The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in humans and persistent infections in animals, enabling widespread environmental reservoirs. Intracellular survival of S. Typhimurium depends on a suite of type III secretion system effectors encoded within Salmonella Pathogenicity Island 2 (SPI-2), many of which also impair host cell migration. Among these, SspH2 is a novel E3 ubiquitin ligase (NEL) effector, but how it inhibits host cell movement has remained unclear. By using a combination of time-lapse microscopy and wound-healing assays, we show that SspH2 is required for S. Typhimurium to fully suppress migration of macrophage-like RAW264.7 cells and that conditional expression of SspH2 alone is sufficient to restrict their movement. SspH2 expression led to a marked reduction in F-actin and fewer cells containing higher-order F-actin structures, indicating cytoskeletal remodeling. In contrast, the catalytically inactive mutant SspH2C580A had no effect, demonstrating that E3 ligase activity is essential for these phenotypes. Knockdown of the host cytoskeletal factor vinculin, a core component of podosomes and focal adhesions, mimicked SspH2's effect on cell migration. When vinculin expression was reduced, SspH2 did not further inhibit migration, suggesting that SspH2 requires vinculin-associated F-actin-rich structures to block cell migration. These findings reveal a new pathway by which S. Typhimurium limits host cell movement, potentially enhancing immune evasion and fitness within the host.