Study On The Effect Of Mycoplasma Hyopneumoniae Nuclease On The Evasion Of Porcine Alveolar Macrophages Extracellular Traps

Mycoplasma hyopneumoniae（Mhp）is the pathogen of mycoplasmal pneumonia of swine（MPS）,which mainly colonizes the lungs.Porcine alveolar macrophages（PAMs）,located on the surface of alveolar cavities,are an important defense against invasion of pathogenic microorganisms.After Mhp enters the body,it inevitably comes into contact with PAMs that reside in the lungs.However,Mhp can exist in the infiltrated lung environment of PAMs for a long time without being easily removed,which proves that Mhp has a mechanism of resisting the action of PAMS.1.Study on the interaction between Mhp and PAMs and the clearance of PAMsIn this study,laser confocal microscopy was used to observe that Mhp could adhere to the surface of primary PAMs after incubation,and could be swallowed by a small amount of primary PAMs.The ability of Mhp high virulent strain 168 to infect primary PAMs was significantly stronger than that of Mhp low virulent strain 168L by q PCR quantitative detection.The CCU₅₀（Color Change Unit）results showed that a considerable number of live Mhp could still be detected after Mhp was incubated with primary PAMs for 36 hours.The results of gentamicin killing test showed that Mhp could not survive in the primary PAMs,suggesting that Mhp mainly survived on the surface of the primary PAMs after interacting with the primary PAMs.Recent studies have found that Macrophages can not only clear pathogens through phagocytosis,but also produce Macrophages extracellular traps（METs）to capture and kill pathogenic microorganisms such as bacteria,fungi and viruses.In this study,by laser confocal microscope observation,DNA fibrous structure could be observed in the primary PAMs treated with Mhp,and the significant expression of citrate histone H4Cit3 related to METs formation was found in the immunofluorescence test.It was proved that Mhp could induce METs structure in primary PAMs.Mhp can survive on the surface of primary PAMs for a long time without being cleared by primary PAMs,suggesting that there may be some mechanisms for Mhp to resist the killing effect of METs on the surface of PAMs.2.Study on the effect of Mhp nuclease on the evasion of METsDue to the simple structure of mycoplasma,its ability to synthesize nucleic acid substances needed for its own growth is limited,so it needs to generate nucleases to degrade exogenous nucleic acids to obtain the raw materials needed for its own growth.DNA is the basic skeleton of METs.Therefore,whether nuclease can degrade METs generated by PAMs and help it resist the killing effect of METs needs further study.The results of agarose gel electrophoresis showed that the supernatant of Mhp and its culture had nucleic acid degradation activity.RT-PCR results found four nuclease-related proteins highly transcripted in Mhp high virulent strain,namely Nuc,Nfo,Exo and Mnu A.In this study,these four proteins were recombined and purified,and the four proteins were incubated with calf thymus DNA respectively.It was found that Nfo protein had the most obvious degradation effect on nucleic acid substrate,and its polyclonal antibody could effectively block the degradation of METs by Mhp.Flow cytometry was used to further analyze the localization of Nfo on the membrane surface of Mhp strain.It was found that there were components that could be recognized by Nfo antibody on the surface of Mhp strain,and the content of Nfo protein in the strong strain was significantly higher than that in the weak strain.The surface localization of Nfo protein was conducive to the interaction between Mhp strain and METs,which suggests that Nfo protein might be a potential virulence factor of Mhp.The nuclease Nfo amino acid sequence was compared with the corresponding sequences of various animal mycoplasmas,and it was found that the nuclease Nfo had high homology（76.8%）in various animal mycoplasmas,including Mycoplasma hyorhinis,Mycoplasma bovis and Mycoplasma ovipneumoniae.It is suggested that the protein may have universal applicability both as a virulence factor and as a target for prevention and control.In summary,this study detected the phagocytosis and killing of Mhp after the primary PAMs was incubated with Mhp,and preliminarily explored the role of nuclease in Mhp’s resistance to METs killing.The possible mechanism of Mhp causing persistent infection in host was analyzed from the perspective of anti-PAMs killing clearance,and a new target was provided for the prevention and control of MPS and the research and development of drugs to treat the disease.