Professional phagocytes undergo shifts in cell shape in response to a variety of cues, and this plasticity requires
the ability to remodel the cytoskeleton in extreme ways. Historically, many new insights into cell biology have
emerged from studying intracellular pathogens that have evolved to survive by modulating the subcellular
organization and function of host pathways. The goal of this proposal is to better understand how mycobacteria
manipulate the host cytoskeleton and the role of the WASH complex, an Arp2/3 nucleation promoting factor, in
this process. We have discovered an ancient mycobacterial effector, EsxM, that promotes changes to the
macrophage cytoskeleton through a putative interaction with WASHC4, a member of the WASH complex. These
changes enhance the dissemination of mycobacterial disease via migrating macrophages. The striking shift in
macrophage morphology and behavior induced by EsxM have led us to probe the extent of actin rearrangements
during infection (Aim 1) and whether the WASH complex may have unknown roles in regulating cell shape and
motility (Aim 2). Although the importance of cytoskeletal rearrangement in migrating cells is established, little is
known about how mycobacterial effectors may manipulate this axis during infection. Additionally, the WASH
complex has been studied primarily for its role in endocytic trafficking, but less is known about its potential role
in macrophage migration, or as a target of intracellular bacteria. Thus, new investigations into the host
cytoskeleton and the WASH complex are needed to understand their roles in cell migration and immunity. The
overall hypothesis of this proposal is that mycobacterial effector EsxM induces changes in the actin
cytoskeleton that lead to enhanced migratory capacity of macrophages via the WASH complex. In order
to test this hypothesis, I will first take advantage of our established zebrafish model which offers genetic
tractability and optical transparency. In Aim 1, I will utilize an established macrophage F-actin reporter zebrafish
line to observe changes in the actin cytoskeleton induced by EsxM in the context of cell migration and innate
immunity. I will combine high resolution imaging, pharmacological treatment, and genetic tools to functionally
test modes of macrophage migration during these processes. In Aim 2 I will test how genetic disruption of the
WASH complex changes macrophage morphology and migration in vivo using zebrafish knockouts. I will also
take advantage of a mouse line in which the WASH complex is disrupted to study its role in mycobacterial
infection in a mammalian model. Overall, these studies will both take advantage of the zebrafish system to
provide insights into the dynamic actin cytoskeleton in real time, and interrogate a role for the WASH complex in
macrophage migration. The completion of the proposed work will represent a significant advancement in our
understanding of how the actin cytoskeleton is altered in the context of infection, and how the WASH complex
regulates macrophage biology beyond endocytic trafficking alone. The findings will highlight the importance of
studying host-pathogen interactions to elucidate the function of host-factors during infection and homeostasis.