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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]

Simple Summary: In this paper, a three-round nested PCR assay with high specificity and sensitivity (2.62 copies/reaction), employing the Pseudomonas plecoglossicida sctU gene, was established. The detection system was used to detect large yellow croaker samples in the Zhejiang and Fujian areas. It was found that March and April were the peak times of visceral white nodules disease, which was inversely related to temperature. Using this detection method, early detection of visceral white nodules disease in large yellow croakers could be realized. The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea. [ABSTRACT FROM AUTHOR]

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, Δ tssH-1 and Δ tssD-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. • Three distinct type VI secretion systems (T6SSs) encoded in P. plecoglossicida fish isolates. • Three T6SSs play different roles in P. plecoglossicida' s virulence or inter-bacterial killing. •. Acquisition of the T6SS-1 enables P. plecoglossicida strains to proliferate and cause disease in the fish host [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY‐P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild‐type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild‐type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker. [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 a facultative Gram-negative bacteria that causes 'visceral granulomas disease' (VGD) in large yellow croaker (LYC; Larimichthys crocea) and several other fish species leading to high economic losses. Currently, there are no commercial vaccines against VGD. Thus, we evaluated the efficacy of a live attenuated vaccine (LAV) made by our laboratory in LYC administered orally in alginate-coated LAV-loaded feeds. To evaluate the tolerance of the LAV in the acidic environment of the stomach, we treated the alginate-coated LAV with a simulated gastric juice without pepsin. There was a 10-fold decrease in the number of viable LAV cells after treatment with the simulated gastric juice (pH 2.0). Despite so, we found a significant increase in LAV cells in the liver and hindgut within 4 h post first feeding of oral vaccine (hpff), which increased to high levels by 24 hpff indicating that LAV cells that survived the stomach acidic environment reached the hindgut for adsorption. Although there was a transient change in the composition of the gut microbiota at 24 hpff, analysis using the Shannon and β-diversity indices showed a similarity in the bacterial composition found before and after vaccination indicating that the LAV had no long-term adverse effect on the gut microbiota. Systemic and mucosal humoral responses were characterized by high serum antibody levels and upregulation of the pig and sign while activation of cell-mediated immunity was characterized by upregulation of MHC-Iα, MHC-IIβ, Prf1 and IFNγ genes. The overall protection in vaccinated LYC was estimated at relative percent survival (RPS) of 60%. Altogether, this study has shown that LAVs can be used as oral vaccines to confer protective immunity without impacting adverse effects on the fish microbiota. • P. plecoglossicida causes 'visceral granulomas disease' (VGD) in large yellow croaker. • This is the first report of a promising live attenuated vaccine against VGD by oral administration. • Oral vaccination confers protective immunity without impacting adverse effects on the fish microbiota. [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]

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen mediating visceral granulomas in many piscine species including the large yellow croaker (Larimichthys crocea) but the underlying mechanisms are unclear. RpoE is an alternative sigma (σ) factor involved in regulated intramembrane proteolytic (RIP) cascade, enabling bacterial pathogens to coordinate the expression of genetic traits associated with stress adaptation and virulence determinants in response to diverse stimuli in vitro and in vivo of the hosts. In this study, genes associated to RIP cascade in P. plecoglossicida were identified and characterized to show various sequence similarities to their counterparts in Escherichia coli and P. aeruginosa. The expression of P. plecoglossicida RIP locus was induced by higher temperatures. Moreover, RNA sequencing approach revealed that RpoE regulated the expression of ∼297 and ∼261 genes at virulent (18 °C) and non-virulent (28 °C) temperatures, respectively. RpoE regulon genes are involved in various processes associated with bacterial signal transduction, membrane homeostasis, energy metabolism and virulence. In particular, RpoE positively controlled expression of csrA encoding an RNA binding protein essential for central carbon metabolism. In addition, P. plecoglossicida RpoE was validated to regulate type VI secretion system (T6SS) expression, bacteria competition, biofilm formation and reproduction in macrophages. Collectively, RpoE-centered RIP cascade appeared to play important roles in control of the expression of genes involved in adaptation in vivo and in vitro niches by thermal sensing in P. plecoglossicida. These results facilitates to reveal the pathogenic mechanisms of P. plecoglossicida causing fish diseases and provides new perspectives to control bacterial infection. [ABSTRACT FROM AUTHOR]