Abstract
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 9L10P 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-9L10P/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-9L10P/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.
Highlights
Intracellular pathogens have evolved with remarkable mechanisms to exploit host cell processes to evade degradation within the lysosomes, which is the first line of defense against microbial invasion
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
Ankyrin B (AnkB) is indispensable for intracellular growth of L. pneumophila within macrophages and protozoa
Summary
Intracellular pathogens have evolved with remarkable mechanisms to exploit host cell processes to evade degradation within the lysosomes, which is the first line of defense against microbial invasion. The intracellular bacterial pathogen Legionella pneumophila is ubiquitous in natural aquatic environments and man-made water systems, where it replicates within various protozoan hosts [1]. Intracellular trafficking and intra-vacuolar proliferation of L. pneumophila within amoebae and human macrophages is very similar, at the cellular and molecular levels [1]. Within both evolutionarily distant host cells, L. pneumophila evades endocytic fusion and intercepts ER-to-Golgi vesicle traffic to remodel its phagosome into a rough endoplasmic reticulum (RER)-derived vacuole [2,3]. The Dot/Icm type IV secretion system [4,5] is required for L. pneumophila to modulate various mammalian and protozoan cellular processes through translocation of ,200 effectors into the host cell, but the role of most of these effectors in the intracellular infection is minimal or not known [2,3,6]
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