KLEBSIELLA PNEUMONIAE VGRG4: A T6SS EFFECTOR THAT MODULATES HOST RESPONSE

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen and a major global public health issue due to the emergence of multidrug-resistant strains. Among its virulence factors is the Type VI Secretion System (T6SS), a macromolecular apparatus that delivers effector proteins into target cells. VgrG proteins form the tip of the T6SS and may contain C-terminal extensions with effector functions. Previously, we demonstrated that the VgrG4 protein from K. pneumoniae strain Kp52.145 contains a conserved 138-amino-acid C-terminal domain (CTD) that interacts with cytoskeletal components, inducing cellular protrusions and actin remodeling in macrophages. Here, we further investigated the role of VgrG4 and its CTD in host-pathogen interaction. RAW 264.7 macrophages were stimulated with recombinant VgrG4 or VgrG4-CTD. Cytoskeletal rearrangement and bacterial localization were analyzed by fluorescence microscopy, while bacterial adhesion, internalization, and survival were quantified by colony-forming unit assays. Reactive oxygen species (ROS) production was evaluated using CellROX staining, cytokine production and gene expression were evaluated by ELISA and RT-qPCR, respectively. Western blotting was performed to assess host signaling pathway components. In addition, a vgrG4 mutant strain was generated by homologous recombination. The mutant was characterized by growth and mucoviscosity assays, and its ability to induce cytoskeletal remodeling was examined in macrophages. Both VgrG4 and VgrG4-CTD promoted marked actin cytoskeleton rearrangement, increased bacterial adhesion, ~3- and ~4-fold; intracellular survival, ~3-fold; and localization within acidic compartments, ~5- and ~6-fold. They also enhanced ROS production, ~2.5-fold, and upregulated IL-6, ~7-fold, and IL-10, ~3-fold for VgrG and ~6-fold for VgrG4-CTD gene expression. In contrast, neither protein altered TNF and IL-1β production or induced changes in NF-kB, PI3K, and Akt. The vgrG4 mutant exhibited growth and mucoviscosity profiles comparable to the wild-type strain but failed to induce cytoskeletal rearrangement in RAW 264.7 macrophages. Together, these findings expand the functional characterization of VgrG4 and provide new insights into the molecular mechanisms underlying K. pneumoniae pathogenesis.