RESEARCH ARTICLE Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legio-nella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowa-zekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma mem-brane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infec-tion, with its inactivation via antibody blocking significantly reducing R. typhi host cell inva-sion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remark