Abstract
Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic “CaaX” motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.
Highlights
Legionella pneumophila is an environmental organism that proliferates within various protists hosts in the aquatic environment[1,2,3]
This has led to a truncation of the last 18 amino acids that included the CaaX farnesylation motif, which is essential for anchoring Ankyrin B (AnkB)-AA100/130b into the Legionella-containing vacuole (LCV) membrane, which is indispensable for its biologic function in decorating the LCV with polyubiquitinated proteins[36, 44]
Host proteasomal degradation is essential for intracellular replication of the Philadelphia-derived Lp02 strain[31], its AnkB homologue does not contribute to decoration of the LCV with polyubiquitinated proteins or intracellular replication[28]
Summary
Legionella pneumophila is an environmental organism that proliferates within various protists hosts in the aquatic environment[1,2,3]. Anchoring of the AnkB effector of strain AA100/130b of L. pneumophila into the LCV membrane is mediated through host-mediated farnesylation of the cysteine residue at the -4 position from the C-terminus within the eukaryotic-like “CaaX” motif[3, 36, 44]. While the C-terminal CaaX motif of AnkB-AA100/130b is indispensable for anchoring the effector to the LCV membrane through host-mediated farnesylation, which is essential for function, the CaaX motif is absent from AnkB-Paris. Compared to the ankB-AA100/130b allele, there is a single nucleotide deletion in the ankB-Paris leading to a frame shift, which results in a truncation of the protein for the last 18 amino acids residues, which include a portion of the third ankyrin repeat domain and the CaaX farnesylation motif. The AnkB lysine residues modified by K11-linked polyubiquitination are conserved in the two AnkB effectors[32]
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