Time-Resolved Label-Free Proteomics of SHK-1 Cells After Renibacterium salmoninarum Inoculation Reveals Early...

Renibacterium salmoninarum, the etiological agent of bacterial kidney disease, is a facultative intracellular pathogen whose interaction with salmonid phagocytic cells remains poorly resolved at the protein level. Here, we aimed to define the temporal protein-abundance architecture of SHK-1 macrophage-like cells after R. salmoninarum inoculation and to test whether this response supports broad canonical cell-death pathway engagement. We used label-free quantitative LC-MS/MS proteomics to profile SHK-1 cells over a 48 h post-inoculation time course. Because the design included a single non-infected T0 baseline, analyses were framed as baseline-referenced post-inoculation comparisons rather than a fully controlled mock time course. Of 6842 proteins retained for statistical modeling, 2254 were strictly differentially abundant in at least one contrast relative to T0 (adjusted p < 0.05 and |log₂FC| ≥ 0.585). Perturbation was strongest at 1-2 h and progressively contracted at later time points. Among 1278 recurrent proteins, k-means clustering resolved four temporal modules capturing coordinated remodeling of lysosomal, immunometabolic, cytoskeletal, stress-response, and antioxidant programs. A curated cell-death panel spanning apoptosis, pyroptosis, necroptosis, ferroptosis, and PANoptosis yielded only three detected markers; CASP3 and MLKL met the strict threshold, whereas ACSL4 remained sub-threshold. Overall, early host-cell remodeling, rather than broad canonical death-program execution, was the predominant proteomic signature of SHK-1 cells during the first 48 h after R. salmoninarum inoculation.