Type IV Secretion System Effectors of Anaplasma phagocytophilum

Primary author: Deirdre Fahy
Co-author(s): Jason Park; Michael Dodd; Kelly Brayton

Primary college/unit: College of Veterinary Medicine
Campus: Pullman


Anaplasma phagocytophilum (Ap) is a tick-transmitted obligate intracellular zoonotic pathogen. Ap achieves intracellular infection by delivering effector proteins into the host cytosol utilizing a Type IV secretion system (T4SS). In contrast to other intracellular pathogens e.g. Legionella pneumophilia and Coxiella burnetii, where 100s of T4SS effectors have been found, only 4 have been identified in Ap. Our goal was to extend the repertoire of known Ap effectors. To predict effectors, we used the recently published T4SS effector prediction software OPT4e to identify Ap proteins containing features characteristic of T4SS substrates. To screen for T4SS translocation, effector candidates were fused to a reporter enzyme, which if translocated to the host cell cytoplasm generates a product detectable by ELISA. To overcome the genetic intractability of Ap, fusion constructs were assayed for translocation by the heterologous T4SS of Legionella pneumophila. Although only 7 of 70 screened proteins were translocated in a T4SS dependent manner, there are likely more effectors since a negative result may be due to the heterologous nature of the reporter system. Three of the translocated substrates localize to the host cell nucleus when ectopically expressed. Bioinformatic analysis identified putative C-terminal nuclear localization signals (NLS) in all three of these T4SS substrates. Deletion of these NLS sequences abrogated both nuclear-specific localization and also T4SS translocation, which is consistent with the requirement for specific C-terminal features in translocation of known effectors. Functional characterization of these and other effectors will inform their role in Ap replication, pathogenesis, and transmission.