Intracellular Behavior Of Nocardia Seriolae And Its Apoptotic Effects On Macrophages

Nocardia seriolae,an opportunistic pathogen,highly intensive breeding,improper breeding management,and deteriorated aquaculture environments may lead to a large proliferation of pathogenic bacteria,which infect fish with hypoimmunity or epidermal damage.The manner in which N.seriolae invades the organism varies slightly among fish species,mainly through the gills,anus,and damaged skin.N.seriolae mainly infects fish species with high economic value such as Perciformes.N.seriolae causes nocardiosis with a long incubation period and disease duration,difficult to be detected in the early stage of infection,high morbidity and mortality rates,which has caused large economic losses to the aquaculture industry.N.seriolae is an intracellular bacterium,but the research on its interaction mechanism with host cells is still blank.To understand the pathogenic mechanism of N.seriolae,and to elucidate the intracellular behavior of N.seriolae invading macrophages,survival in the cells,and its apoptotic effect on macrophages,the infection mode of mouse macrophage RAW264.7 infected with N.seriolae was established.Laser confocal co-localization,inverted fluorescence microscopy colocalization,transmission electron microscopy,and flow cytometry were conducted to observe the intracellular behavior of N.seriolae-infected macrophage RAW264.7.The levels of reactive oxygen species and nitric oxide released and the expression of inflammatory factors at the m RNA level were detected to determine the cellular inflammatory and immune responses induced by N.seriolae.Dual fluorescence,mitochondrial membrane potential assay,LDH cytotoxicity assay,DNA Ladder,and the expression of apoptosis-related factors at the m RNA level were detected to determine the apoptotic effect on macrophages infected with N.seriolae.To further observe and verify the intracellular behavior and apoptotic effects on fish macrophages,primary macrophages originating from head kidney of Micropterus salmoides were isolated and identified by specific staining and mpeg1 gene amplification.Macrophage activity was detected by CCK-8 assay and oxygen respiration burst,and morphological and quantitative changes in bacteria and cells during infection were observed by inverted fluorescence microscopy and flow cytometry.Double-fluorescence,LDH release,and mitochondrial membrane potential detection were performed to explore the effect of N.seriolae on the apoptosis of fish macrophages.Laser confocal and inverted fluorescence microscopy revealed that N.seriolae entered macrophages at 2 hours post-inoculation(hpi),were phagocytosed by macrophages at 4～8 hpi,and induced the formation of multinucleated macrophages by severe fusion at 12 hpi.The results of the immune response showed that N.seriolae inhibited the production of reactive oxygen species and releases large amounts of nitric oxide,which persisted in macrophages during infection.The expression levels of the inflammatory factors IL-1β and IL-6 showed an increasing trend,while TNF-α showed no significant changes,indicating that the cells produced an inflammatory response.Flow cytometry,evaluation of mitochondrial membrane potential,release of lactate dehydrogenase,and observation of the ultrastructure of macrophages revealed that apoptosis was induced in the early infection stage and inhibited in the middle and later periods of infection.Additionally,the expression of Bcl-2,Bax,Cyto-C,Caspase-3,Capase-8,and Caspase-9 was induced at 4 hpi and then decreased at 6–8 hpi,indicating that N.seriolae infection induced the activation of extrinsic and intrinsic apoptotic pathways in macrophages,followed by the inhibition of apoptosis to survive inside the cells.Furthermore,high-purity macrophages were isolated from the head kidney of M.salmoides and identified as macrophages after specific staining and amplification of specific genes.The optimal culture conditions were determined to be 1640,1 %penicillin-streptomycin,and 1 % fetal bovine serum.The ability of macrophages to burst oxygen respiration was significantly increased by LPS stimulation(P < 0.05).Similar to the intracellular behavior of N.seriolae infecting mouse macrophages RAW264.7,the bacteria were phagocytosed by M.salmoides head kidney primary macrophages at 2 hpi,the cells become rounded,the rate of adherence was reduced at4 hpi,the bacteria multiplied and surrounded the cells at 6 hpi,and the cells died 8 h after infection.The results of apoptosis-related experiments were also similar to above studies,which showed that the apoptosis rate of macrophages increased,the release of LDH increased and the mitochondrial membrane potential decreased at the beginning of the infection;with the extension of the infection time,the apoptosis rate decreased,the release of LDH and the decrease of mitochondrial membrane potential decreased,indicating that N.seriolae also played a role in promoting and then inhibiting apoptosis of fish macrophages.In conclusion,a model of N.seriola-infected macrophages was established in this study,which comprehensively and systematically revealed the intracellular behavior of N.seriola-invading macrophages,phagocytosis,and changes in the number and morphology of macrophages.N.seriolae may induces apoptosis of macrophages in early stage of infection,followed by the inhibition of apoptosis to survive inside the cells.This study lays a research foundation for further understanding the pathogenesis of nocardiosis and prevention and control of the disease.