Activation of the Salmonella enterica serovar Typhimurium translocated glycerophospholipid:cholesterol acyltransferase SseJ by the small GTPase RhoA

Abstract

<italic>Salmonella enterica</italic> serovar Typhimurium translocates a glycerophospholipid:cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated and converts cholesterol to cholesterol ester upon binding the small GTPase, RhoA. This study shows that SseJ is regulated similarly to cognate eukaryotic effectors and only the GTP-bound form of RhoA, RhoB, RhoC, but not other Rho GTPases stimulates enzymatic activity. Using NMR and biochemistry this work demonstrates that SseJ competes effectively in binding with eukaryotic effectors and specifically competes for a similar RhoA binding surface as Rhotekin, ROCK, and PKN1. The RhoA surface that binds SseJ includes the regulatory switch regions that controls activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. We discovered that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full <italic>Salmonella</italic> virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase further defines how intimately host pathogen interactions have co-evolved through similar and divergent evolutionary strategies.