The Preliminary Study Of The Antimicrobial Mechanism Of QseC Quorum Sensing Inhibitor Br-LED209

ObjectiveThe wide spread of antibiotic resistant bacteria in the world has become a nightmareto human beings and also is a leading cause of death in severe infection patients. Nearlyall significant bacterial infections in the world are becoming resistant to commonly usedantibiotics. The main problem of antibiotic used in clinical infection treatment isinevitably inducing bacterial resistance. Therefore, the discovery and development of newantibiotic drugs, with low resistance, is a new trend for new antibiotic agent research.Quorum sensing system (QSS) is a communicating system which exists broadly amongbacteria. QseC has very important role in gram-negative bacteria. It is an importantregulation system of virulence factors secretion, invasion capacity and pathogenicity. Ithas been reported that selectively block binding of signals to QseC could consequentlyinhibit QseC-mediated activation of virulence gene expression. Meanwhile, blocking QseC does not directly influence pathogen growth, therefore may not induce drugresistance. However, the antibiotic mechanism of QseC inhibitor remains not understood.Our objective is to study the possible antibacterial mechanism of QseC inhibitors, as wellas explore new strategy on antibiotic research.Motheds1. The synthesis and characterization of QseC selective inhibitor Br-LED209:Using ara-acetylaminobenzene sulfonyl chloride and para-bromoaniline as raw material togive4-amino-N-(4-bromophenyl)benzenesulfonamide through acylation and deacylationdeprotected. Then using the resulting product and phenyl isothiocyanate to affordN-(4-bromophenyl)-4-(3-phenyl thiourea)benzenesulfonamide, named Br-LED209. Thechemical structure of Br-LED209was confirmed by MS,1H NMR and13C NMRdata.2. The effect of Br-LED209on the growth of bacteria in vitro: The MICs ofBr-LED209to Salmonella typhimurium, EHEC and Shigella flexneri were determined bymicrodilution method. To determine the growth curve, Br-LED209was added to bacteriacultures to final concentrations of10μM,50μM and200μM respectively. OD600valueswere measured by automatic growth curve analysis apparatus. Levofloxacin was used aspositive control.3. The antibacterial activity of Br-LED209on mice infected with S. typhimurium:BALB/c mice were infected by the intraperitoneal administration of approximately1.0×108colony-forming unit(CFU) of S. typhimurium. The mice were treated orally withBr-LED209(20mg/kg)3huors pre and post infection. Livers, spleens, lungs and kidneyswere harvested24hours post infection, homogenized and plated on agar plates for bacterialcounts to evaluate the effect of Br-LED209on bacterial burden of infected mice. Theprotective effect of Br-LED209on the livers and spleens of infected mice was evaluatedby HE staining.4. The impact of Br-LED209on virulence gene expression in S. typhimurium: Toevaluate whether Br-LED209could block the QseC quorum sensing system of S.typhimurium, the expression level of virulence genes was detected by quantitativereal-time PCR. RNA was extracted from overnight culture grown aerobically in LB medium in the absence or presence of200μM Br-LED209follow the instruction ofbacterial RNA isolation kit.5. The preliminary study of antimicrobial mechanism of Br-LED209: Toevaluate the effect of Br-LED209on the flagella motility of S. typhimurium, motility assaywas performed in the plates containing LB medium with0.3%agar. Bacteria were spottedonto the medium and halo sizes were measured after incubated at37°C for16h. Toassess the effect of Br-LED209on the invasion capacity and replication capacity of S.typhimurium of epithelial cells, HeLa cells were infected for1,6and12hours. Then thecells were lysed with1%Triton X-100and the lysate were diluted and plated on agarplates to determine CFU. Macrophages were infected with S. typhimurium. The culturesupernatant was collected1h after infection to detect the amount of released LDH and tocalculate the pyroptotic rate of macrophages. Fluorescent dye staining assay wasperformed to determine the percentage of PI positive cells by counting cell numbers offour random visual fields to evaluate the effect of Br-LED209on macrophages pyroptosis.The active form of caspase-1and IL-1β was measured by western blotting to determinethe the effect of Br-LED209on the function of inflammasome. Macrophages wereinfected with S. typhimurium for12hours. Then the cells were lysed with1%TritonX-100and the lysate were diluted and plated on agar plates to determine the number ofCFU to evaluate the effect of Br-LED209on the intracellular survival of S. typhimurium.Resluts1. The QseC selective inhibitor Br-LED209was successfully synthesized:Br-LED209synthesized is yellow solid. The melting point of Br-LED209is116~120°C.The purity of Br-LED209is higher than95%. The result of MS show that the molecularweight of Br-LED209is consistent with the theoretical value. NMR data demonstratedthat the chemical structure of Br-LED209was correct.2. Br-LED209does not hinder the growth of bacteria in vitro: The results of MICshowed that the Br-LED209does not have antibacterial activity at the concentration of256μg/mL. The results of growth curve demonstrated that Br-LED209does not hinder thegrowth of bacteria in vitro at10μM to200μM. These results suggest that Br-LED209 does not have significant bacteriostasis.3. Br-LED209significantly reduced the bacterial burden of infected mice: BALB/cmice were infected by the intraperitoneal administration of S. typhimurium. The mice weretreated orally with Br-LED209(20mg/kg)3huors pre and post infection. Livers, spleens,lungs and kidneys were harvested24hours post infection for bacterial counts. The resultsshow that bacterial number of livers, spleens, lungs and kidneys of control group were6.03×107CFU,1.04×108CFU,1.02×106CFU and8.02×105CFU, respectively. And thebacterial number of livers, spleens, lungs and kidneys of Br-LED209group were1.01×107CFU,2.06×107CFU,1.68×105CFU and1.33×105CFU, respectively. There wassignificant difference between control and Br-LED209group(P<0.001). Meanwhile,Br-LED209treatment could reduce pathological damage of organs of infected mice. Theabove results suggest Br-LED209treatment could reduce bacterial virulence and decreasethe survival and replication capacity of S. typhimurium in infected mice. We suppose thatthe above effect of Br-LED209may be related to its inhibition of bacterial virulence andactivation of innate immune system.4. Br-LED209diminished the expression of QseC-dependent virulence genes:Blocking QseC quorum sensing system of S. typhimurium may affect the expression ofQseC-dependent virulence genes. The expression level of virulence genes was detected byquantitative real-time PCR. The result of quantitative real-time PCR demonstrated thatBr-LED209at200μM could block the QseC quorum sensing system of S. typhimuriumand diminish the expression of QseC-mediated virulence factors such as flhDC, sifA andsopB(P<0.001).5. Br-LED209could inhibit the flagella motility and decrease the invasion capacityand replication capacity of S. typhimurium: The above results demonstrated thatBr-LED209could diminish the expression of flagellar gene, invasion related andreplication related gene. Therefore, we observed the effect of Br-LED209on the flagellarmotility and the invasion and replication capacity of S. typhimurium of HeLa cells. Theresult of motility assay demonstrated that Br-LED209could inhibit the flagella motility ofS. typhimurium (P<0.05). The result of HeLa cell invasion assay showed that Br-LED209 could decrease the invasion capacity and replication capacity of S. typhimurium(P<0.001).6. Br-LED209could suppress S. typhimurium infection induced macrophagepyroptosis and promote the clearance of intracellular bacteria: In order to explore whycould Br-LED209reduce bacterial burden of infected mice, we evaluated the immuneresponse of macrophages on S. typhimurium. The result showed that LDH released frommacrophage increased after infection. This indicated that S. typhimurium infecton couldinduce macrophage pyroptosis. Br-LED209treatment could decrease the amount ofreleased LDH and inhibit S. typhimurium induced macrophage pyroptosis(P<0.001). Thepercentage of PI positive cells detected by fluorescent dye staining assay confirmed theabove result(P<0.001). In addition, the result of macrophages infection assaydemonstrated that Br-LED209could reduce the intracellular survival of S.typhimurium(P<0.001). The above results suggest that Br-LED209could suppress S.typhimurium infection induced macrophage pyroptosis and promote the clearance ofintracellular bacteria.7. Br-LED209could inhibit the activation of caspase-1and IL-1β in infectedmacrophages: In order to further explore how could Br-LED209suppress macrophagepyroptosis, we detected the maturation of inflammsome activation related proteincaspase-1and IL-1β. The result of western blotting showed that Br-LED209could depressthe expression of activated caspase-1and IL-1β(P<0.001). This suggests that Br-LED209could inhibit the activation of inflammasome, and may recover the clearance ability ofinfected macrophage through its suppression of macrophage pyroptosis.Conclusion1. Br-LED209could diminish the expression of QseC-mediated key virulencegenes, and subsequently inhibit the flagella motility and reduce the invasion capacity andreplication capacity of S. typhimurium.2. Br-LED209could inhibit S. typhimurium infection induced pyroptosis andrecover the clearance ability of infected macrophage. These effects may be related to itsinhibition of the expression of flhDC, sifA and sopB.