ApaH Diadenosine Polyphosphatase from Legionella pneumophila

Legionella pneumophila (LP), the causative agent of Legionnaires' disease, is a Gram‐negative bacterium and facultative intracellular parasite. The Nudix diadenosine polyphosphatase, NudA, is a virulence factor in LP, and a related nonNudix diadenosine polyphosphatase, ApaH, is a virulence factor in E. coli and acts additively with the E. coli NudA for virulence. Thus LP apaH was cloned using PCR and an apaH knockout mutation was created in LP. While the LP apaH knockout mutation had no apparent phenotype in studies of growth at 25° C or intracellular virulence, a double apaH/nudA knockout mutant could not be created, likely because this dual mutation is lethal, thus indicating a role for apaH in LP. With this in mind, we subcloned apaH for overexpression to study the enzyme, but the resultant protein was insoluble, so we once again subcloned the gene, this time using the maltose binding protein (MBP) as a solubility tag. We were successful in obtaining a soluble fusion protein with diadenosine polyphosphatase activity. Due to technical difficulties using the MBP as an affinity tag, ApaH is currently being purified using more traditional purification methods including size exclusion and ion exchange chromatography. Once the ApaH diadenosine polyphosphatase has been purified, it will be characterized further enzymatically.