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Pseudomonas plecoglossicida is a Gram-negative bacterium that causes chronic granulomatous diseases in fish. However, the molecular mechanisms underlying its pathogenicity are poorly understood. Here, we identified a novel two-component system (TCS) named PvgAS in P. plecoglossicida , which regulates the expression of genes including the type VI secretion system-1 (T6SS-1), a key virulence factor, as well as the type III secretion system (T3SS) gene cluster and several genes associated with metabolic pathways. We showed that PvgAS is essential for bacterial virulence in large yellow croaker (LYC), an important aquaculture species, by controlling T6SS-1 expression, which is crucial for tissue colonization and intracellular survival. In contrast, T3SS was dispensable for P. plecoglossicida virulence in this model. We also confirmed T6SS-1 gene regulation by PvgAS at the transcriptional and translational levels using RNA-seq, RT-qPCR, lacZ reporter, and western blot assays. Our study reveals a regulatory and functional role of PvgAS in P. plecoglossicida pathogenicity and suggests that PvgAS may be a potential target for new therapeutic interventions against this bacterium. • Identified a novel two-component system PvgAS in Pseudomonas plecoglossicida. • PvgAS regulates type III and type VI secretion systems and bacterial virulence. • T3SS is dispensable but T6SS-1 is essential for P. plecoglossicida pathogenicity. • PvgAS is a potential target for developing new therapeutic interventions. [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is a Gram-negative fish pathogen that affects the large yellow croaker (LYC; Larimichthys crocea) and several other economically important fish species. The bacterium can cause systemic infections in LYC, resulting in visceral granulomas disease (VGD) with high mortality in cultured populations. Despite the severity of this disease, the LYC industry lacks an effective vaccine for VGD. We have constructed a highly attenuated P. plecoglossicida mutant by deleting a virulence gene (tssD-1) from the wild-type (WT) strain XSDHY-P. We then investigated the safety and efficacy of the mutant strain Δ tssD-1 in LYC juveniles as a live attenuated vaccine candidate. LYCs were vaccinated with Δ tssD-1 by two intraperitoneal injections per fish, and then challenged with P. plecoglossicida XSDHY-P by the immersion route. Vaccination of Δ tssD-1 induced a significantly higher level of P. plecoglossicida -specific serum IgM and increased expression of CD8α and MHCIα, and resulted in a relative percentage survival of 86.3%. The safety of Δ tssD-1 was demonstrated because no mortality or clinical signs were found in a LYC group injected with a high dose (1.8 × 106 CFU per fish) of Δ tssD-1. This is the first report of a defined live attenuated strain providing protective immunity in LYC against P. plecoglossicida. • Attenuated Pseudomonas plecoglossicida strain Δ tssD-1 is safe and immunogenic as a fish vaccine. • Δ tssD-1 induces both humoral and cellular immune responses. • Δ tssD-1 provides protective immunity against P. plecoglossicida infection. [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is an important pathogenic bacterial species in aquaculture, causing visceral granulomas in large yellow croaker (Larimichthys crocea) and several other economically important fish species. The GacS/GacA is a two-component regulatory system (TCS) primarily found in Pseudomonads for its role in virulence, quorum sensing, and biofilm formation. To investigate the potential role of GacS/GacA system of P. plecoglossicida , a gacS deficient mutant (Δ gacS) was generated based on a clinical isolate XSDHY-P. Compared to the wild-type (WT) strain, the Δ gacS strain showed almost no growth defect in vitro but was impaired in swimming motility and biofilm formation. Furthermore, the Δ gacS strain exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system. In a cell model originating from the head-kidney of large yellow croaker, the Δ gacS showed lower capacities of adhesion, invasion, and intracellular survival compared to the WT strain. In addition, the virulence of the Δ gacS and WT bacterial strains were evaluated in large yellow croaker. The results showed that the deficiency of gacS drastically decreased bacterial tissue loads and substantially attenuated P. plecoglossicida virulence in fish. Furthermore, a comparative transcriptome was performed between the Δ gacS and WT strains by RNA-sequencing (RNA-seq). Transcriptomic analysis results revealed 25 upregulated and 273 downregulated genes in the Δ gacS strain compared with the WT strain, and the downregulated genes were mainly involved in flagellar assembly, metabolism, bacterial chemotaxis, and biofilm formation, which are consistent with their respective phenotypic traits. These findings will provide functional insight into the role of GacS/GacA in P. plecoglossicida. • The Δ GacS was impaired both in swimming motility and biofilm formation compared to the P. plecoglossicida WT strain. • The GacS plays a contributing role in the adhesion an invasion to LYC-hK, and in immune escaping of P. plecoglossicida. • Deletion of the GacS gene drastically attenuated the virulence of P. plecoglossicida to fish. • The results of the comparative transcriptomic analysis are consistent with their respective phenotypic traits. [ABSTRACT FROM AUTHOR]