The Mechanism Of EGCG Against Streptococcus Pneumoniae Pneumolysin And Sortase A

Streptococcus pneumoniae(S.pneumoniae,the pneumococcus),a common Gram-positive bacterial pathogen,is the major causative agent of community acquired pneumonia(CAP).The pneumonia causes ~1 million deaths of children younger than 5 years of age in developing cuuntries annually.Persons at higher risk for invasive pneumococcal disease include children,the elderly and the immunocompromised individuals.Pneumococcal infection causes various diseases,including sinusitis,otitis media,pneumonia,sepsis and meningitis.Recently,the antibiotics-resistant bacteria emerge increasingly and spread worldwide,which makes the research for drugs based on new anti-infection strategies to be urgent.With the in-depth understanding of bacterial pathogenesis,people realize the importance of anti-virulence strategy.This approach is to target functions essential for infection,such as virulence factors required to cause host damage and disease,and interfere the pathogenesis of bacteria.Traditional Chinese Medicine is an important resource for development of novel drugs.In this study,we have identified one natural compound,Epigallocatechin gallate(EGCG),with the activity of simultaneous inhibition of pneumoccal pneumolysin(PLY)and sortase A(SrtA).EGCG,a major component of green tea catechins,possesses diverse biological properties,including anti-oxidant,anti-inflammatory and anti-carcinogenic effects.The result of antimicrobial susceptibility test showed that EGCG exhibited little antiS.pneumoniae activity,with a minimal inhibitory concentration(MIC)? 2234 ?M(1024 ?g/ml).The pore-forming toxin PLY,an important virulence factor,plays a crucial role in the pathogenesis of pneumococcus.PLY monomers oligomers to form transmembrane pores in cholesterol-rich membranes to result in the intracellular components leakage and cell lysis.PLY cytotoxicity,which is attributed to its cytolytic activity,is closely associated with the development of invasive pneumococcus disease.In this study,we obtained PLY recombinant protein with high purity via prokaryotic expression and purification.The results of hemolytic activity inhibition assay and western blot showed that EGCG did not affect the expreesion of PLY in S.pneumoniae,but could directly inhibit the hemolytic activity of purified PLY.In the co-incubation system of PLY and human alveolar epithelial cells(A549),the addition of EGCG could significantly attenuate PLY-mediated cell injury.In the PLY oligomerization system,the addition of EGCG could previously reduce the formation of PLY oligomers.The results above indicated that EGCG neutralized PLY's pore-forming activity by direct interaction.SrtA,another critical virulence determinant for the pathogenesis of S.pneumoniae,is a membrane-localized transpeptidase which is widely found in Gram-positive bacteria.Many virulence-associated surface proteins are covalently anchored to the cell wall by SrtA which recognizes a conserved carboxylic sorting motif,LPXTG,and cleaves it between the threonine and glycine residues.The activity of Srt A is directly associated with pneumococcal pathogenicity.In this study,the purified SrtA?N81 and NanA proteins were obtained via prokaryotic expression and purification.The antisera with high titer respectively against SrtA?N81 and NanA were obtained via immulizing mice with the purified proteins.The inhibitory effect of EGCG against SrtA was evaluated using a fluorescence resonance energy transfer(FRET)assay.The peptidase activity of SrtA?N81 was significantly inhibited following pre-incubation with EGCG in a dose-dependent manner.The expression of SrtA in S.pneumoniae was not affected with the addition of EGCG in the culture medium of pneumococci,but the production of NanA on the cell wall was obviously reduced compared to control.When S.pneumoniae co-cultured with EGCG,the biofilm formation was significantly inhibited.Furthermore,the adhesion of S.pneumoniae to human epithelial cells(Hep2)was significantly inhibited with the addition of EGCG in the co-culture system of pneumococci and Hep2 cells.The results above indicated that EGCG inhibited SrtA's transpeptidase activity by direct interaction.Mice were intranasally inoculated with S.pneumoniae strain D39 following treatment with EGCG or PBS as a control.The survival analysis revealed that EGCG could postpone the death of S.pneumoniae infected mice.Bacterial survival in the lungs of infected mice treated with EGCG was significantly reduced compared with the control.Gross inspection and histopathological analysis revealed that the pathological injury in the lungs of infected mice treated with EGCG was remarkably alleviated compared with the control.Furthermore,the lung wet/dry weight ratio of mice treated with EGCG was significantly decreased compared with the control,which indicated an alleviation of pulmonary edema.Functional analyses from in vivo and in vitro suggest that EGCG can inhibit toxin activity of PLY and SrtA through interaction and,subsequently,weaken the virulence of S.pneumoniae.Finally,the preferential binding modes of EGCG with PLY and SrtA were determined by molecular dynamics simulations based on the docking results.The binding model of EGCG with PLY revealed the binding of EGCG to the cleft between domains 3 and 4 in PLY via hydrogen bonding and hydrophobic interactions,and the strong interactions with Ser256,Glu277,Tyr358 and Arg359,respectively.The binding model of EGCG with SrtA revealed the binding of EGCG to the “activity” region of SrtA via hydrogen bonding and hydrophobic interactions,and the strong interactions with Thr169,Lys171 and Phe239,respectively.Site-directed mutagenesis of PLY and SrtA were performed to produce the mutants(PLY-E277 A,PLY-Y358 A,PLY-R359 A SrtA?N81-T169 A,SrtA?N81-K171 A and SrtA?N81-F239A),and the inhibitory effects of EGCG on their activities were assessed.The results showed that EGCG did not effectively inhibit the cytolytic activities of PLY varants or the peptidase activities of Srt A variants,which indicated that the mutations in the aforementioned residues impaired the interactions of EGCG with PLY and SrtA.This further verified the computational biology results.In conclusion,these findings provide an important experimental basis for the elucidation of the mechanism of natural component against bacterial infection and,lay foundation for the development of anti-virulence agents against S.pneumoniae specially targeting PLY and SrtA.