Eukaryotic protein mimicry as an infection strategy for Ehrlichia chaffeensis

Ehrlichia chaffeensis (E. ch.) expresses the TRP120 multifunctional effector on the surface of infectious dense-cored ehrlichiae which is known to play a role in phagocytic entry, but a cognate host receptor has not been identified. We recently reported that E. ch. activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled (Fzd) family of receptors. In this study, we used immunofluorescence microscopy to demonstrate very strong colocalization between E. ch. and Fzd2, 4, 5, 7, and 9, as well as coreceptor LRP5 1-3 h post infection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by ELISA and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. ch. infection demonstrating complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. ch. infection. This document reports the first example of bacterial mimicry of Wnt pathway ligands and investigates a pathogenic mechanism with potential for targeting by antimicrobial therapeutics.