AMPylation is a newly discovered posttranslational modification used to stably modify proteins with AMP by virulence factors of several pathogens. In all described AMPylation factors, a conserved functional motif called Fic (filamentation induced by cyclic adenosine monophosphate) with the sequences (HPFx[D/E]GN[G/K]R) catalyzes the enzymatic reaction whereby the invariant histidine residue is essential. Biochemical modification by the AMP moiety alters the activity of the target molecules, suggesting that this signaling mechanism represents a novel posttranslational modification with functions similar to other well established modifications, such as phosphorylation, ubiquitylation and glycosylation (27). The L. pneumophila effector AnkX is known to interfere with host membrane trafficking pathways via a mechanism that is dependent upon a Fic motif located in its N-terminal domain. However, the two most important questions about the activity of AnkX have not been addressed. First, it is not clear whether AnkX possesses AMPylation activity. Second, the cellular targets of AnkX are unknown. Our recent studies have revealed some highly intriguing properties of AnkX: The self-AMPylation activity of AnkX requires one or more factors of eukaryotic origin. Moreover, we observed that AnkX is toxic to yeast and such toxicity can be suppressed by overexpressing components of the complex involved in docking vesicles to the cis-Golgi compartment. Therefore, we hypothesize that one or more host factors are required to activate the AMPylation activity of AnkX and that AnkX targets one or more proteins involved in anterograde membrane transport between the endoplasmic reticulum (ER) and the Golgi apparatus. In the proposed study, we plan to: 1. Identify and characterize the host factor(s) required for the AMPylation activity of AnkX. 2. Analyze the effect of AnkX on host signaling pathways and intracellular growth L. pneumophila. We also plan to analyze the putative co-factor(s) by studying their roles in cellular processes and by identifying the relevant binding proteins. These studies will shed light on the putative AMPylation enzymes that do not require a Fic motif, which could lead to novel lines of research into this exciting field. PUBLIC HEALTH RELEVANCE: AMPylation of host proteins is employed by many important pathogens. Thus, understanding of how this bacterium affects host cell processes could lead to clues in our study of other diseases associated with abnormalities of these processes. Our study can provide novel strategies for the prevention and/or treatment of infections caused by Legionella pneumophila other important pathogens.