The Functional Study Of Mitochondrial Targeting Glutamyl Endopeptidase From Nocardia Seriolae

China is a big country in aquaculture.With the rapid development of aquaculture,aquatic diseases cast a shadow on aquaculture industry.In recent years,fish nocardiosis has become a serious aquatic diseases in aquaculture.When aquatic animals are in weak condition and immunocopromised state,pathogens can infect them through gills,feeds or wounds.The symptoms of the disease are anorexia,abdominal distention,and diffuse distribution of dense granulomas in the internal organs.Due to lack of effective control measures,fish nocardiosis caused excessively high mortality and infection rate and brought enormous loss in aquaculture.Nocardia seriolae has become the main pathogen of fish nocardiosis recently,which can affect about 39 kinds of marine fish and freshwater species.However,the pathogenic mechanisms and the virulence factors of N.seriolae are still unclear.By analyzing the genome of N.seriolae ZJ0503,we found a Glutamyl endopeptidase homolog encoded by ORF6296 and named it as Glu NS.Protein Glu NS was predicted to be a mitochondrial targeting secretory protein and speculated to be a potential virulence factor of N.seriolae.In this study,Glu NS gene was cloned and its function was analyzed according to its' sequence.Glu NS was verified whether or not to be a secretory protein by identifying the extracellular products of N.seriolae using mass spectrometry.Recombinant plasmids of Glu NS were constructed to study the subcellular localization and pathogenicity of Glu NS in fish cells.Glu NS-deleted and complemented strains were constructed by homologous recombination to investigate the changes of bacterial pathogenicity both in fish and cells.Through this study of Glu NS which is a mitochondrial targeting secretory protein from N.seriolae,it will clarify the function of Glu NS on the pathogenesis of nocardial infection,promote a deeper understanding of the pathogenic mechanism of N.seriolae,and provide a theoretical basis for the prevention and treatment of fish nocardiosis.The main results and conclusions are as follows:1.Glu NS gene was cloned successfully,and found that the protein encoded by Glu NS genewas a glutamyl endopeptidase homolog without any obvious signal peptide site,whichbelongs to Streptomyces group.Glu NS was identified as secretory protein by massspectrometry and co-located with mitochondria in fish cells,and it also inferred that thefunction of Glu NS was relative to mitochondria.The overexpression experiment foundthat at 48 hour post transfection(h.p.t.)with pc DNA-Glu NS,the cells became round,thenucleus shrinkage and apoptosis body appeared in the cells.The activity of caspase-3rose significantly and ??m was degraded obviously.These results confirm that Glu NScan induce apoptosis in fish cells.2.By using the homologous recombination,the NS-?Glu NS strain and NS-c?Glu NS strainwere constructed,and their characteristics were compared with those of wild strain(N.seriolae ZJ0503).The results showed that there was no difference in the gene deletionin the liquid culture,but in the solid culture,the deletion strains showed growthretardation.3.In order to further explore the pathogenic of NS-?Glu NS strain.(1)At fish level,hybirdsnakehead(C.maculata × C.argus)were challenged with the NS-?Glu NS strain andNS-c?Glu NS strain by intraperitoneal injection to measure the median lethal dosen(LD50).The LD50 of NS-?Glu NS strain was higher than that of the wild strain with asignificant difference,while there was no distinct difference between the NS-c?Glu NSstrain and wild strain.These results showed that the deletion of Glu NS reduced thepathogennicity of N.seriolae,suggested Glu NS is a pivotal virulence gene of N.seriolae.To futher investigate the ability of intracellular survive and cell infection of Glu NS-deleted N.seriolae.(2)In the aspet of cell,the effects of Glu NS on N.seriolae inducedrespiratory burst and cell infection were studied.The results showed that Glu NS hadlittle effect on the escape of N.seriolae from macrophage phagocytosis and inhibition ofmacrophage respiration;and Glu NS gene isn't closely related to the infection ability ofN.seriolae.In summary,Glu NS may not exert its virulence factor function by affectingmacrophage function and infecting host cells.