Staphylococcus Epidermidis Color Aminoacyl-trna Synthesis Enzyme And Arlrs Of Two-component Signal Transduction Systems As Antibacterial Targets

Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes that rarely causes pyogenic infections in healthy individuals.However, during the past two decades S.epidermidis has emerged as one of the major pathogens in nosocomial infections.The primary pathogenicity trait of S. epidermidis is associated with its ability to form biofilms on surfaces of medical devices,limiting severely the efficacy of many conventional antibiotics,and biofilms may also protect the bacteria against attacks from the host defence system.In parallel,the appearance of multi-drug resistant S.epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals,which urgently require design novel antibiotics against staphylococcus infections,especially in relation to biofilm development.The aim of this study is to look for the drug targets against S.epidermidis,which is consisted of two major parts:one is the discovery of lead compounds against S. epidermidis;the other is the discovery of molecular basis of biofilms formation in clinical isolates and the evaluation of feasibility of the potential anti-biofilm drug target.In this study,we have found three inhibitors of the S.epidermidis tryptophanyl-tRNA synthetase that have bacteriostatic activites.Besides,we investigated the role of TCS ArlRS in the regulation of biofilm formation in S. epidermidis,and find ArlRS is an anti-biofilm drug target.Our findings will contribute to the development of anti-staphylococci drugs.Part 1.S.epidermidis tryptophanyl-tRNA synthetase as an antibacterial targetAmong bacterial essential proteins,prokaryotic aminoacyl-tRNA synthetases (ARSs) have attracted attention as potential antibacterial targets.6 As a group,ARSs are universal enzymes that exist in all living organisms because they catalyse the attachment of amino acids to transfer RNAs(tRNAs),which are the adaptors required for the information flux from the messenger RNA templates to the polypeptide chains.Tryptophanyl-tRNA synthetase(WRS) belongs to ARS class I family.S.epidermidis WRS(SeWRS) and cytoplasmic human WRS(HWRS) share very low sequence similarity,which may be a potential drug target.In this work,we apply an approach combining structure-based discovery in silico with biochemical and biological experiments in vitro to screen S.epidermidis WRS inhibitors.A 3D structural model of SeWRS was constructed by using the homologous modelling approach using the B.stearothermophilus enzyme as the reference.SBVS resulted in the identification of 111 candidates as potential SeWRS inhibitors using SPECS chemical lead-compound database.Among them,three compounds were potent inhibitors in vitro,blocking the activity of SeWRS(IC₅₀ are between 15.1～42.2μM, K_d are between 3.76～13.9μM) as well as the growth of S.epidermidis(MIC values are between 6.25～100μM),while showing low inhibition of the human tryptophanyl-tRNA synthetase and displaying low cytotoxicity to mammalian cells. These compounds are good leads to develop new antibiotics.Part 2.Study of the two component signal transduction system ArlRS of S.epidermidis,a potential anti-biofilm targetThe primary pathogenicity trait of S.epidermidis is associated with its ability to form biofilms on surfaces of medical devices,limiting severely the efficacy of many conventional antibiotics,and biofilms may also protect the bacteria against attacks from the host defence system.Thus,the discovery of anti-biofilm drugs is one of the strategies to combat with S.epidermidis infections.Biofilm formation is a very complicated process,which is composed of two steps: single bacterial cells attaches to the material surface;intercellular adhesion of the bacterial cells forms multiple layers structures.So far many genes are found to be involved in the biofilm formation,including several functional genes(atlE,ica,aap, bap,etc.) and some regulatory genes(sarA,sigB,agr,etc.).Previous studies have revealed the potential role of some two-component signal transduction systems(TCSs) in regulating biofilm formation.So far,sixteen pairs of TCSs have been found in S.epidermidis,in which ArlRS attracts our attentions.In this part of the study,we investigate the molecular basis of the regulation of biofilm formation by ArlRS,which may contribute to the discovery of new anti-biofilm targets.Firstly,we constructed an arlS gene deletion mutant of the S.epidermidis strain 1457,which was named with WW06.Several methods,including PCR and RT-PCR, were used to confirm the arlS gene deletion and the successful construction of WW06.By western blot,we found that the translation of ArlR protein was deficient in the strain of WW06.Thus,what we got is an ArlRS deficient strain.The growth curve of WW06 was similar to that of SE1457;however,biofilms were no longer forms in the mutant.We further investigated the molecular mechanism of biofilm-forming ability loss in WW06,and found that the initial attachment ability remained in this mutant.By using the methods of DNA microarray and real-time RT-PCR,we found that the transcription of several genes involved in the second step of biofilm formation,such as icaADBC,sigB,sarA,etc,decreased in WW06.The biofilm forming phenotype can be restored by overexpressing icaADBC in WW06, but not by overexpressing sigB,indicating that ArlRS regulates biofilm formation through the regulation of icaADBC.ArlR can bind to the promoter region of ica operon,which was confirmed by electrophoretic mobility shift assay(EMSA), indicating ArlRS directly regulates the transcription of icaADBC.We further investigated the relationship between ica transcription and arlRS transcription in the 9 ica⁺/BF^- S.epidermidis strains,and found that the transcription of ica operon in these nine strains was dramatically lower that in SE1457.In these S.epidermidis strains,seven displayed transcriptional loss of arlR,and two of them showed lower arlR transcription than SE1457.Based on these findings,we think two component signal transduction system ArlRS is an anti-biofilm target.Two strategies were used in the present study.Our findings will contribute to the development of anti-staphylococci drugs.