Date on Master's Thesis/Doctoral Dissertation
Microbiology and Immunology
Microbiology and Immunology, PhD
Abu Kwaik, Yousef
Committee Co-Chair (if applicable)
legionella pneumophila; ankyrin domain; intracellular pathogen
Legionella pneumophila is an aquatic organism capable of intracellular replication within a wide range of protozoan hosts and within human macrophages where it causes Legionnaires’ Disease. L. pneumophila manipulates a variety of host cell processes by translocating, into the host cell cytosol, more than 300 effector proteins via the type IVB secretion system (T4SS). The AnkB effector from the AA100/130b strain (AnkB-AA100) contains a C-terminal CaaX motif that rapidly becomes farnesylated upon translocation into the host cell and anchored to the cytosolic face of the Legionella-containing vacuole (LCV) membrane, which is essential for intracellular replication. We show that a homolog of AnkB from the Paris strain (AnkB-Paris) has a frameshift mutation that truncates the C-terminus eliminating the CaaX motif, but creating a unique KNKYAP sequence that resembles a eukaryotic di-lysine ER retention motif (KxKxx). AnkB-Paris localizes to the cytosolic face of the LCV membrane most likely through the ER retention motif. Phylogenetic analyses indicate that the AnkB-Paris allele is common among environmental isolates and is positively selected specifically for the di-lysine motif. Ectopic expression of AnkB-Paris results in a perinuclear distribution and trans-rescues the ankB mutant of the AA100/130b strain for intravacuolar replication. The trans-rescue is dependent on an intact di-lysine ER-retention motif, which most likely enables anchoring of AnkB to the ER-derived LCV membrane. AnkB contains a N-terminal eukaryotic F-box domain that recruits the cellular ubiquitylation machinery by interacting with the host Skp1 protein resulting in decoration of the LCV with polyubiquitylated proteins. Proteins targeted for ubiquitylation during infection are likely bound by ankyrin domains in the C-terminus of AnkB. Based on the crystal structure, we identified four residues within the ankyrin domains likely involved in binding specific substrate proteins. Mutation of these residues did not affect protein folding but resulted in loss of accumulation of ubiquitylated proteins around the LCV and a severe defect in intracellular replication, similar to the ankB null mutant. Overall, we identified a di-lysine ER retention motif in the C-terminus of AnkB-Paris and 4 substrate binding residues within the 3 ankyrin domains of AnkB-AA100 both of which are essential for biological function.
Perpich, John David, "Divergent evolution of di-lysine ER retention vs. farnesylation motif-mediated anchoring of the ankb virulence effector." (2018). Electronic Theses and Dissertations. Paper 2978.