Investigating the Role of the GTPase Arl8b in Plasma Membrane Repair of Mtb-Infected Macrophages

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 DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is an extraordinarily successful pathogen that infects 8.6 million people yearly and causes 1.3 million deaths. The principal host cell of Mtb is the lung macrophage (M). It is not well understood how in some cases the internalized Mtb remain sequestered and are ultimately killed and in other cases persist and escape recognition by the immune system. We have shown that membrane repair mechanisms are extremely important in determining the outcome of infection. Membrane repair maintains the integrity of the Mφ plasma membrane after infection of M with avirulent Mtb, a requisite step for apoptosis of infected Mφ, which kills the pathogen. Virulent Mtb, in contrast, block membrane repair and instead actively induce necrosis, supporting spread of infection and escape from immune control. Mechanistically both avirulent and virulent Mtb induce microdisruptions of the host M plasma membrane. These plasma membrane lesions are repaired in M infected with avirulent Mtb, but in the case of virulent Mtb, necrosis ensues because the bacillus inhibits plasma membrane repair. The goal of this project is the delineation of membrane repair in Mtb-infected M, which will lead to increased understanding of the protective host response mechanism and of the critical steps, by which virulent Mtb escape host containment. A major component of the membrane repair mechanism is Arl8b, a small GTPase involved in lysozyme motility and trafficking. In Aim 1 we will use a cell line model (HeLa cells) to (a) determine the role of Arl8b and (b) identify additional interacting effector molecules in plasma membrane repair. In Aim 2 we will test whether Arl8b and the validated effectors are required for plasma membrane repair of Mtb-infected M and whether virulent Mtb subverts their function. The described studies are directed towards therapeutic interventions to enhance the host anti-mycobacterial response (host-directed tuberculosis therapy).