Localization of the PelC and PelE effectors to the <i>Legionella</i> -containing vacuole through host-mediated prenylation and their role in intracellular proliferation

<i>Legionella pneumophila</i> is an intracellular pathogen that replicates in macrophage and protozoan hosts within an endoplasmic reticulum (ER)-derived phagosome that evades the endosomal-lysosomal degradation pathway. The Dot/Icm Type IV Secretion System (T4SS) of <i>L. pneumophila</i> injects ~368 effector proteins into eukaryotic host cells to manipulate various cellular processes, remodeling macrophages and protozoan hosts into proliferative niches. However, most <i>L. pneumophila</i> effectors are dispensable for intracellular bacterial proliferation. Many of the effectors contain eukaryotic-like domains and motifs, such as the C-terminal "-CaaX" prenylation motif. In <i>L. pneumophila</i> strain AA100/130b, there are seven effectors harboring C-terminal -CaaX motif, designated as <u>p</u>renylated <u>e</u>ffectors of <i>Legionella</i> (Pels). Ectopic expression of Pels in mammalian cells results in distinct membrane localization, yet their subcellular localization and their role in the infection remain unknown. Here, we show spatial localization of the seven Pels to the cytosolic face of the LCV in a T4SS-dependent manner, and the conserved cysteine residue of the -CaaX motif is required for this spatial localization. Null mutations in <i>pelC</i> and <i>pelE</i> resulted in a significant reduction in intracellular replication in human monocyte-derived macrophages (hMDMs) and <i>Acanthamoeba polyphaga</i>, but not in <i>Vermamoeba vermiformis</i>, exhibiting host tropism. The <i>ΔpelC</i> and <i>ΔpelE</i> mutants exhibited significant failure in remodeling of the LCV by the ER and a significant increase in trafficking of their vacuoles through the endosomal-lysosomal degradation pathway within human macrophages. Our findings indicate that the PelC and PelE effectors, which hijack the highly conserved eukaryotic prenylation machinery, are required for LCV biogenesis and for intracellular replication.