Evolutionarily conserved IL-22 participates in gut mucosal barrier through its receptors IL-22BP, IL-10R2 and IL-22RA1 during bacterial infection in teleost.

IL-22 is a critical cytokine of epithelial mucosal barrier. In humans, IL-22 signals through a heteroduplex receptor consisting of IL-22R and IL-10Rß. In fish, IL-22 and its receptors homologues have been cloned in a number of species, however, no studies have been reported how the receptors are involved in IL-22 transduction. For this purpose, in this study we identified IL-22 and its soluble receptor IL-22BP and transmembrane receptors IL-22RA1 and IL-10R2 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1, respectively). WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were relatively conserved in the evolutionary process, sharing the same conserved domains as their higher vertebrate homologues. When the fish were infected with the Aeromonas hydrophila, the expression of WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were significantly induced in the gut. The co-IP assay showed that WR-IL-22 not only interacted with WR-IL-22BP, but also with WR-IL10R2 and WR-IL22RA1. When introduced in vivo, WR-IL-22 activated the JAK1-STAT3 axis and protected the gut mucosa from A. hydrophila infection. However, overexpression of WR-IL-22BP or knockdown of transmembrane receptors WR-IL10R2 and WR-IL22RA1 significantly inhibited the activation of WR-IL-22-mediated JAK1-STAT3 axis and promoted bacterial colonization in the gut. These results provided new insights into the role of IL-22 and its receptors in the gut mucosa barrier and immune response in teleost.

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 ΔSsp1 was created. ΔSsp1 bears an in-frame deletion of the Ssp1 gene. A live infection study in crucian carps showed that, compared to CCL1, ΔSsp1 infection exhibited increased Occludin expression, reduced intestinal permeability and tissue dissemination capacity, and attenuated overall virulence in vivo. However, ΔSsp1 showed no differences in the biofilm formation, swimming motility, and resistance to environmental stress. These lost virulence capacities of ΔSsp1 were restored by complementation with the Ssp1 gene. Global transcriptome analysis and quantitative real-time RT-PCR showed that compared to CCL1 infection, ΔSsp1 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 ΔSsp1 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.