Identification And Functional Study Of Lipoate-protein Ligase Mhp-LplJ In Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae(Mhp)is the pathogen of Mycoplasmal pneumonia of swine(MPS).Although the mortality rate of MPS is low in production practice,the proliferation and adhesion of Mhp in the respiratory tract leads to depressed defense ability of the respiratory system,and is easy to cause secondary infection of other respiratory pathogens,resulting in porcine respiratory disease complex(PRDC).As a result,the case fatality rate is greatly increased and serious economic losses are caused.In view of Mhp clinical chronic infection is increasingly serious and biosynthesis is limited,our laboratory group has carried out a series of researches on the key enzymes in Mhp growth and metabolism,laying the foundations for Mhp infection prevention and developing new drug targets.In the previous research project about enzymes related to Mhp lipoic acid metabolism,researchers verified the biological activity of the putative lipoate-protein ligase Mhp-Lplthrough in vitro experiments with the Glycine cleavage system H protein(Gcv H)as the substrate.And also analyzed the crystal structure and structural domain of Mhp-Lpl.In the further study on lipoic acid metabolism system of Mhp,dihydrolipoamide dehydrogenase(PdhD)was identified as another lipoate-dependent substrate of Mhp.PdhD is the E3 subunit of Mhp pyruvate dehydrogenase complex(PDH).Its normal lipoylation is essential for the catalytic activity of PDH.The results of ligation assays in vitro indicated that Mhp-Lplcan not complete the lipoylation modification of PdhD,which proves that there are other lipoic acid metabolism-related enzymes in Mhp.In addition to Mhp-Lpl,there is an open reading frame MHP?RS01680 encodes another putative lipoate-protein ligase in the genome of Mhp through bioinformatics analysis.Because of the high similarity between the amino acid sequence of MHP?RS01680 and the lipoateprotein ligase LplJ of Bacillus subtilis,the protein encoded by MHP?RS01680 is named as MhpLplJ.The results of sequence analysis and structural simulation showed that Mhp-LplJ has the typical structure of lipoate-protein ligase,conserved catalytic active site and lipoyl binding motif.The results in vitro showed that Mhp-LplJ has an extensive lipoate ligase activity against Mhp Gcv H,PdhD and the lipoate-dependent protein of Escherichia coli(E.coli),dihydrolipoic acid transacetylase(Dihydrolipoamide acetyltransferase,Ace F)and dihydrolipoyl succinyltransferase(Dihydrolipoyllysine-residue succinyltransferase,Suc B).The lipoate ligase activity of Mhp-LplJ was further verified by cell-based lipoylation assay with the lipoylation deficient E.coli strain as research model.Except for the lipoate liagse activity,Mhp-LplJ also showed specific octanoate ligase activity against PdhD in the study of modification activity tests based on Mhp PdhD substitute strain.The octanoate ligase activity was also further verified by in vitro experiments.Considering that PdhD,as the E3 subunit of pyruvate dehydrogenase complex(PDH),plays an important role in the growth,metabolism and energy transfer of Mhp.This study attempted to affect the growth and metabolism of Mhp by inhibiting the lipoate modification activity of Mhp-LplJ on PdhD.The lipoic acid analogs 8-bromooctanoic acid(8-Br O)and 6,8-dichlorooctanoate(6,8-diClO),which have different affinity activities with Mhp-LplJ,were selected for further study.Ligation assays in vitro showed that 8-Br O and 6,8-diClO have different degrees of interference on the function of Mhp-LplJ.Further growth inhibition experiments showed that the lipoate analogs inhibit Mhp growth in a dose-dependent manner.And compared with 6,8-diClO,8-Br O with higher affinity to Mhp-LplJ has a more obvious inhibitory effect.The PdhD lipoylation level of Mhp cultured with lipoic acid analogs at different concentrations also showed that the growth rate of Mhp was positively correlated with the degree of lipoate modification of PdhD.All of these phenomena indicate that when the lipoate ligase activity of Mhp-LplJ against PdhD was interfered,the growth rate of Mhp was significantly inhibited,and reflect that Mhp-LplJ has the potential to be explored as a novel drug target for the treatment of Mhp infection.The experimental results of this study show that E3 subunit of PDH,PdhD,is a lipoatedependent substrate of Mhp,and its lipoylation modification is completed by the novel lipoateprotein ligase Mhp-LplJ.The activity interference of Mhp-LplJ can significantly inhibit the growth of Mhp.It is proved that Mhp-LplJ plays an important role in the lipoic acid metabolism of Mhp.The in-depth studies on Mhp-LplJ are of great significance to further understanding the growth and metabolism characteristics of Mhp,and to develop new therapeutic drugs for MPS.