| Pasteurella multocida（P.multocida）is a gram-negative pathogen and can be divided into five capsular serotypes（A,B,D,E and F）according to different capsular antigens,which can cause diseases in poultry,livestock,and even humans.In cattle,P.multocida type A mainly leads to respiratory diseases and pneumonia,causing huge economic losses to the cattle industry.At present,the prevention and treatment of the disease caused by bovine P.multocida mainly depends on vaccines and antibiotics.However,there is only vaccine of P.multocida type B for bovine hemorrhagic sepsis,no effective vaccine for P.multocida type A in China,and the cross-protection effect of vaccines between different serotypes is low.Furthermore,antibiotics resistance and environmental pollution have brought great challenges to the prevention and treatment of the disease infected by P.multocida.Therefore,the development of safer and more effective compounds against P.multocida infection has important theoretical and practical significance.The previous study of our laboratory found that after the infection of P.multocida type A strains CQ2（Pm CQ2）,a large number of differentially expressed genes（DEGs）were mainly enriched in amino acid metabolism-related pathways such as serine and tryptophan according the results of RNA-seq analysis of mouse lung tissue,and the content of free amino acids in lung tissue of mice changed significantly before and after infection.Additionally,exogenous L-serine lowers the secretion of inflammatory cytokines in lung tissue of mice and macrophage infected by Pm CQ2,but has no direct inhibitory effect on Pm CQ2,suggesting L-serine may resist Pm CQ2 infection by affecting the host immune response.However,the specific mechanism remains unknown.In addition,the tryptophan metabolic pathway was also significantly enriched after Pm CQ2 infection,especially its downstream metabolite-melatonin metabolic pathway remodeling,including the significant up-regulation of melatonin metabolism-related enzymes,and the decreased melatonin content in lung tissue and serum,indicating melatonin plays an important role in the process of Pm CQ2 infection.Therefore,this study mainly explored the specific role and mechanism of L-serine and tryptophan metabolite-melatonin in the process of Pm CQ2 infection.The main results are as follows:1.L-serine combats P.multocida infection by regulating macrophage-and/or neutrophil mediated inflammation2 mg/kg of exogenous L-serine could significantly improve the survival rate of mice,and reduce the bacterial colonization,the lung lesions as well as inflammatory responses of mice during Pm CQ2 infection.Additionally,analysis of the results of transcriptomic sequencing of mouse lung tissue indicated that macrophages were the most significantly enriched immune cells after Pm CQ2infection.Then,the mouse alveolar macrophages were depleted by liposomes clorophosphate,it was found the anti-inflammatory effect of L-serine was inhibited,but not completely inhibited,and neutrophil compensation increased.Next,neutrophils were depleted using an anti-Ly6G monoclonal antibody,consistently,the anti-inflammatory effect of L-serine was partially inhibited,and macrophage compensation increased.Finally,after simultaneously removing alveolar macrophages and neutrophils,the results of immunohistochemistry and ELISA showed that the inhibitory effect of Lserine on the hyperinflammatory response induced by Pm CQ2 infection is completely abolished.The above results indicate that L-serine lowers the inflammatory response mediated by macrophages and neutrophils.2.L-serine inhibits macrophage polarization towards pro-inflammatory M1 type by reprogramming macrophage transcription and metabolismTo explore the inhibitory mechanism of L-serine on Pm CQ2-induced macrophage inflammation,the cellular function analysis was performed.It was found that the addition of 10m M L-serine did not affect the cell activity and NO production of macrophages,but significantly inhibited the secretion of inflammatory cytokines IL-1β,TNF-α,IFN-γ,and IL-17 in macrophage infected by Pm CQ2（MOI=1）or activated with classical LPS+IFN-γ（1μg/m L+20 ng/m L）.The specific signaling pathway was further analyzed by Western blot,and it was found that L-serine significantly inhibited the activation of inflammasome NALP1,AIM2,NLRP3（Caspase-1）,NLRC4 and the expression of HIF-1α.The transcriptomic sequencing of macrophage found that L-serine reprograms the transcription of macrophages,and the DEGs are mainly involved in the biosynthesis and metabolism of substances（amino acids）.The RT-PCR validation results were consistent with the transcriptome sequencing,indicating that L-serine might mainly affect the metabolic process of macrophages.Then,metabolomic analysis of macrophages was performed,and found that L-serine treatment resulted in a large number of differential metabolites,including lower levels of glucose,isomaltose,lactose,cellobiose,fructose,etc.The KEGG functional enrichment analysis showed that L-serine mainly affects the metabolism of galactose,starch,sucrose,fructose and mannose in inflammatory macrophages.Further experiments on cellular respiration found that 10 m M L-serine significantly inhibited the glycolysis process of macrophages,while having little effect on their oxidative phosphorylation level,indicating that 10 m M L-serine inhibits the polarization of macrophages towards the M1 type by inhibiting glycolysis process.3.Tryptophan metabolite-melatonin against P.multocida infectionTo explore the role of tryptophan metabolite-melatonin in Pm CQ2 infection,in this study,the preventive effect of melatonin was first explored.Exogenous melatonin（30 mg/kg,60 mg/kg and120 mg/kg）was supplemented by intraperitoneal injection for 6 or 7 consecutive days,followed by intraperitoneal inoculation of Pm CQ2（2.2×105 CFU）.The therapeutic effect of melatonin was then explored,after infected with 2.2×105 CFU of Pm CQ2 by intraperitoneal injection,the mice received intraperitoneally melatonin（30 mg/kg,60 mg/kg and 120 mg/kg）for four times,first 30min after Pm CQ2 infection,the next three times every 6 h.It was found melatonin（30 mg/kg,60mg/kg and 120 mg/kg）significantly improved the survival rate of mice infected by Pm CQ2,decreased the bacterial colonization in the lungs of mice,and reduced the secretion of inflammatory cytokines in lung tissue and serum,and the combination of prevention and treatment has better anti-infection effect.4.Tryptophan metabolite-melatonin inhibits Gram-negative pathogens by targeting citrate synthase R300This study further found that melatonin can directly inhibit the growth of Pm CQ2 in vitro,and has a broad-spectrum antibacterial effect.Transcriptome sequencing results of bacteria found that DEGs were mainly enriched in membrane transport and carbohydrate metabolism-related pathways.Additionally,melatonin can increase the permeability of bacterial cell membranes,causing the leakage of macromolecular substances such as proteins and sugars.The results of scanning electron microscopy and transmission electron microscopy further found that melatonin causes bacterial cell membranes to appear folds,depressions,holes and cavitation,indicating that melatonin damages the integrity of bacterial cell membranes.The results of bacteria metabolomic analysis showed that a large number of differential metabolites were mainly enriched in the tricarboxylic acid cycle pathway,and the two upstream substrates,acetyl-Co A and pyruvate,were up-regulated after melatonin treatment,while the downstream product,citric acid,was down-regulated.Further experiments found that melatonin inhibits the activity of citrate synthase,the key enzyme in this process.Subsequently,the citrate synthase（glt A）gene deletion strain（MG1655Δglt A）and the complementing strain（MG1655Δglt A/Pglt A）were constructed,and the in vitro antibacterial experiment was performed again.It was found that compared with MG1655 and MG1655Δglt A/Pglt A,melatonin did not affect the membrane permeability of MG1655Δglt A,indicating that melatonin inhibits the growth and reproduction of bacteria by reducing the content of citric acid via inhibiting the activity of citrate synthase.To explore how melatonin specifically affects citrate synthase,type II citrate synthase was expressed and purified by prokaryotic expression system.Compared with NADH(binding energy:1.77×10-4 mol/L),a known inhibitor of type II citrate synthase,melatonin was found to be more sensitive to type II citrate synthase of negative bacteria(binding energy:7.62×10-5 mol/L),but hardly binds to type I citrate synthase（commercial product as a control）by surface plasmon resonance analysis（LSPR）.Then,homology modeling and computer molecular docking analysis found that melatonin can form hydrophobic interactions with four amino acid residues（N361,L301,M302 and H265）of type II citrate synthase,and form hydrogen bond with D363,R300,P263 and R307,in which the forces of R300,D363 and H265 are stronger than others,and R300>D363>H265.Therefore,point mutations at these three sites were made,and then mutants CS（R300A）,CS（R300A,D363A）and CS（R300A,D363A,H265A）were expressed and purified.Finally,LSPR analysis found that the affinity of these three mutants to melatonin was significantly down-regulated,which were 1.83×10-4,2.65×10-4 and 2.83×10-4 mol/L,respectively,indicating that melatonin directly targets the R300,D363 and H265 sites of gram-negative bacteria type II citrate synthase,and the R300 site may play a major role.The above results show that L-serine regulates the polarization state of macrophages by reprogramming transcription and metabolism to inhibit Pm CQ2-induced hyperinflammation,providing a new nutritional strategy for the prevention of pathogenic bacterial infections;and tryptophan metabolite-melatonin inhibits the enzymatic activity of gram-negative bacteria type II citrate synthase by targeting the R300 site,reducing citric acid synthesis and thus affecting bacterial growth and reproduction to play an anti-Pm CQ2 infection role,which provides a new target and research idea for the prevention and control of bacterial infectious diseases and the development of new antibacterial drugs. |
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