Abstract
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.
Highlights
Exploitation of eukaryotic cellular processes is essential for proliferation of intracellular microbial pathogens
Identification of L. pneumophila C-terminal CaaX motifcontaining proteins Our previous study showed that host cell prenylation of Ankyrin B (AnkB) anchors it to the membrane of the Legionella-containing vacuolar (LCV), and that the three host enzymes (PFTase, IcmT, and RCE-1) involved in prenylation and processing of the prenylated C-terminus are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB (Price et al, 2010b)
The U937 human macrophage cell line was infected with the WT L. pneumophila strain AA100, the ankB mutant or the ankB mutant complemented with the native ankB allele or a substitution variant allele defective in prenylation
Summary
Exploitation of eukaryotic cellular processes is essential for proliferation of intracellular microbial pathogens. Co-evolution and adaptation of L. pneumophila to the intracellular lifestyle within ameba in the aquatic environment is believed to have played a major role in its ability to exploit evolutionarily conserved eukaryotic processes that enables its proliferation within human alveolar macrophages (Molmeret et al, 2005; Franco et al, 2009) Within both evolutionarily distant host cells, L. pneumophila evades endocytic fusion and intercepts ER-to-Golgi vesicle traffic to remodel its phagosome into an ER-derived vacuole (Kagan and Roy, 2002; Molmeret et al, 2005; Shin and Roy, 2008; Isberg et al, 2009). The F-box domain of AnkB interacts with the host Skp component of the SCF1 ubiquitin ligase complex and functions as a platform for the docking of polyubiquitinated proteins to the Legionella-containing vacuolar (LCV) membrane within human cells, Acanthamoeba, and Dictyostelium discoideum (Dorer et al, 2006; Habyarimana et al, 2008; Price et al, 2009, 2010a; Al-Khodor et al, 2010a,b; Lomma et al, 2010)
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