T6SS-1 mediates cytosolic access and modulates immune responses in Pseudomonas plecoglossicida infection of Larimichthys crocea macrophages.

Abstract

Intracellular survival is a key virulence trait of Pseudomonas plecoglossicida, the agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea). However, the bacterial determinants that support persistence within host phagocytes remain incompletely defined. Here, we investigated the contribution of the type VI secretion system-1 (T6SS-1) to macrophage infection using a ΔtssD-1 mutant and a head kidney-derived macrophage-like cell line (LYC-hk). Gentamicin protection assays showed that ΔtssD-1 bacteria entered macrophages at levels comparable to wild type at 0 h post-infection, but failed to undergo sustained intracellular replication at later time points, whereas a chromosomally complemented strain restored this phenotype. Differential permeabilization microscopy indicated that T6SS-1 is required for phagosomal rupture and exposure of bacteria to the host cytosol, and transmission electron microscopy confirmed cytosolic localization of wild-type and complemented bacteria but not the mutant. Time-resolved transcriptomic profiling revealed a shared early inflammatory signature in response to both strains, followed by divergence at 4-8 h post-infection. Wild-type infection was associated with distinct expression patterns, including strong induction of the osmotic stress marker lrrc8a, which was supported by RT-qPCR validation. Together, these results identify T6SS-1 as a key determinant of phagosomal rupture and cytosolic access in P. plecoglossicida macrophage infection and link this intracellular transition to distinct host transcriptional signatures.