| Congresso Brasileiro de Microbiologia 2023 | Resumo: 163-2 | ||||
Resumo:Klebsiella pneumoniae (KP) is an opportunistic pathogen that is of concern to public health systems around the world, as multi-resistant isolates are frequently identified. One of its virulence factors is the Type VI Secretion System (T6SS), a macromolecular complex that may translocate effector proteins. VgrG proteins are structural components of the tip of T6SS, but may also contain a variable C-terminal extension with an effector role. In a previous study, we identified that at least 100 KP isolates present a VgrG containing a conserved C-terminal extension of 138 amino acids, although its function is not yet known. Among them, there is the VgrG4 protein from Kp 52145 strain. We showed that VgrG4-CTD interacts with cytoskeletal proteins and induces the remodeling of actin filaments in macrophages. The aim of this project is to further characterize the role of VgrG4 and VgrG4-CTD in the modulation of host-pathogen interaction. RAW 264.7 macrophages were stimulated with the recombinant proteins VgrG4 or VgrG4-CTD. Fluorescence microscopy was performed to visualize the cell cytoskeleton. Reactive oxygen species (ROS) production was analyzed by CellRox stain on the Cytell imaging system. The production of cytokines was measured by immunoenzymatic assays (ELISA). Control and stimulated macrophages were infected with KP 52.145 strain at MOI 50:1. Adhesion, internalization, and bacterial survival rates were measured by counting colony-forming units. The first results show that both proteins seem able to induce alterations in the actin cytoskeleton but do not seem to induce ROS production. Furthermore, we observed that both proteins affect the adhesion and survival rate of KP in macrophages, but have no influence on bacterial internalization by macrophages. Lastly, none of the proteins was able to induce the production of the pro-inflammatory cytokine TNF-α. Although preliminary, these data bring interesting insights for the functional characterization of the VgrG4 protein for KP-mediated infection, contributing to understanding the molecular mechanisms involved in the host-pathogen interaction.
Palavras-chave: opportunistic pathogen, T6SS, VgrG4, protein-protein interaction, host-pathogen interaction Agência de fomento:CNPq, FAPERJ, CAPES, FIOCRUZ | |||||