Anaplasma phagocytophilum Ankyrin A Protein (AnkA) Enters the Nucleus Using an Importin-β-, RanGTP-Dependent Mechanism

Abstract

Anaplasma phagocytophilum, a tick-borne obligately intracellular bacterium of neutrophils, causes human granulocytic anaplasmosis. Ankyrin A (AnkA), an effector protein with multiple ankyrin repeats (AR) is injected via type IV-secretion into the host neutrophil to gain access to the nucleus where it modifies the epigenome to promote microbial fitness and propagation. AR proteins transported into the host cell nucleus must use at least one of two known eukaryotic pathways, the classical importin β-dependent pathway, and/or the RanGDP- and AR (ankyrin-repeat)-dependent importin β-independent (RaDAR) pathway. Truncation of the first four AnkA N-terminal ARs (AR1-4), but not other regions, prevents AnkA nuclear accumulation. To investigate the mechanism of nuclear import, we created point mutations of AnkA N-terminal ARs, predicted to interfere with RaDAR protein import, and used importazole, a specific inhibitor of the importin α/β, RanGTP-dependent pathway. Nuclear colocalization analysis shows that nuclear localization of AnkA is unaffected by single AR1-4 mutations but is significantly reduced by single mutations in consecutive ARs suggesting RaDAR protein nuclear import. However, AnkA nuclear localization was also decreased with importazole, and with GTPγS. Furthermore, A. phagocytophilum growth in HL-60 cells was completely suppressed with importazole, indicating that A. phagocytophilum propagation requires a β-importin-dependent pathway. A typical classical NLS overlapping AR4 was subsequently identified suggesting the primacy of the importin-α/β system in AnkA nuclear localization. Whether the mutational studies of putative key residues support RaDAR NLS function or simply reflect structural changes that diminish engagement of an AR-NLS-importin pathway needs to be resolved through careful structure-function studies.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium

“…Our collective analyses indicate that Ot Rvh genes can form a functional P-T4SS, despite the pervasive mobile element-induced gene shuffling in Ot genomes. While no Ot rvh transported effector has been described to date, it is highly likely that Ot utilizes the rvh T4SS during host cell infection, as secreted proteins that interact with the T4SS gatekeeper, RvhD4, have been described for R. typhi ( 59 – 61 ), R. rickettsii ( 60 , 62 ), Anaplasma marginale ( 63 ), Anaplasma phagocytophilum ( 64 – 70 ) and Ehrlichia chaffeensis ( 66 , 71 – 74 ).…”

Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum

“…Bacterial proteins are also secreted via the type IV secretion system (T4SS) 10 . For example, the protein AnkA is secreted and crossed the vacuolar membrane to reach the nucleus of host cells, to decrease the generation of ROS species 50 . This protein is also able to bind to nuclear proteins, ATC-rich sequences, and transcriptional regions of the CYBB locus.…”

Metagenome diversity illuminates the origins of pathogen effectors

“…The rvh T4SS is odd in its design ( 42 , 43 ), with specialized duplications of some components hypothesized to autoregulate effector secretion ( 44 , 45 ). Effectors have been experimentally characterized for species of Ehrlichia ( 46 – 49 ), Anaplasma ( 50 – 55 ), and Rickettsia ( 56 – 58 ). As the rvh T4SS is present in purportedly free-living Mitibacteraceae and Athabascaceae, Schön et al ( 28 ) proposed that these rickettsiae utilize the rvh T4SS for killing congener microbes, provided their genomes harbor candidate rvh effectors with characteristics similar to effectors in other T4SS and type VI secretion system (T6SS) killing machines ( 59 , 60 ).…”