Mechanisms Of MnmE Protein Regulating Pathogenicity And Environmental Adaptation Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae（APP）is a Gram-negative bacterium,a member of the genus Actinobacillus in the Pasteurellaceae family.It was the main cause of pleural pneumonia in pigs.The disease is characterized by hemorrhagic necrotizing pneumonia and fibrinolytic pleurisy,with a high rate of infection and fatality.Therefore,it is very important to clarify the growth characteristics of APP under different conditions and analyze the related t RNA modifying enzymes for understanding the molecular mechanism of APP growth and rapid response to environmental pressure.Under the background that the country advocates non-resistant breeding,only by creating effective prevention and control preparations can diseases be fundamentally controlled.In previous work,we have demonstrated that the signaling molecule（p）pp Gpp can regulate the phenotype and virulence of APP,but have not yet conducted research on the target protein in vivo.First,verify whether MnmE is the target protein of（p）pp Gpp in APP,and perform isothermal titration calorimetry（ITC）.The results show that the affinity between MnmE and（p）pp Gpp is about 5 times that of GTP,indicating that MnmE is also the target protein of（p）pp Gpp in APP.Therefore,a series of biological characteristics analysis and determination,intracellular hydrogen peroxide（H₂O₂）content,ribosome map,protein synthesis rate and pathogenicity were carried out by constructing gene mutant strains（Δmnm E）and reincarnation strains（Cmnm E）.The function of MnmE from different sources and its induced phenotype provide a theoretical basis.Through the determination of growth curves,the results showed thatΔmnm E had disordered growth（enhanced/weakened）when 5 amino acids were absent.Transmission electron microscopy（TEM）,survivability,biofilm and stress ability were used to determine the growth and survival of APP,andΔmnm E decreased the anti-stress ability in vitro.The viability ofΔmnm E in adverse environments was explored by simulating in vitro hyperosmotic pressure and oxidative stress tests.The results showed thatΔmnm E had reduced osmotic pressure and oxidative stress tolerance.In order to further study the mechanism of MnmE to explore the effect on APP,we conducted intracellular H₂O₂ content,ribosome profiling,protein synthesis rate and pathogenicity experiments of three strains.The results showed thatΔmnm E increased intracellular hydrogen peroxide（H₂O₂）.Compared with WT,Δmnm E reduced the distribution of ribosome size subunits and inhibited the rate of protein synthesis.The pathogenicity test and LD₅₀ determination of the G.mellonella showed that showed that the mutation of mnm E reduced the virulence of APP by about 1/6 times that of WT.In this study,we further validated the function of MnmE in APP at the translational level.Based on label-free quantitative proteomic data,100 differentially expressed proteins（36 up-regulated and 64 down-regulated）were identified inΔmnm E compared to WT.Many proteins associated with protein modification,metal ion transport,and stress were down-regulated.Nineteen metabolic pathways were affected,which are critical for the growth of APP.We conclude that MnmE from APP is essential for cell growth,stress adaptation,protein synthesis,and virulence.The binding of MnmE to（p）pp Gpp in APP was verified by isothermal titration calorimetry（ITC）.The results showed that the affinity between MnmE and（p）pp Gpp was about5 times that of GTP,which proved that MnmE in APP was the target protein of（p）pp Gpp.Therefore,in order to further study the mechanism of MnmE’s influence on the growth and biological characteristics of APP,a series of studies were conducted on its growth characteristics,intracellular hydrogen peroxide（H₂O₂）content,ribosome assembly,protein synthesis rate and pathogenicity based on the construction of gene mutant（Δmnm E）and complement strain（Cmnm E）.The results showed that compared with WT,the growth ability ofΔmnm E was disordered（enhanced/weakened）when five amino acids were deleted,and the growth changed greatly;the content of intracellular hydrogen peroxide（H₂O₂）inΔmnm E increased by 1.5 times;At the same time,the mutation of mnm E inhibited the distribution of APP ribosome size subunits and reduced the rate of protein synthesis;the pathogenicity test was carried out through the G.mellonella model to explore its virulence changes,and the results showed that the mutation of mnm E caused APP pathogenicity to be about 1/6 of that of WT,and the pathogenicity was significantly reduced.In order to study the resistance ability of different APP strains to adverse external environment,survival ability,biofilm and stress ability tests were conducted.The results showed thatΔmnm E’s survival ability was 1/2 times that of WT.ΔThe osmolality and oxidative stress tolerance of mnm E were significantly decreased（about four orders of magnitude）compared with that of the wild strain.The results showed that the viability ofΔmnm E was 1/2 times that of WT;at the same time,the survival ability ofΔmnm E mutant under the adverse environment of high osmotic pressure and oxidative stress was simulated in vitro.The results showed that the osmotic pressure and oxidative stress tolerance ofΔmnm E were significantly reduced by about four orders of magnitude compared with wild strains.Based on the above results to further verify the function of MnmE in APP,label-free quantitative proteomics was carried out to analyze the differential proteins between WT andΔmnm E,and bioinformatics analysis was carried out.The results showed that a total of 100 proteins were differentially expressed（36 up-regulated and 64 down-regulated）,and mutations in mnm E caused many proteins related to protein modification,metal ion transport and stress to be down-regulated,resulting in 19 metabolic pathways affected,which are critical for the growth of Actinobacillus pleural pneumoniae.In conclusion,MnmE from Actinobacillus pleuropneumoniae is essential for cell growth,stress adaptation,protein synthesis and virulence.To sum up,the results of this study show for the first time that MnmE is the target protein of the semaphore molecule（p）pp Gpp in APP,and can also participate in the ribosome assembly of APP,protein synthesis and its biofilm formation ability.It also affects the tolerance and pathogenicity of APP,and regulates its growth under the nutrient environment of amino acid deletion.This result provides a reference for further elucidating the pathogenic mechanism of APP.