Abstract: Objective: Dentifying bacterial effector proteins secreted by Mycobacterium marinum during infection of macrophages provides a theoretical foundation for elucidating its intracellular survival mechanisms and addressing drug resistance issues in non-tuberculous mycobacteria. Methods: Mycobacterium marinum infection of RAW 264.7 mouse macrophages was used to identify bacterial-derived secreted proteins systematically via liquid chromatography-tandem mass spectrometry. This was complemented by bioinformatics approaches, including gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and protein-protein interaction analysis. Results: A total of 1,629 bacterial secreted proteins were identified in the supernatant of RAW 264.7 cells infected with Mycobacterium marinum. GO terms predominantly enrich biological processes such as bacterial metabolism and stress responses, as well as molecular functions including enzyme activity. KEGG pathway enrichment analysis identified the RNA degradation pathway as the most significantly enriched pathway, involving nine secreted proteins. The key molecules identified through PPI analysis primarily relate to ribosomal function. Conclusion: This study systematically elucidates for the first time the types of proteins secreted by Mycobacterium marinum within host macrophages and their functional characteristics. These proteins are significantly enriched in key pathways such as bacterial metabolism and RNA degradation. Concurrently, effector proteins of Mycobacterium marinum, such as MMAR_1032 and MMAR_1052, may function as pivotal hub molecules in the pathogenesis and progression of non-tuberculous mycobacterial infections.

Key words: Mycobacterium marinum infection, Mycobacterium marinum secreted protein, macrophage, proteomics