The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the 9L 10P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB- 9L 10P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB- 9L 10P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.
Legionella pneumophila is abundantly found in the aquatic environment within various protozoa and can cause a severe pneumonia called Legionnaires' disease when it invades human macrophages in the lung. The ability of L. pneumophila to invade and proliferate within macrophages and protozoa is dependent on the translocation of specific proteins into the invaded cell via a specialized secretory device, and these proteins modulate various host cell processes. Of these translocated proteins, AnkB is indispensable for intracellular growth of L. pneumophila within macrophages and protozoa. Here we show that AnkB is essential for establishing a favorable intracellular replicative niche by promoting the decoration of the Legionella containing vacuole (LCV) with polyubiquitinated proteins. The AnkB effector achieves this by mimicking the action of host cell F-box proteins, a highly conserved component of the SCF ubiquitin ligase complex that is found in both unicellular organisms and mammalian cells. Our study provides new insights into the ability of intracellular pathogens to hijack evolutionarily conserved host cell processes through molecular mimicry to establish a favorable replicative niche within various hosts and to cause disease in mammals.