RasGAP and Crk bind Anaplasma phagocytophilum effector AteA at tyrosine-phosphorylated EPLYA motifs

Host cell signaling networks play critical roles in responding to bacterial infections. Intracellular pathogens manipulate these signals to establish a replicative niche in the host cell. Vector-borne rickettsial pathogens, such as Anaplasma phagocytophilum, encounter the dual challenge of interfacing with cell signaling in both the mammalian host and the tick vector Ixodes scapularis. A central feature that enables A. phagocytophilum survival in both mammals and ticks is the type 4 secretion system (T4SS), which secretes effector proteins into the host cell to manipulate biological processes. Only a few A. phagocytophilum T4SS effectors have been experimentally tested, primarily in mammalian cells. While AteA has been identified as the first T4SS effector known to be essential for A. phagocytophilum survival in the tick, its molecular targets in the tick remained unknown. Here, we investigated AteA interactions with tick cell proteins and a possible mechanism of AteA activation. AteA contains multiple EPIYA-like short linear motifs with the consensus sequence EPLYA. Other known bacterial EPIYA motif effectors, including AnkA from A. phagocytophilum, target mammalian phosphotyrosine signaling proteins. We found that AteA is tyrosine-phosphorylated at EPLYA motifs by the non-receptor tyrosine kinases Src and Abl in eukaryotic cells and during tick infection. Protein pull-down assays revealed that Ixodes SH2 domain-containing proteins, RasGAP and Crk, preferentially interact with phosphorylated AteA EPLYA motifs. RasGAP and Crk have interconnected roles in cell signaling, suggesting that A. phagocytophilum targets these pathways during infection of the arthropod vector.IMPORTANCETick-borne diseases are on the rise globally, linked to changes in climate, land use, and human-animal interactions. Interrupting vector-borne transmission requires understanding how the vector and pathogens interact. Recent studies have shown ticks detect and respond to pathogens through cellular immune and stress signaling pathways. We previously demonstrated that the tick-borne bacterium Anaplasma phagocytophilum encodes a tick-specific secreted effector, AteA, that is essential for the pathogen's survival in the tick. Here, we showed that AteA is phosphorylated by tick cell kinases, which promotes binding to host cell SH2 domain-containing proteins that are integral to cellular signaling networks. Understanding how these interactions affect signaling pathways in the tick will be essential if interventions targeting the vector are to be developed.