The RACK1 Signaling Scaffold Protein Selectively Interacts with Yersinia pseudotuberculosis Virulence Function

Abstract

Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins

“…Some bacterial virulence factors simply require scaffold proteins to reach their destination within host cells or to become enzymatically active, while others target the host scaffold proteins to suppress defenses. Yersinia species secrete the TTSS effector YopK (for Yersinia outer protein K), which binds to the Receptor for Activated C Kinase1 in mammals (Thorslund et al, 2011). It is hypothesized that this interaction blocks phagocytosis, allowing efficient extracellular proliferation of the bacteria.…”

Impact of the N-Terminal Secretor Domain on YopD Translocator Function in Yersinia pseudotuberculosis Type III Secretion

“…To begin with, the cytotoxicity assay is not capable of detecting subtle defects in Ysc-Yop T3SS functionality (12,17,19). In addition, Yersinia-induced cytotoxicity and antiphagocytosis are phenotypically unlinked; highly attenuated yopE point mutants are still cytotoxic with respect to HeLa cell monolayers, whereas bacteria defective in antiphagocytosis are always avirulent (1,75,77). Accordingly, for use as an indirect measure of Yersinia-mediated antiphagocytosis, we believe the viability assay to be a superior tool to ascertain T3SS function.…”

Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components

“…YopK is translocated into host cells and regulates T3SS translocation by interacting with the proteins YopB and YopD (36,41). YopB and YopD are both integral transmembrane proteins and form the pore, or translocon complex, through which other Yops are injected into the host cell.…”