The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence

Abstract: The Gram-negative bacterium Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to establish infections. The T3SS translocates a diverse set of effector proteins directly into the host cells. The coordinate action of the translocated effectors blocks the innate immune system of the host and ensures extracellular proliferation of the bacterium. YopE is an essential effector that disrupts the actin cytoskeleton of infected host cells. This cytotoxicity is caused by the inactivation of RhoGTPases by the GTPase Activating Protein (GAP) activity of YopE. YopE was demonstrated to inactivate the RhoGTPases Rac1 and RhoA in vivo. However, Rac1 and RhoA inactivation was not a prerequisite for cytotoxicity or virulence. Thus, YopE must have additional targets during infection. Surprisingly, avirulent yopE mutants had lost the control of Yop expression in the presence of target cells and they all overtranslocated effectors. It appeared as if translocated YopE was able to control Yop expression and effector translocation via a feedback inhibition mechanism. This feedback inhibition was dependent on functional GAP activity. Translocation control could also be mediated by exogenous GAP activity, suggesting that effector translocation control might be a general property of all bacterial GAP proteins. Besides YopE, the regulatory protein YopK was also found to be involved in the effector translocation control process. Clearly, as demonstrated in virulence, the roles for YopE and YopK are intimately related.                       Further, YopE possesses a membrane localization domain (MLD) required for proper localization. A yopE?MLD mutant had lost the feedback inhibition of YopE expression and was avirulent. Hence, the effector translocation control of YopE requires both proper localization as well as functional GAP activity.                                           In addition, fish keratocytes were established as a novel model system for Y. pseudotuberculosis infections. YopE was found to be the sole effector responsible for cytotoxicity towards the keratocytes. Further, induction of cytotoxicity required fully native YopE protein which indicated that the keratocytes would be useful as a sensitive model system for further studies of YopE mediated phenotypes.In summary, this thesis work has sought to unravel the multiple functions of translocated YopE. A novel role was elucidated where Yersinia utilizes translocated YopE to control the process of effector translocation into host cells. This regulatory control was connected to virulence in the mouse model of disease. Thus, perhaps YopE should be considered also as a regulatory protein besides being a classical effector.