Aeromonas hydrophila Ssp1: A secretory serine protease that disrupts tight junction integrity and is essential for host infection

Aeromonas hydrophila is a Gram-negative bacterial pathogen with a broad host range, including fish and humans. In this study, we examined the function of a secretory serine protease (named Ssp1) identified in pathogenic A. hydrophila CCL1. Ssp1 possesses a trypsin-like serine protease domain and contains two conserved PDZ domains. Recombinant Ssp1 protein (rSsp1) treatment increased intestinal permeability by downregulating and redistributing tight junction protein Occludin in intestinal Caco-2 cells in vitro. Western blot demonstrated that rSsp1 treatment in Caco-2 cells resulted in marked increases in the expressions of myosin light chain kinase (MLCK) and phosphorylated myosin light chain (p-MLC). For virulence analysis, an isogenic CCL1 mutant delta Ssp1 was created. delta Ssp1 bears an in-frame deletion of the Ssp1 gene. A live infection study in crucian carps showed that, compared to CCL1, delta Ssp1 infection exhibited increased Occludin expression, reduced intestinal permeability and tissue dissemination capacity, and attenuated overall virulence in vivo. However, delta Ssp1 showed no differences in the biofilm formation, swimming motility, and resistance to environmental stress. These lost virulence capacities of delta Ssp1 were restored by complementation with the Ssp1 gene. Global transcriptome analysis and quantitative real-time RT-PCR showed that compared to CCL1 infection, delta Ssp1 promoted the expressions of antimicrobial molecules (MUC2, LEAP-2, Hepcidin-1, and IL-22). Finally, CCL1 infection caused significant dysbiosis of the gut microbiota, including increased Vibrio and Deefgea compared to delta Ssp1 infected fish. Taken together, these results indicate that Ssp1 is essential for the virulence of A. hydrophila and is required for the perturbation of intestinal tight junction barrier.

Automated summary

The secretory serine protease Ssp1 in Aeromonas hydrophila plays a crucial role in its pathogenicity by increasing intestinal permeability and disrupting tight junctions. The mutant strain delta Ssp1 exhibited reduced virulence, but showed no differences in biofilm formation, swimming motility, and resistance to environmental stress.