and Thr459) of CHO (molecule in blue), which is the natural receptor of pneumolysin, except for the distance between the respective ligands and Thr459/Leu460. (B) Modeling of the distance between ligands, CHO and BSS, and Thr459/Leu460 of pneumolysin as a function of time. The average distance between CHO and Thr459/Leu460 residues is 1.50 nm, and the average distance between BSS and Thr459/Leu460 is 4.02 nm. These results are consistent with those of the binding free energy calculation. The results of the binding free energy calculation show the binding energy of CHO and Thr459/Leu460 is 1.64 and 1.32 kcal/mol and 1.04, 0.52 kcal/mol with BSS. The reason for this is that the distance between BSS and Thr459/Leu460 is longer than that of CHO and Thr459/Leu460.
Figure 3
Figure 3. β-sitosterol protects against S. pneumoniae infection.
(A) Survival curves of mice infected with S. pneumoniae D39. Mice infected with wild type bacteria were treated at two different time points before infection or postinfection at one hour with β-sitosterol (BSS-1 and BSS-2) or with the solvent (controls) and the mortality of the mice was monitored daily for 5 days. Mice infected with the pneumolysin deficient mutant was established as a control. Note the significant protection achieved by both β-sitosterol concentrations. **p < 0.01. The numbers of bacteria recovered from lungs of differently treated infected mice. Lungs of mice infected for 48 hours were obtained; ground tissues were plated onto bacteriological media to enumerate the bacterial counts. (B) The numbers of bacteria recovered from lungs which were treated with BSS before infection for one hour. (C) The numbers of bacteria recovered from lungs which were treated with BSS after infection for one hour. All experiments were done in triplicate and similar results were obtained in three independent experiments. **p < 0.01. (D) The pathology of lungs of infected mice. Note that in untreated mice infected with D39 WT, the majority of the airspace was obliterated by inflammatory cell infiltrates. Infected mice treated with β-sitosterol showed much less such damage.

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