Mechanism Of Mhp597 Nuclease In Promoting The Infection Of Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae(M.hyopneumoniae)is the pathogen of mycoplasmal pneumonia of swine,and often infects pigs together with other pathogens.After infected,the tracheal epithelial cells and alveolar epithelial cells will be destroyed,and typical inflammatory lesions and tissue necrosis will be present.Like many other mollicutes,Mycoplasma cannot synthesize de novo pyrimidine and purine bases,and have to degrade and assimilate metabolic precursors from the host by excreting nucleases for their growth and replication.Moreover,mycoplasmal nucleases have the ability to degrade the Neutrophil extracellular traps(NETs)and induce the apoptosis of host cells,which indicate nucleases may play an important role in the process of M.hyopneumoniae infection.M.hyopneumoniae could produce three nucleases namely Mhp597,Mhp109 and Mhp379.We expressed all these three nucleases after TGA correction of the genes and obtained the purified recombinant proteins with nickel affinity chromatography.One microgram of double-stranded DNA(dsDNA),single-stranded DNA(ssDNA),plasmid DNA or RNA could be digested completely in 15 s by 1 μg of rMhp597,which suggesting rMhp597 has a very strong ability to degrade nucleic acid,whereas the activity is limited under the temperature from 17-57 ℃ and Ca2+ and Mg2+ dependent.Comparing to rMhp597,rMhp109 and rMhp379 showed mild degrading activities with active temperature range 17-47 ℃.Neutrophil traps pathogens by releasing neutrophil extracellular traps(NETs)which is consisted of DNA skeleton,and pathogenic bacteria has evolved a kind of specific adaptations to evade neutrophil by digesting NETs with nuclease.In this study,NETs induced by M.hyopneumoniae could be destroyed by rMhp597,rMhp109 and rMhp379,and the NETs-digestion ability of rMhp597 was higher than the other two recombinant nucleases.The culture supemant of M.hyopneumoniae could also digest NETs,which could be blocked by polyclonal antibody of rMhp597,confirming the nucleic acid degradation activity of Mhp597 secreted in the supermant by Mhyopneumoniae.Further quantitative real-time PCR assay demonstrated that NETs destruction could efficiently protect M.hyopneumoniae from being trapped by neutrophils,indicating that NETs-destruction is a kind of mechanism of the immune evade of M.hyopneumoniae.Indirect immunofluorescence showed rMhp109,rMhp379 and rMhp597 were able to adhere to the cytomembrane of porcine alveolar macrophages(PAMs)and were able to induce the apoptosis of PK15 cell,and reduce their viability apparently in a dose-dependent manner.Quantitative real-time PCR assay of the cytokines mRNA in PAMs treated with siRNA or specific inhibitors TAK-242、ST2825、Bay11-7082 showed that rMhp597 could vigorously up-regulated IL-1β、IL-8 and TNF-α via the signal pathways of TLR4、MyD88 and NF-κB。Further studies showed that both rMhp597δTM and rMhp597δ315-377 retained the abilities of cyto-adhesion,cyto-toxicity and up-regulating the expression of proinflammatory cytokines,indicating the deficiency of N-terminal transmembrane region and C-terminal strongly basic polar region has no effect on these biological functions of Mhp597.Besides,rMhp597 down-regulated the expression of type Ⅰ IFN(IFN-α/β)and promoted the multiplication of porcine reproductive and respiratory syndrome virus(PRRSV)rather than rMhp109 and rMhp379,while all of them couldn’t promoted the multiplication of porcine circovirus 2.The qPCR assay showed that the expression level of IFN-α/β-expression associated molecular TLR3,TLR7,TLR8,TLR9 and IRF3 are also suppressed by rMhp597,indicating that rMhp597 may regulate IFN-α/βthrough TLR3,TLR7,TLR8,TLR9 and IRF3 in PAMs cells.Studies on the truncated proteins rMhp597δTM and rMhp597δ315-377 proved that the deficiency of the two regions has no effect on immunosupression function of rMhp597.In conclusion,all of rMhp109,rMhp379 and rMhp597 possess the abilities of digesting nucleic acids,cyto-adhesion and cyto-toxicity,and are able to protect M.hyopneumoniae from being trapped by neutrophils by destroying NETs.Mhp597 is an important virulence protein secreted by M.hyopneumoniae with very high nuclease activity and is able to up-regulate the expression of pro-inflammatory cytokines,down-regulate the expression of IFN-α/β and promote the replication of PRRSV.