RalF-Mediated Activation of Arf6 Controls Rickettsia typhi Invasion by Co-Opting Phosphoinositol Metabolism

Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P2, by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P2 for R. typhi entry. PI(3,4,5)P3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism.