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bioRxiv · 10.64898/2026.04.30.721992

The GspCD-dependent type II secretion system drives necrotizing soft tissue infection by Aeromonas hydrophila

Tonosaki, Y.·Yamazaki, K.·Owada, S.·Yamaguchi, K.·Kashimoto, T.
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Abstract

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. IMPORTANCENecrotizing soft tissue infections (NSTIs) are rapidly progressive, life-threatening bacterial infections, and Aeromonas hydrophila is an important causative agent. Here, we show that the GspCD-dependent type II secretion system (T2SS) drives A. hydrophila virulence in a murine NSTI model. Transposon mutants in gspC or gspD exhibited impaired extracellular protein secretion, reduced hemolytic activity, attenuated tissue damage, decreased bacterial proliferation and dissemination, and markedly reduced lethality. Comparative analysis further indicated that T2SS disruption alters the extracellular virulence landscape rather than affecting a single toxin alone. These findings provide in vivo evidence that coordinated T2SS-dependent secretion is a central determinant of severe A. hydrophila soft tissue infection.

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Tonosaki, Y., Yamazaki, K., Owada, S., Yamaguchi, K., Kashimoto, T.. 2026-05-04. The GspCD-dependent type II secretion system drives necrotizing soft tissue infection by Aeromonas hydrophila. https://doi.org/10.64898/2026.04.30.721992

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