Identification of a Nocardia seriolae secreted protein targeting host cell mitochondria and inducing apoptosis in fathead minnow (FHM) cells

Abstract

Nocardia seriolae, is a Gram-positive, partially acid-fast, aerobic, and filamentous bacterium. This bacterium is the main pathogen of fish nocardiosis. A bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503 showed that ORF3141 encoded a secreted protein with a signal peptide at the N-terminate which may target the mitochondria in the host cell. However, the functions of this protein and its homologs remain unknown. In this study, we experimentally tested the bioinformatic prediction on this protein. Mass spectrometry analysis of the extracellular products from N. seriolae showed that ORF3141 was a secreted protein. Subcellular localization of the ORF3141-GFP fusion protein revealed that the green fluorescence protein co-localized with the mitochondria, while ORF3141{Delta}sig-GFP (with the signal peptide deleted) fusion protein was evenly distributed in the whole cell of fathead minnow (FHM) cells. Thus, the N-terminate signal peptide had a significant impact on mitochondrial targeting. Notably, the expression of ORF3141 protein changed the distribution of mitochondria from perinuclear halo into lumps in the transfected FHM cells. In addition, apoptotic features were found in the transfected FHM cells by overexpression of ORF3141 and ORF3141{Delta}sig proteins, respectively. Quantitative assays of mitochondrial membrane potential value, caspase-3 activity and apoptosis-related gene mRNA expression suggested that cell apoptosis was induced in the transfected FHM cells. In conclusion, the ORF3141 was a secreted protein of N. seriolae that targeted host cell mitochondria and induced apoptosis in FHM cells. This protein may participate in the cell apoptosis regulation and plays an important role in the pathogenesis of N. seriolae.\n\nAuthor summaryNocardia seriolae is the causative pathogen responsible for fish nocardiosis. This facultative intercellular bacterium, adapts to survive and colonize by evading intracellular killing after being engulfed with macrophages in the host. Despite considerable economic losses caused by N. seriolae in fish infection, the pathogenic mechanism and specific virulence factor of this bacterium remain ambiguous. In this study, the characteristic of ORF3141 protein function was investigated by subcellular localization and its possible contributions on the ability of N. seriolae to induce apoptosis in transfected fathead minnow (FHM) cells was investigated. Here, we confirmed that ORF3141 was a secreted protein that targeted host cell mitochondria and induced cell apoptosis in FHM cells. Interestingly, after deleting the signal peptide, ORF3141{Delta}sig protein was evenly distributed in the whole host cell and did not co-localize with the mitochondria which could also induce cell apoptosis. Thus, the N-terminate signal peptide played an important role in mitochondrial targeting, and the domain part without the signal peptide had a critical relationship with cell apoptosis. These results demonstrated that ORF3141 mays act as a potential virulence factor that induces apoptosis in fish cells. This protein is significant to elucidate the pathogenic mechanism of N. seriolae and this study mays provide beneficial insight to prevent and treat fish nocardiosis.