Tonosaki, Y.; Yamazaki, K.; Owada, S.; Yamaguchi, K.; Kashimoto, T.

Necrotizing soft tissue infections (NSTIs) are fulminant bacterial diseases characterized by rapid tissue destruction, systemic deterioration, and high mortality. Aeromonas hydrophila is an important causative agent of NSTIs, but the system-level bacterial mechanisms that coordinate tissue destruction, in vivo expansion, dissemination, and host lethality remain incompletely understood. Here, we investigated the contribution of the GspCD-dependent type II secretion system (T2SS) to A. hydrophila pathogenesis using transposon mutants, extracellular protein analyses, and a mouse NSTI model. Mutants carrying transposon insertions in gspD and gspC showed defective secretion of a FLAG-tagged truncated AerA construct and markedly reduced hemolytic activity in culture supernatants. Comparative analysis of extracellular proteins further showed that disruption of gspC altered the extracellular protein landscape, with reduced abundance of multiple known or predicted virulence-associated factors, including AerA, Ahh, lipase, and metalloprotease. In the mouse NSTI model, both mutants exhibited attenuated virulence, including reduced serum markers of tissue injury, less severe histopathological damage, impaired in vivo expansion and dissemination, and decreased lethality. These defects were more pronounced in the gspC mutant than in the gspD mutant. Together, these findings show that the GspCD-dependent T2SS functions as a coordinated extracellular secretion system that drives tissue destruction, in vivo expansion, dissemination, and lethal outcome during A. hydrophila NSTI.