PROJECT SUMMARY There is a fundamental gap in our understanding of the complex processes that govern the interactions between Mtb and macrophages. The overall objective of this application is to investigate how the novel biophysical phenomenon of phase separation impacts biological processes, specifically in the context of Mtb infection. A detailed knowledge of the molecules that recognize and respond to pathogens is required to reveal how cells fight infection; therefore, there is a critical need to understand how phase separation may influence or control innate immune responses. Mycobacterium tuberculosis (Mtb) is an incredibly successful and deadly human pathogen that infects one-quarter of the world's population. While interaction of Mtb bacilli and macrophages activates numerous innate immune pathways, we have a limited understanding of how these complex networks of host sensing molecules are regulated to work cooperatively. Furthermore, only a small subset of the many secreted effectors used by M. tuberculosis have well-characterized functions. Recent studies have illuminated the biological and cellular importance of liquid-liquid phase separation, a process by which proteins condense into discrete droplets to alter their localization and function in a cell. Several proteins involved in the host response to M. tuberculosis infection, like cGAS, TBK1, p62, and LC3, have been found to phase separate in vitro, but how in vivo phase separation impacts host responses to infection is unknown. Preliminary studies have found that these and other innate immune proteins form circular puncta in M. tuberculosis-infected cells that resemble phase separated droplets. The central hypothesis of this proposal is that upon infection, pathogen- sensing and post-translational modifications induce phase separation of host proteins and that Mtb modulates these condensation events with its own phase-separating PE/PPE proteins. Here, a combination of novel optogenetics tools, live cell fluorescent imaging, and host and bacterial genetics will be employed to probe the biological consequences of phase separation of host proteins (Aim 1) and Mtb proteins (Aim 2). In addition, directed and unbiased genetics approaches will be used to probe how post-translational modifications, and especially ubiquitination in particular, contributes to phase separation during Mtb infection (Aim 3). This approach is innovative in that it uses novel tools to specifically and precisely modulate phase separation in order to link this biophysical process with meaningful cellular phenotypes. The proposed research is significant because it will greatly expand our understanding of how macrophages destroy Mtb and advance efforts to combat Mtb infection via enhancing host responses.

PROJECT NARRATIVE The proposed research is relevant to human health because Mycobacterium tuberculosis (Mtb) is the number one infectious disease killer in the world. The ability of innate immune cells to kill or control Mtb infection is critical for disease outcomes. Therefore, I aim to understand how this process is regulated in hopes of one day developing therapeutics designed to enhance macrophages' antibacterial responses and control infection in human patients. The proposed research is relevant to the part of NIH's mission that pertains to making fundamental discoveries that will help prevent and treat disease.