Molecular Mechanisms of Bacterial Toxins Targeting the Actin Cytoskeleton

With the climate change-related emergence of new pathogens and the reemergence of old, once-controlled pathogens, as well as in light of rapidly developing antibiotic resistance, society is in urgent need of a more thorough understanding of the bacterial pathogenicity and its major mediators – toxins and toxic effector proteins (effectors). The actin cytoskeleton is a common target for numerous bacterial effectors. However, the mechanisms by which many actin-targeting bacterial toxins exhibit their pathogenicity remain poorly understood. The long-term goal of the proposal is to decipher the in-depth molecular and cellular mechanisms of bacterial effectors targeting the actin cytoskeleton to i) enable alternative ways of targeting pathogens and ii) get a deeper understanding of the actin cytoskeleton per se. The current proposal is directly relevant to the NIH mission as it focuses on three families of bacterial effectors, all produced by human pathogens. Furthermore, the proposal is of interest for a general understanding of human physiology as each of the above toxins reveals novel properties of the actin cytoskeleton. Thus, Aim 1 focuses on deciphering the novel hitherto unprecedented ability of VopF and VopL effectors produced by Vibrio cholerae and Vibrio parahaemolyticus to potentiate actin processive polymerization at the pointed ends. Aim 2 addresses the properties of SipA, an effector from Salmonella enterica that exerts a novel mode of binding to actin, bestowing the pathogen an ability to adhere to and invade the polarized host cell. In Aim 3, the proposal will decipher the mechanisms of actin-dependent membrane remodeling by Legionella pneumophila effectors. Understanding these and related processes is particularly important given that all intracellular pathogens depend heavily on hijacking host membrane organization to survive and thrive inside the affected cell. The research strategy for the toxin characterization will be based on using several highly complementary experimental approaches. The effects of the toxins on actin dynamics in bulk and at the single-filament level will be combined with cell biology and structural biology approaches. Specifically, bulk actin dynamics will be monitored via modifications of the pyrene-actin polymerization approach. The effects of the toxins on the actin dynamics at the single-filament level will be characterized by total internal reflection fluorescence microscopy, which will be enforced by microfluidics (in collaboration with Dr. Shekhar) for deciphering the mechanisms employed by VopF/VopL toxins (Aim 1). A modification of this technique will be used to decipher the mechanisms of a membrane-reorganizing protein MavH (Aim 3). The structures of VopF with the actin pointed end (Aim1), and SipA bound along the filament side (Aim 2) will be characterized by the cryoEM reconstruction in collaboration with Dr. Egelman’s group. Fluorescence anisotropy will be used to describe the strengths of the interactions between actin and the effectors. The proposed study is significant and innovative as it fills major gaps in our understanding of the toxicity of several life-threatening pathogens and reveals novel ways of operation of the actin cytoskeleton.