| Streptococcus pneumonia(S.pn)is a gram-positive microorganism,an important pathogen that causes many infections.It colonizes in the upper respiratory tract and guides a range of life-threatening diseases such as pneumonia,otitis media,bacteremia and meningitis.It is one of the major pathogens leading to community-acquired pneumonia and hospitalization.The incidence is high among infants,seniors and patients,including conditions such as splenomegaly,chronic diseases and immunodeficiency diseases.Traditional antibiotics are considered the first choices for the clinical treatment of S.pn infection,but with the emergence of tolerances due to the global antibiotic abuses,bacterial resistances have in fact affected the evolution and spread of S.pn.The ?-lactam antibiotic resistance is widely distributed,penicillin-insensitive S.pn isolates in 2004 has reached 35%,in addition to quinolone and macrolide antibiotics resistance strains appeared by the case at the same time.S.pn as a new class of drug-resistant bacteria,and even have been resistant to vancomycin.Pneumolysin(PLY)is one of the major virulence factors elaborated by S.pn;this toxin is a member of the cholesterol-dependent cytolysins(CDCs).Engagement of cholesterol induces the formation of a multi-subunit complex by PLY that lyses host cells by forming pores on the membrane.At present,the pathogenicity of PLY has been very clear.PLYas an essential virulence factor of S.pn,can penetrate the physical defenses of the host,stimulate host cell apoptosis,possesses inflammatory properties and activate complement system.In addition to erythrocytes,PLY can also act on the epithelial cells,macrophages,with immune escape and damage DNAdouble strand.The pathogenicity of S.pn strain that could not express PLY was significantly reduced,which was shown to be attenuated after infection with experimental animals.It was proved that PLY could enhance its pathogenicity in pathogen-induced invasive infection.The vital role PLY playing in pneumococcus pathogenesis makes this virulence factor one of the most promising targets for the treatment of S.pn infection.The active ingredients of medicinal plants provide a wide range of sources and alternative spaces for the development of pharmaceutical compounds.According to the anti-virulence strategy,drug screening for pathogens' major virulence factors is also mainly dependent on natural compounds.In this study,S.pn PLY was selected as the objective of the study.Two natural compounds,?-sitosterol and verbascoside,were screened from the active ingredients of various medicinal plants.The results were analyzed by erythrocyte lysis test,minimal inhibitory concentration test,immunoblot analysis,oligomerization analysis,computational biology analysis based on protein 3D structures,protective effects on lung epithelial cells,and protection against streptococcus pneumoniae mice model.Then we investigated the abilities and mechanisms of the two drugs to neutralize the biological toxicity of PLY.The anti-virulence of natural compounds was used to control pathogen infection,the survival pressure of antibiotics on microbes was reduced and the progress of bacterial multidrug resistance evolution was impeded.In the study,the purified recombinant protein(r PLY)was used,in more than ten natural compounds we got ?-sitosterol and verbascoside,which could directly neutralize r PLY erythrocyte lysis at very low doses.The drugs co-cultured with S.pn wild-type strain D39,when the drugs' concentrations > 2048 ?g/ml,the growthes of bacteria were not affected,indicating that ?-sitosterol and verbascoside did not have antibacterial activities against pneumococcus.The bacteria in the co-culture broth were harvested to conduct Western Blot tests.The specific blots of PLY were homogeneous.The results show that the expression of PLY is not affected by the increase of drug concentrations,which means the drug does not affect thetranscription and translation of ply gene.In order to further explore the mechanisms of the two compounds to inhibit the activity of PLY,the structural model of PLY was constructed by using molecular homologous protein as a template for molecular docking and molecular dynamics simulation.The structures were respectively docked with ?-sitosterol and verbascoside,the dominant conformation was selected and the molecular dynamics calculations were performed on the results.The results of the evaluation data indicate that the free PLY is unfettered in solution and that the protein exhibits a weaker flexibility after binding to verbascoside,indicating that the PLY residues are more rigid due to binding to verbascoside,which binds to the PLY domain 3 and domain 4,plays a role in the process of protein oligomerization.The stability at the binding sites was mainly provided by the residues Asp471,Asn470,Glu277,Tyr358 and Arg359.In ?-sitosterol-PLY complex,the relative interaction between the compound and PLY residues Thr459,Leu460 is hindered by the relatively large steric hindrance of the alkyl chain of ?-sitosterol C25.The distance between Thr459 and Leu460 is longer than the one between protein and its natural receptor cholesterol,but as a cholesterol structure analog,?-sitosterol binds to PLY in a cholesterol way.PLY is a cholesterol-dependent cytotoxin,which has been studied in depth.High concentrations of PLY can spontaneously assemble in solution to form oligomers without the cell membranes or cholesterol.We analyzed the pretreatment of drug-protein mixed samples by high performance liquid chromatography(HPLC).The results show that ?-sitosterol does not affect the self-oligomerization of PLY,while that of verbascoside significantly inhibits the formation of PLY oligomers.The conclusion is consistent with our calculated biology results.S.pn D39 causes pulmonary epithelial cell apoptosis,and PLY can directly cause lung epithelial cells,macrophages and other necrosis.PLY is genotoxic and can induce DNA damage without serious damage to the host cell membrane,resulting in discrete ?-H2 AX lesions in the nucleus.In this study,PLY and A549 cells co-cultured samples were mixed with ?-sitosterol and verbascoside,respectively,using laser confocal instrument to observe the stained fluorescence of the cells,and to assess the release of lactate dehydrogenase(LDH).The results show that both compounds could significantly inhibit the injury of A549 cells at low concentration and decrease the necrosis rate of target cells.Immunofluorescence assay shows that the frequency of DNA damage in A549 cells exposed to PLY is significantly reduced in the presence of the test drugs.These results indicate that by inhibiting the oligomerization of PLY in the host membrane or preventing its initial binding to membrane cholesterol,its cytotoxicity to eukaryotic epithelial cells can be inhibited and the genetic toxicity is reduced.After the establishment of streptococcus pneumoniae pneumonia mice models,dosing regimens were designed by the half-lifes,then to investigate the treatment effects.The results show that the treatment of ?-sitosterol and verbascoside could significantly increase the survival rates of mice with lethal pneumonia and reduce bacterial colonization in infected mice,which is beneficial to the removal of pathogens and alleviate the experiment mice lung tissue congestion,inflammation and injury symptoms.In conclusion,?-sitosterol binds to the PLY cholesterol binding sites to inhibit the toxin binding to the membranes;while verbascoside binds to PLY oligomerization-related sites to restrain the formation of oligomers.Thus they inhibit the cytotoxicity and genotoxicity of toxins,protect the target organ epithelial cells,relieve the damage caused by invasive infection,and provide protection for the body,so as to have an effective anti-infective effect without antibacterial activity.These views provide complements to the pharmacological activitives of the ingredients in medicinal plants,at the same time,provide theoretical basises and referential compounds for anti-infective agents. |
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