Antibacterial Effects Of Traditional Chinese Medicine Monomer On Streptococcus Pneumoniae Via Inhibiting Pneumococcal Histidine Kinase (VicK)

ObjectiveTwo-component systems (TCSs) VicR/VicK is one of TCSs in S.pneumoniae essential for pneumococcal survival. VicR/K can regulate theintegrity of bacterial cell wall and is associated with drug resistance. VicKhistidine kinase, responding to various environmental stimuli thentransferring it to the response regulator, have recently attracted attention asdrug targets for antimicrobials.Recently, there are many reports in screening of antimicrobial drugstargeted VicK, We have also previously obtained six inhibitors that targetedthe HATPase domain of VicK in S.pneumoniae which had effectiveantibacterial activity. Traditional Chinese medicine (TCM) is an importantsource of new drugs. Previously, we have obtained96potential VicKinhibitors from the TCM monomer database by virtual screening. This studywas carried out to investigate the bactericidal activity of these compoundsand the mechanisms of the antibacterial activities. MethodsThe full-length S. pn VicK protein was expressed in Escherichia coli(E. coli) by prokaryotic expression technology, the conservation of VicK in6different serotypes of S. pn was analysed by Western blot. The ATPaseactivity of the full-length VicK protein was detected by using Kinase-Glo Luminescent Kinase Assays. We screened compounds inhibited the ATPaseactivity of VicK protein for subsequent experiments in vivo, Finally, five ofthem were further studied using a standard MIC, MBC and cytotoxicityassay. Synergic antimicrobial activity of the five compounds and PEN wasalso investigated in vitro and vivo. Mouse sepsis model by S.pn (D39) wassuccessfully established by intraperitoneal infection of D39, and the survivaltime of the infected mice treated with compounds was monitored. Micewere challenged with S. pn19F, or19A to establish Local infection modeland the bacterial loads in nasal washes and lung washes were estimatedafter administration of drugs. Trace checkerboard dilution method was usedto detect the synergistic effect with other antibiotics, Finally, the mechanismof compounds against bacteria was studied from several aspects, such asbacterial morphology, biofilm formation.ResultsThe inhibition rate of five Compounds on kinase activity was greaterthan50%, The MICs of5’-(Methylthio)-5’-deoxyadenosine, octanal2,4-dinitrophenylhydrazone, deoxyshikonin, kavahin, and dodecyl gallateagainst S. pneumoniae were37.1,38.5,17,68.5, and21μg/mL, respectively. These compounds showed no obvious cytotoxicity effects on Vero cells，andthey increased the survival time of mice infected by S. pneumoniae.Meanwhile, five compounds exerted antimicrobial effects onmultidrug-resistant S. pneumoniae clinical strains，MRSA, Streptococcusmutans， Streptococcus mitis， False streptococcus pneumoniae andStreptococcus pyogenes. Moreover, they can inhibit cell division andbiofilm formation at sub-MIC. In addition, Deoxyshikonin and dodecylgallate enhanced the antimicrobial activity of penicillin in vivo and in vitro，and effectively cleared nasopharyngeal and lung colonization caused bydifferent penicillin-resistant pneumococcal serotypes.ConclusionOur result suggests that five novel inhibitors of VicK had significantantimicrobial activity and enhanced the antimicrobial activity of otherantibiotics, They are good candidates against multidrug-resistant S.pneumoniae infection.