Streptococcus Pneumoniae Endopeptidase O Enhances Host Innate Immunity

Objective:Infectious diseases are the most severe global challenges we are facing today or tomorrow.Due to the wide-spread of drug-resistant bacteria and emerging pathogens,existing anti-microbial agents are becoming harder and harder to counter these challenges.However,the development of new agents cannot meet the growing need of clinical practice,therefore novel strategy is in desperate need.Immunostimulator targeting Toll-like receptors has been proven to be effective for promoting the bacteria clearance,and showed its potential in preventing and treatment against bacterial infection.Trained immunity is a novel discovered immune phenomenon,which is also called innate immune memory.When the host encountered with some specific microbials or compounds,the innate immune system is 'trained' to produce a memory-like effect,and facilitates the host with enhanced anti-infection protection.Evidence showed that TLRs are related with the induction of trained immunity.Streptococcus pneumoniae endopeptidase O,PepO,is a novel virulence protein which is capable of induce host innate immune response via TLR2 and TLR4,thus is a possible anti-infection agent with high potentialMethods:1.PepO promotes the phagocytosis and bactericidal activity via autophagy induced by TLR2 and TLR4 activationThe recombinant PepO protein and its punctate mutant is produced by molecular cloning and affinity chromatography.The truncated mutants were constructed according to the prediction of the domain and the binding of PepO to TLR2/TLR4 was confirmed by immunoprecipitation.The autophagy markers in peritoneal derived macrophages(PDM)treated with Pepo were detected by immunoblot and immunofluorescent microscopy to monitor the induction of autophagy.The D39?pepO mutant was constructed by homologous recombination,and compared the autophagy induced by WT D39 and D39?pepO mutant.The relationship between autophagy phagocytosis and bactericidal activity was confirmed by counting the in cellular bacteria after infecting the PepO treated PDM with WT D39,S.aureus 502A and P.aeruginosa PAK,and the colocalization of LC3 and bacteria.It was further confirmed by assessing the phagocytosis and bactericidal activity of PepO treated PDM after the inhibition of autophagy by Bafilomycin A1.The phagocytosis and bactericidal activity and autophagy is assessed in PepO treated tlr2,tlr4 and tlr2/tlr4 deficient PDM to unveil the effect of TLR2 and TLR4 in PepO promoting bacteria clearance and inducing autophagy.The signal pathway participating the process of Pepo inducing autophagy is assessed by detecting the signaling molecules by immunoblot.The potential of PepO promoting bacteria clearance is confirmed by establishing pneumonia model in PepO treated tlr2,tlr4,tlr2/tlr4 and macrophage deficient mice2.PepO induce trained immunity by manipulating glycolysisThe bone marrow derived macrophage(BMDM)is cultured ex-vivo and then treated with PepO to detect the glycose consumption.The induction of trained immunity is monitored by the assessment of phagocytosis and bactericidal activity after 1 day of PepO treatment and 3 days of rest.The H3K4me3 methylation and H3K27Ac acetylation of the TNF-? and IL-6 promotor were detected by Chromatin Immunoprecipitation in PepO trained immunity to confirm the epigenetic reprogramming of PepO induction.The effect of glycolysis is confirmed by inhibition with inhibitor BPTES.The phagocytosis and bactericidal activity and epigenetic modulation of TNF-? and IL-6 were assessed in PepO treated tlr2 deficient BMDM to monitor the effect of TLR2 in PepO induced trained immunityResults:1.PepO induce autophagy and promotes the phagocytosis and bactericidal activity of macrophage1)The recombinant PepO protein,truncated mutant PepO1-430,PepO431-630 recombinant protein and D39?pepO mutant strain were successfully prepared.The binding sites with TLR2 and TLR4 are proved to be in the M13 N domain of 3-383 amino acid residue.2)After PepO treatment of mouse PDM,the expression of autophagy markers ULK-1 and beclin 1 increased significantly,the conversion of LC3 I to LC3 II was significantly enhanced,and the distribution of LC3 in the cytoplasm was transformed from a scattered form to an aggregation form,suggesting that PepO can induce enhanced autophagy in mouse macrophages.The co-localization of LC3 and S.aureus in PepO treated PDM was significantly increased comparing to untreated PDM(p<0.05),suggesting PepO-induced autophagy is related with phagocytosis.After inhibiting autophagy with Bafilomycin A1,PepO's enhancement of macrophage phagocytosis and bactericidal effect was significantly reduced(p<0.05;p<0.01)3)After stimulation with PepO,tlr2,tlr4 and tlr2/tlr4 deficient PDM observed that the intracytoplasmic LC3 spots were significantly less than wild-type PDM(p<0.05),suggesting that TLR2 and TLR4 are involved in the regulation of autophagy induction by PepO.After PepO stimulation,the autophagy-related signaling molecule mTOR showed a biphasic change,in early phase the phosphorylation level elevated while in late phase the phosphorylation was suppressed.AMPK returned to the baseline level after an early rise.Therefore,it is speculated that AMPK mainly plays a role in the early stage of PepO stimulation,and the role of stimulating late mTOR is more important4)The phagocytosis and bactericidal activity of PepO-treated tlr2 or tlr4 single-defective PDM were significantly enhanced compared to the untreated group(p<0.05),but the phagocytosis and bactericidal effect of PepO-treated tlr2/tlr4 double-defective PDM was not significantly different from the untreated group(P>0.05).After PepO treatment in tlr2/tlr4 deficient mice,there was no significant difference in lung bacterial load(p>0.05),indicating that both TLR2 and TLR4 are involved in the enhancement of PepO-induced bacterial clearance of macrophages.After autophagy was inhibited,the phagocytosis and bactericidal activity of PepO-treated tlr2-deficient PDM showed no significant difference from that of the untreated group(p>0.05),but the phagocytosis and killing effect of tlr4-deficient PDM was still higher than that of the untreated group(p<0.05),indicating that there are other TLR2-related mechanisms involved in the induction of macrophage bacterial clearance by PepO5)The phagocytosis and bactericidal activity of PepO-treated PDM on S pneumoniae D39,S.aureus 502A,and P.aeruginosa PAK were significantly enhanced(p<0.05),and in a dose dependent manner,suggesting that PepO induced a species-independent protection against bacteria.In vivo Studies have shown that after PepO treatment,the bacteria load in lungs of S.pneumoniae D39 and multidrug-resistant P.aeruginosa was significantly reduced(p<0.05),suggesting that PepO can increase the host's resistance to respiratory bacterial infections.After the removal of macrophages in mice by chloride phosphate liposomes,there was no significant difference in bacteria load between the PepO-treated mouse and the PBS-treated group(p>0.05),suggesting that the PepO induced bacteria clearance enhancement was mediated by macrophages.2.PepO induce trained immunity by manipulating glycolysis1)After PepO treatment,the glucose consumption of BMDM increased significantly compared with the PBS treatment group(p>0.05),suggesting that PepO is involved in the regulation of macrophage metabolism.2)After PepO training,the H3K4me3 methylation and H3K27Ac acetylation in the promotor region of TNF-?and IL-6 elevated significantly,indicating the epigenetic reprogramming induced by PepO treatment;3 days after PepO treatment,the phagocytosis and bactericidal activity against S.aureus of BMDM is significantly higher than PBS treated BMDM,suggesting that PepO efficiently induced trained immunity in BMDM3)After pretreatment of BMDM with the glycolysis inhibitor BPTES to block the glycolysis pathway,the phagocytosis of PepO-treated BMDM was significantly lower than that of the glycolysis un-inhibited group Meanwhile,the H3K4me3 methylation and H3K27Ac acetylation in the promotor region of TNF-?and IL-6 were significantly lower than those in the glycolysis un-inhibited,this proved that glycolysis was involved in the process of PepO-induced training immunity4)After PepO training,the phagocytosis of tlr2-deficient BMDM was still partially enhanced(p<0.05),but there was no significant difference in phagocytosis compared with the PBS group(p>0.05),suggesting that TLR2 and PepO induced training immunity Establishment;and in tlr2-deficient BMDM,BPTES pretreatment still significantly reduces the enhancement of PepO training on phagocytosis(p<0,05),suggesting that there are other mechanisms besides TLR2 that induce training immunity via glycolysisConclusion:In this study,we explored the role of TLR2/TLR4 dual ligand protein PepO as an immunomodulator to enhance innate immune defense and its molecular mechanism.We found that PepO promoted the phagocytosis and bactericidal activity of macrophages by inducing autophagy,and therefore led to the enhancement of anti-bacteria defense in host pulmonary tract.We confirmed that this effect of PepO relied on both TLR2 and TLR4 and is species-independent,which is effective against multiple pathogens.On the other hand,PepO induces enhanced glycolysis of macrophages through TLR2/TLR4,thereby inducing macrophages to establish training immunity,thereby long-term enhancing the responsiveness of the innate immune system to invading pathogens.The above results indicate that PepO can significantly enhance the host's innate immune defense and is a protein immunomodulator with potential therapeutic and preventive value.