A conserved interaction between the effector Sca4 and host clathrin suggests additional contributions for Sca4 during rickettsial infection.

Intracellular bacterial pathogens deploy secreted effector proteins that manipulate diverse host machinery and pathways to promote infection. Although many effectors carry out a single function or interaction, there are a growing number of secreted effectors capable of interacting with multiple host factors. However, few effectors secreted by arthropod-borne obligate intracellular Rickettsia species have been linked to multiple host targets. Here, we investigated the conserved rickettsial secreted effector Sca4, which was previously shown to interact with host vinculin in donor cells to promote cell-to-cell spread in the model Rickettsia species R. parkeri. We discovered that Sca4 also binds the host cell protein clathrin heavy chain (CHC, CLTC) via a conserved segment in the Sca4 N-terminus. In mammalian host cells, ablation of CLTC expression or chemical inhibition of endocytosis reduced R. parkeri cell-to-cell spread, indicating that clathrin promotes efficient spread. Unexpectedly, the contribution of CHC to spread was independent of Sca4 and appeared restricted to the recipient host cell, suggesting that the Sca4-clathrin interaction regulates another aspect of the infectious lifecycle. Indeed, R. parkeri lacking Sca4 or expressing a Sca4 truncation unable to bind clathrin had markedly reduced burdens in tick cells, hinting at a cell type-specific function for the Sca4-clathrin interaction. Sca4 homologs from diverse Rickettsia species also bound clathrin, suggesting that the function of this novel effector-host interaction may be broadly important for rickettsial infection. We conclude that Sca4 has multiple targets during infection and that rickettsiae may manipulate host endocytic machinery to facilitate several stages of their life cycles.