The A naplasma phagocytophilum effector AmpA hijacks host cell SUMO ylation

Summary SUMO ylation, the covalent attachment of a member of the small ubiquitin‐like modifier ( SUMO ) family of proteins to lysines in target substrates, is an essential post‐translational modification in eukaryotes. Microbial manipulation of SUMO ylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMO ylation. Here, we demonstrate that the obligate intracellular bacterium, A naplasma phagocytophilum , utilizes an effector, AmpA ( A . phagocytophilum post‐translationally m odified p rotein A ) that becomes SUMO ylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH 1387) localizes to the A. phagocytophilum ‐occupied vacuolar membrane ( AVM ). Algorithmic prediction analyses denoted AmpA as a candidate for SUMO ylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein ( GFP )‐tagged AmpA . SUMO ylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP ‐ AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly‐ SUMO ylated, which was consistent with the observation that AmpA colocalizes with SUMO 2/3 at the AVM . Only late during the infection cycle did AmpA colocalize with SUMO 1, which terminally caps poly‐ SUMO 2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA ‐mediated knockdown of U bc9 – a necessary enzyme for SUMO ylation – slightly bolstered A . phagocytophilum infection, pharmacologically inhibiting SUMO ylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP ‐ AmpA served as a competitive agonist against native AmpA in infected cells, while lysine‐deficient GFP ‐ AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMO ylated during infection, representing a novel tactic for A . phagocytophilum survival.