| Staphylococcus aureus(S.aureus)is an important Gram-positive pathogen.S.aureus can cause skin and soft tissue infections,as well as life-threatening diseases such as pneumonia,osteomyelitis,sepsis,and infective endocarditis.Due to the widespread use of antibiotics,the problem of S.aureus resistance to antibiotics has attracted attention,especially the emergence and prevalence of methicillin-resistant S.aureus(MRSA).MRSA strains are resistant to nearly allβ-lactam antibiotics.However,β-lactams has been empirically used for clinical treatments of infectious diseases caused by Gram-positive bacteria.For MRSA infections,which are not initially recognized,β-lactams are not only ineffective in the treatment of infections but also likely contributing to poor outcomes by enhancing the pathogenicity of MRSA.Several studies have revealed that sub-inhibitory concentrations ofβ-lactam antibiotics can promote S.aureus pathogenicity by increasing the expression of alpha-toxin,Panton–Valentine leukocidin(PVL),enterotoxins,or staphylococcal protein A(SpA)in vitro.The contributions of certain altered virulence factors to MRSA pathogenesis in vivo and the molecular mechanisms underlyingβ-lactam-modulated MRSA pathogenesis remain largely unknown.Therefore,study on the molecular mechanism ofβ-lactam induced MRSA virulence gene expression and its role in promoting MRSA pathogenicity will support the reasonable choice of antibiotics in clinical practice.Staphylococcal accessory(sar)and accessory gene regulators(agr)have been recognized to play central roles in the regulation of S.aureus virulence.SarA is a 14.7 kDa pleiotropic global regulator that binds to the specific motifs in the promoter regions of target genes,modulates the expression of approximately 120 genes in S.aureus via agr-dependent or-independent pathways,involving bacterial colonization,pathogenesis,biofilm formation and drug resistance.Lipoproteins(Lpps)are important proteins anchored to the bacterial membrane.S.aureus encodes 55–70 putative Lpps,which can stabilize cell membrane,transport substance,and transmiss signals.Some lipidated Lpps can be secreted,and bind to the Toll-like receptor(TLR)2 of the host immune cells to activate TLR2-dependent innate immune system and promote inflammatory response,thereby enhence bacterial pathogenicity.More than 30%of the Lpps in S.aureus are hypothetically conserved proteins with unknown functions.And near 21%of the Lpp genes in S.aureus are named lipoprotein-like genes(lpl).N315 carries 12(21%)hypothetical Lpls.Among these Lpls,nine proteins are specific to theνSaα.Studies have shown that some Lpls may be closely related to the expression of inflammatory factors and the pathogenicity of S.aureus,but the exact biological function and regulation mechanism of Lpls remain unclear.In this study,we found a cluster of lpl genes in MRSA could be induced by sub-inhibitory concentrations ofβ-lactams,the regulation mechanisms ofβ-lactams induce Lpls and its role in the pathogenicity of MRSA were investigated.The main contents and results are as following:1.Expression of MRSA Lpls upregulated in response to sub-inhibitory concentrations ofβ-lactam induction1)β-Lactam induction and identification of MRSA Lpls.MRSA strains were induced by different kinds of antibiotics.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)revealed that a protein band of approximately 30 kDa was upregulated afterβ-lactams induction.The protein band was analyzed through liquid chromatography tandem mass spectrometry(LC-MS/MS).Three putative Lpls,namely,SA2273,SA2274,and SA2275 were identified,which are encoded by a consecutive lpl gene cluster.Reverse-transcription polymerase chain reaction(RT-PCR)detection revealed that sa2275,sa2274,and sa2273 were co-transcribed and constitute an operon.2)Transcriptional and translational analysis of MRSA Lpls induced byβ-lactam antibiotics.Quantitative RT-PCR(RT-qPCR)detection showed that the mRNA levels of sa2275,sa2274,and sa2273 were upregulated in N315 after treatment with sub-inhibitory concentrations of OXA.Western blot results showed that the expression and secretion of Lpls in MRSA can induced by sub-inhibitory concentrations ofβ-lactams in a dose-and time-dependent manner.2.Role and mechanism of β-lactams increasing the expression of lpl by the global regulator SarA1)Role of SarA in mediating the increasing expression of MRSA lpl byβ-lactams.RT-qPCR revealed that the expression levels of sarA,agrA,and RNAIII were significantly upregulated in OXA-treated N315 compared with those in the untreated samples.And Western blot analysis indicated thatβ-lactam-stimulated expression of Lpls in MRSA may be controlled by SarA through an agr-independent pathway.2)The mechanism of SarA in mediating the increasing expression of MRSA lpl byβ-lactams.We constructed a reporter vector containing the lpl promoter-controlled lacZ gene(pOS1-lpl~P)and performedβ-galactosidase assay by transforming pOS1-lpl~P into the MRSA strains N315 and N315ΔsarA.The results showed that SarA can significantly affect the expression of lacZ.Western blot analysis showed that SarA controlled Lpls expression in response toβ-lactam antibiotic induction.We analyzed the binding motif of SarA in the promoter regions of lpl and discovered a typically predicted SarA box(5`-ATTTAAT-3`).Electrophoretic mobility shift assay(EMSA)showed that S.aureus SarA can directly bind to the lpl cluster promoter region,and regulate the expression of lpl in the presence ofβ-lactams.3.β-Lactam-induced Lpls triggered TLR2-dependent proinflammatory cytokine production in vitro and in vivo1)Lpls triggered TLR2-dependent proinflammatory cytokine production by macrophages.To determine whether Lpls contribute to innate immune stimulation,we constructed lpl deletion strains and lpl-overexpressing strain for macrophage infection.Results showed that production of IL-6 and TNF-αin mouse RAW264.7 macrophages significantly decreased after treatment with lpl deletion strains compared with those of wild-type strains administered.And macrophages were stimulated with purified lipidated SA2275-his and unlipidated SA2275-his(-sp)proteins.Results showed that lipidated Lpls are needed for the recognition of TLR2 receptors to trigger immune response.2)MRSA Lpls enhanced proinflammatory cytokine production in mice.Animal experiments further demonstrated thatβ-lactam-induced Lpls triggered TLR2-dependent proinflammatory cytokine production in mice.4.β-Lactam-stimulated Lpls promoted the pathogenesis of MRSA1)Lpls enhanced the bacterial burden in mouse kidneys.To investigate whetherβ-lactam-stimulated Lpls enhanced the colonization of MRSA,we determined the bacterial burden in the organs of a mouse bacteremic model.BALB/c mice were infected intravenously with pGFP plasmid-transformed N315Δlpl and N315 for 5 days,and bacterial colonization was tracked,the bacterial loads in the kidneys of N315-infected mice were also significantly higher than those of N315Δlpl-infected ones.These results indicate that Lpls plays an important role in the colonization of MRSA-infected mouse kidneys.2)Lpls enhanced the skin abscess formation in mice.Mouse subcutaneous infection model results confirmed thatβ-lactam-stimulated Lpls aggravated host TLR2-dependent MRSA infections.In conclusion,we demonstrated that a lpl cluster(sa2275,sa2274,sa2273)of MRSA was upregulated in response to sub-inhibitory concentrations ofβ-lactam induction.β-Lactams stimulate the expression of SarA,which directly binds to the lpl cluster promoter region and upregulates Lpls expression in MRSA.The increased Lpls in MRSA significantly promotes TLR2-dependent signaling pathway activation and results in inflammatory response by triggering proinflammatory cytokine levels in vitro and in vivo,thereby possibly contributing to bacterial pathogenicity by inducing host immune responses and promoting bacterial colonization.Our findings suggest that antibiotics should be rationally selected according to the results of drug susceptibility.As the drug is metabolized in the patient,sub-inhibitory concentrations ofβ-lactams are not only ineffective in treatment but also possibly contribute to poor outcomes by enhancing the pathogenicity of MRSA. |
