Obligatory intracellular bacterium Anaplasma phagocytophilum AnkA regulates actin dynamics and spatiotemporal bacterial release.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes an emerging infectious disease, human granulocytic anaplasmosis. It undergoes a biphasic developmental cycle inside membrane-bound vacuoles within the host human neutrophils, maturing from a proliferating reticulate cell form to an infectious dense core (DC) form that is subsequently spontaneously released from host cells to initiate a new infection cycle. However, how A. phagocytophilum coordinates growth and release is unknown. Here, we found localized cortical F-actin disruption occurs where Anaplasma-containing vacuoles abut on the plasma membrane to release bacteria. Disruption of actin filaments by cytochalasin D and latrunculin B induced unrestrained release of almost all intracellular A. phagocytophilum from host cells, which were significantly less infectious than spontaneously released bacteria. A. phagocytophilum AnkA, a type IV secretion system (T4SS) effector, was found to localize in the cell periphery with cortical F-actin. By immunoprecipitation followed by mass spectrometry, AnkA was found to interact with actin, α-actinin 4 (Actn4) involved in actin cross-linking, and gelsolin for actin filament remodeling. shRNA-knockdown of Actn4 or gelsolin, enhanced release of premature A. phagocytophilum. Glutathione S-transferase (GST)-tagged C-terminus of AnkA (AnkA-C) interacted with actin and gelsolin, whereas the N-terminus (AnkA-N) interacted with Actn4. In vitro pyrene-actin polymerization assay showed that GST-AnkA-C has stronger actin polymerizing activity than GST-AnkA or GST-AnkA-N. Ectopically expressed GFP-AnkA-N localized to the plasma membrane and induced membrane ruffling, whereas GFP-AnkA-C colocalized with and enhanced stress fiber formation. These results demonstrate that AnkA is the first example of bacterial molecules interacting with gelsolin and Actn4. The result suggests that by colocalizing with cortical F-actin and controlling F-actin dynamics and cross-linking, AnkA regulates spatiotemporal release of A. phagocytophilum. The current study unravels a new paradigm of retention/release mechanism of intracellular pathogen regulated by a T4SS effector.