Structure and functions of a bacterial Type IV effector complex

Project Summary Bacterial pathogens use “effector” proteins as part of their virulence strategies to subvert host functions and promote bacterial intracellular survival, proliferation and persistence. These effectors operate individually or collectively to modulate cellular processes, usually in a functionally integrated manner. Bacterial effectors may have antagonistic or convergent activities on cellular pathways or regulate each other’s activities (as “meta-effectors”). How multiple effector activities are coordinated spatially and temporally in host cells remains largely unknown. In our efforts to characterize mechanisms of intracellular proliferation of the intracellular bacterium Brucella abortus, we have uncovered a physical interaction between three Type IV secretion effectors - BspF, BspH and BspI - suggesting that they form a multi- effector complex when delivered into host cells. BspF is a putative acetyltransferase that localizes to the recycling endosome and interacts with the host protein ACAP1, a GTPase activating protein (GAP) of the endocytic GTPase Arf6, thereby inhibiting Arf6 activity and retrograde vesicular transport to promote Brucella intracellular growth. BspH and BspI remain poorly characterized but contain structural Armadillo (ARM) repeats and a GAP domain, respectively, suggesting protein-protein interactions for BspH and host GTPase modulation capabilities for BspI. Consistently, we found that BspI directly interacts with the host GTPases Rac1 and Rab5. Moreover, we found that BspF, BspH and BspI interact in a complex associated with early endosomal structures. We will test the hypothesis that these effector proteins intrinsically form a multi-effector platform that coordinates host GTPase-modulating effector activities on endosomal transport to promote bacterial intracellular growth. Aim 1 will determine key aspects of the structure and assembly of the BspF:H:I complex, using computational, biochemical and structural biology approaches. Aim 2 will i) determine the function of BspI and of the BspF;H:I complex in Brucella’s intracellular cycle and ii) define functional interactions between BspF, BspH and BspI. The proposed research will address seminal questions towards establishing a new paradigm of integration of bacterial effector functions with broad impact to the field of bacterial pathogenesis.