Divergent evolution of Di-lysine ER retention vs. farnesylation motif-mediated anchoring of the AnkB virulence effector to the Legionella-containing vacuolar membrane.

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.

Selective requirement of the shikimate pathway of Legionella pneumophila for intravacuolar growth within human macrophages but not within Acanthamoeba.

Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids.

Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila.

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III-VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

Host-mediated post-translational prenylation of novel dot/icm-translocated effectors of legionella pneumophila.

The Dot/Icm type IV translocated Ankyrin B (AnkB) effector of Legionella pneumophila is modified by the host prenylation machinery that anchors it into the outer leaflet of the Legionella-containing vacuole (LCV), which is essential for biological function of the effector in vitro and in vivo. Prenylation involves the covalent linkage of an isoprenoid lipid moiety to a C-terminal CaaX motif in eukaryotic proteins enabling their anchoring into membranes. We show here that the LCV harboring an ankB null mutant is decorated with prenylated proteins in a Dot/Icm-dependent manner, indicating that other LCV membrane-anchored proteins are prenylated. In silico analyses of four sequenced L. pneumophila genomes revealed the presence of eleven other genes that encode proteins with a C-terminal eukaryotic CaaX prenylation motif. Of these eleven designated Prenylated effectors of Legionella (Pel), seven are also found in L. pneumophila AA100. We show that six L. pneumophila AA100 Pel proteins exhibit distinct cellular localization when ectopically expressed in mammalian cells and this is dependent on action of the host prenylation machinery and the conserved cysteine residue of the CaaX motif. Although inhibition of the host prenylation machinery completely blocks intra-vacuolar proliferation of L. pneumophila, it only had a modest effect on intracellular trafficking of the LCV. Five of the Pel proteins are injected into human macrophages by the Dot/Icm type IV translocation system of L. pneumophila. Taken together, the Pel proteins are novel Dot/Icm-translocated effectors of L. pneumophila that are post-translationally modified by the host prenylation machinery, which enables their anchoring into cellular membranes, and the prenylated effectors contribute to evasion of lysosomal fusion by the LCV.