Immunization With Peptide Mimicking Lipoteichoic Acid Protects Mice Against S.aureus Systemic Infection And Pretreatment Of Pam3CSK4 Attenuates The Inflammatory Response With MRSA

Part Ⅰ Immunization with peptide mimicking lipoteichoic acid protects mice against S.aureus systemic infectionBackground Due to the enormous capacity of Staphylococcus aureus(S.aureus)to acquire antibiotic resistance,it becomes imperative to develop vaccines for decreasing the risk of its infection.Lipoteichoic acid(LTA)is a conserved component in S.aureus.However,it has not been used as a vaccine candidate due to its property of its thymus-independent antigen(TI antigen).Objective In this study,we investigated the protection effect of peptide mimcking epitope of LTA in mice systemic infected with S.aureusMethods Based on a sequence mimic LTA,GHKEDRQWCQHS,we synthesized a linear peptide(named L2-3,sequence:HSGHKEDRQWCQHSGG)and a multiple antigenic peptide(MAP,named MAP2-3)which is carrying four copies of a L2-3 peptide.The antigenicity of L2-3 and MAP2-3 was identified by ELISA.BALB/c mice and C57BL/6 mice were immunized with MAP2-3 or MAPctrl,respectively.Serum samples were detected during the immunization.After immunization with MAP2-3 five times,all mice were infected with S.aureus.The survival rate was determined in lethal-dose S.aureus challenge model and the bacterial burden in organs was measured in systemic infection model.The percent of memory B cells(IgG+B cells and IgG1+B cells)were analyzed by flow cytometry.Results Linear-peptide L2-3 reacts with anti-LTA mAb specifically.MAP2-3,a tetra-branched,multiple antigenic peptide binds to anti-LTA mAb and polyclonal antibodies against S.aureus,respectively.After immunization with MAP2-3 five times,antiserum recognized MAP2-3 reacts with S.aureus and LTA,respectively.Compared with MAPctrl immunization or blank control,MAP2-3 immunization induce more IgG class antibodies,including IgG1,IgG2a and IgG2b.The percentage of IgG1+B cells is significant increased in splencoytes from MAP2-3 immunized mice compared with that from MAPctr1 mice or naive mice.Immunization with MAP2-3 decrease the bacteria burden in organs of BALB/c and C57BL/6 mice,and significant prolonged their survival time after S.auerus lethal-dose challenge.Levels of IFN-γ,IL-17A/F,IL-4 and CCL3 are increased in spleen from MAP2-3-immunized mice after S.aureus systemic infection.Conclusion Immunization with MAP2-3,a multiple antigenic peptide that mimics the epitope of LTA,elicits efficient humoral response against to S.aureus and protects mice against S.aureus systemic infectionPart Ⅱ Pretreatment of Pam3CSK4 Attenuates The Inflammatory Response in Mice Infected with Methicillin-resistant Staphylococcus aureusBackground Although pro-inflammatory cytokines play an important role in mediating the initial host defense against bacteria,excessive production of inflammatory cytokines(also named cytokine storm)is correlated with sepsis that can cause tissue damage,multiple organ failure and even death.Previous works indicate that pretreatment with TLR ligand,such as LPS,PGN and single phosphorus acyl lipid A(MPLA)can attenuate the inflammatory response,improve the survival rate in different infection model.Objective In this study,we investigated the protection effect of Pam3CSK4 pretreatment in mouse model with S.aureus systemic infection and explored the mechanism of the attenuation proinflammatory response by Pam3CSK4 pretreatment.Methods The mice were injected with Pam3CSK4,a TLR2 agonist,via tail vein or abdominal cavity once daily for two consecutive days.The control mice were administrated with sterile PBS.In lethal-dose MRS A challenge,all mice were then challenged with the lethal dose of MRSA 1×108 CFU/each 24 h after the second Pam3CSK4 injection.The survival rate of mice after MRSA challenge was observed.In S.aureus systemic infection model,all mice were challenged with live MRSA(ATCC43300 at 2×107 CFU/each)1 day after the second pretreament.The bacterial burden in serum and organs were detected,respectively.The level of cytokines in serum,kidney and spleen were detected by ELISA or real-time PCR,respectively.The relative expression of TLRs,NOD2,IRAKs or Tollip in spleen was detected by real-time PCR.The expression of phosphorylated NF-κBp65,IRAK-M and Tollip in spleen were detected by Western Blot,respectively.Results Pretreatment of Pam3CSK4 protected the mice against infection of methicillin-resistant Staphylococcus aureus and enhanced the bacterial clearance in bacteremia model.Pro-inflammatory cytokines,such as TNF-α,IL-6,IL-1β,and CCL3 significantly decreased,whereas the relative expression of anti-inflammatory cytokines IL-10 and TGF-β increased in splenocytes from the Pam3CSK4-treated mice.The attenuation of inflammation response is possibly due to the down-regulation of TLR2 expression and downstream molecules such as IRAK-1 and IRAK-4,two key components of NF-κB signaling pathway.Moreover,the up-regulation of IRAK-M and Tollip,two negative effectors of NF-κB pathway,also contributes to the protective effect of Pam3CSK4 treatmentConclusion Pretreatment of Pam3CSK4 inhibits TLR2 signaling pathway and attenuates the inflammatory response in mice infected with methicillin-resistant Staphylococcus aureus.