Acquisition of a type VI secretion system is critical for Pseudomonas plecoglossicida induced granulomas in fish internal organs

Pseudomonas plecoglossicida is one of the most prevalent pathogens affecting the large yellow croaker (LYC; Larimichthys crocea), causing a disease characterized by granuloma formation in the spleen, kidney and liver. Previously we discovered two active type VI secretion systems (T6SSs) in P. plecoglossicida XSDHY-P (a fish pathogenic strain). However, whether these systems are involved in bacterial pathogenesis remains unclear. In this work, we screened all putative T6SSs encoded in XSDHY-P genome and investigated their roles in virulence and interbacterial competition. Bioinformatic analysis suggests that XSDHY-P encodes three distinct T6SS gene clusters (PP T6SS-1, PP T6SS-2 and PP T6SS-3), and PP T6SS-1 was probably acquired independently through horizontal gene transfer. To examine if these T6SSs are associated with virulence, we generated XSDHY-P isogenic mutants ΔT6SS-1, ΔT6SS-2, ΔT6SS-3, ΔtssH-1 and ΔtssD-1, and performed fish infection assays to evaluate their virulence in LYC model. The deletion of PP T6SS-1, tssH-1 or tssD-1 drastically attenuated P. plecoglossicida virulence, and these mutants were unable to induce granulomas in the fish host. In addition, we investigated the roles of the three PP T6SSs in interbacterial competition through an in vitro bacterial competition assay. We found that P. plecoglossicida killed competitor bacteria in a PP T6SS-2-dependent manner. Together, these results indicate that T6SSs in P. plecoglossicida fish isolates are important for bacterial fitness both in vivo and in vitro, and that PP T6SS-1 has the anti-eukaryotic function, and is a key virulence determinant, whereas PP T6SS-2 is antibacterial, and might be important for competing with host commensal bacteria during infection.