Identification and characterization of the PAAR-1 gene in Pseudomonas plecoglossicida: Insights into bacterial phenotypes and host immune responses in Large Yellow Croaker (Larimichthys crocea)

Pseudomonas plecoglossicida is a pathogen bacterium responsible for visceral white spot disease (VWND) in Large Yellow Croakers (Larimichthys crocea), causing significant economic losses in commercial fish farms. The proline-alanine-alanine-arginine repeats protein (PAAR) is a core component of the spike structure in the type VI secretion system (T6SS), which injects toxic effectors into host and contributes to bacterial virulence. However, the role of the PAAR gene in P. plecoglossicida and its impact on bacterial infection and host immune responses remain unexplored. In this study, PAAR-1 was identified for the first time in P. plecoglossicida as an effector gene within the T6SS-1 gene cluster, which is regulated and secreted by T6SS-1. The P. plecoglossicida mutant strain (ΔPAAR-1) and its complementary strain (C-ΔPAAR-1) were constructed for subsequent investigation. Compared to the wild-type strain, ΔPAAR-1 exhibited reduced biofilm formation, adhesion, total antioxidant capacity, and secretion of T6SS core protein Hcp-1. In vitro, ΔPAAR-1 showed decreased survival rates in Large Yellow Croaker macrophage cell line (LYC-FM) due to impaired oxidative stress tolerance. In vivo, infection with ΔPAAR-1 led to a significant reduction in mortality, bacterial colonization, and the formation of spleen nodules in Large Yellow Croakers. Comparative transcriptome analysis revealed that PAAR-1 predominantly influences the host Toll-like receptor (TLR) signaling pathway and apoptosis by upregulating the expression of plasma membrane-associated TLRs, including TLR1, TLR2, and TLR5, while downregulating the expression of endosomal TLRs like TLR3, TLR7, TLR8, and TLR9, along with its downstream molecules such as MyD88 and TRAF3. Additionally, knockout PAAR-1 downregulates apoptosis-related genes including AP-1, NF-κB, FAS-L TNFα, Caspase8, and FAS-L. Real-time quantitative polymerase chain reaction (RT-qPCR) further confirmed these findings. Furthermore, the proportion of apoptotic cells was significantly lower in the ΔPAAR-1 infected LYC-PKM cells. These results indicate that PAAR-1 is involved in regulating TLR signaling pathway and apoptosis in Large Yellow Croaker. This study provides the first identification of the core T6SS-1 gene PAAR-1 in P. plecoglossicida, offering valuable insights into its pathogenic mechanisms and presenting a potential target for attenuated vaccine development.