Regulation Studies Of Wall Teichoic Acid On The Virulence Factors Expression In CA-MRSA

Methicillin-resistant Staphylococcus aureus(MRSA)is resistant to almost all the clinically used β-lactam antibiotics,which poses a great challenge to global public health security.MRSA infections have occurred primarily under exposure to hospitalassociated risk factors(HA-MRSA)for a long time since its discovery in 1961.However,community-associated MRSA(CA-MRSA)has emerged as an important cause of infections over the last 3 decades.Unlike most strains of HA-MRSA,CAMRSA strains are generally resistant to lower concentrations of β-lactam antibiotics and exhibit higher virulence and often cause disease in otherwise healthy individuals.An in-depth understanding of the molecular basis for the enhanced virulence of CAMRSA strains may help to discover novel strategies to combat CA-MRSA infections.Phenol-soluble modulins(PSMs)are a family of highly expressed cytolytic peptide toxins of CA-MRSA strains,which are strictly and positively regulated by staphylococcal accessory gene regulator(agr)activity.PSMs mainly include PSMα,PSMβ and δ-toxin.Deletion of psmα operon(coding PSMα)in CA-MRSA strains dramatically reduce bacterial virulence in different animal models of infection,which points to a strategy of inhibiting PSMα production to control the severity of CAMRSA infections.Wall teichoic acid(WTA)is a cell wall polymer of S.aureus,which is initially catalyzed by non-essential TarO.Like PSMs,WTA is also highly expressed in CA-MRSA strains and thus enhances the virulence of CA-MRSA.In addition,the lack of WTA re-sensitizes MRSA to β-lactams.Thus,inhibition of WTA biosynthesis has also drawn significant interest as a strategy against MRSA infections.Although considerable progress has been made regarding the important function of WTA in Staphylococcal infections,its role in virulence gene expression is unknown.In this study,we fused the psmα promoter of CA-MRSA USA300(LAC)with the galactosidase coding gene lac Z to construct a psmα-lac Z transcriptional fusion reporter gene system,and performed a high-throughput screening of the ability of3987 compounds(2395 FDA-approved drugs,1199 clinically tested drugs and 393 bioactive agents)to inhibit psmα-lac Z,we identified that tunicamycin was a potential inhibitor for the psmα promoter activity of USA300.Further studies showed that,in addition to the inhibition of psmα,another virulence factor Sp A was also inhibited by tunicamycin.We next determined that the inhibitory effect of tunicamycin on psmα3and Sp A expression was resulted from the blockage of WTA synthesis by TarO inhibition,and gene expression changes caused by WTA inhibition are largely depends on the activation of two component system Vra RS.Importantly,blockage of WTA synthesis in USA300 not only led to decreased virulence gene expression,but also dramatically reduced the virulence of USA300 in Galleria mellonella and mouse infection models.Subsequently,using fluorescence microscopy assays,we found that defect of WTA synthesis in USA300 resulted in the delocalization of septum protein PBP2,which possibly disrupted the functional integrity of PBP2 and further reduced the resistance of USA300 to PBP2 selective β-lactams and PBP2 transglycosylase inhibitor moenomycin.Additionally,upon treatment with epicatechin gallate(a compound that also delocalized septum protein PBP2),genetic depletion of pbp2 or inhibition by PBP2 selective β-lactams,all these conditions led to the inhibition of Sp A and psmα3 while the activation of Vra RS regulatory function and showed a similar virulence gene expression pattern with WTA abrogation of USA300.Importantly,we determined that a PBP2-selective β-lactam cefuroxime with low doses significantly attenuates the virulence of USA300 against Galleria mellonella despite this pathogen being highly resistant to this drug.We found that absence of PBP2 a coding gene mecA inhibited the PBP2 delocalization ability of tunicamycin and reduced the activation of Vra RS regulatory function by TarO inhibitors.Our studies also possibly suggested that Lipid II accumulation on the outer cell wall is the potential explanation for the activation of Vra RS regulatory function.Additionally,we concluded in this study that the regulatory effect of WTA on virulence gene expression was not only occurred in USA300 but also existed in other CA-MRSA strains including USA400 and 2011-137.In conclusion,in this study we revealed a novel regulatory axis,WTA/PBP2/Vra RS,for controlling virulence genes expression in CA-MRSA.Our results also underscored that chemical genetics is a powerful method for uncovering new biological pathways amenable to pharmacological modulation.As maintenance of cell wall homeostasis appears to be important for S.aureus infections,more indepth knowledge about the role of cell wall integrity signaling pathway in regulating virulence gene expression may help to elucidate the pathogenic success of CA-MRSA and would provide an opportunity to develop novel strategies for the treatment of CAMRSA infections.