The intracellular pathogen Legionella pneumophila modulates the activity of host
GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella
protein DrrA post-translationally modifies the
GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during
infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved
tyrosine residue in the switch II region of Rab1 was detected during
infection. In addition, a modification to an adjacent
serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector
protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a
phosphocholine moiety to Rab1. This
phosphocholine transferase activity used
CDP-choline as a substrate and required a conserved
histidine residue located in the FIC domain of the AnkX
protein. During
infection, AnkX modified both Rab1 and Rab35, which explains how this
protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of
Rab GTPases represents a mechanism by which bacterial FIC-domain-containing
proteins can alter host-cell functions.