Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

Legionnaires' disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L.pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L.pneumophila genes with high similarity to the Escherichia coliK(+) transporters. These three genes were expressed by L.pneumophila and have been designated kupA, kupB and kupC. Investigation using the L.pneumophilakup mutants revealed that kupA is involved in K+ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L.pneumophila were complemented by the expression E.colitrkD/Kup gene in trans, a bona fide K+ transporter encoded by E.coli. Collectively, our data indicate that KupA is a functional K+ transporter expressed by L.pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions.