Investigating the role of liquid-liquid phase separation in the interaction between Mycobacterium tuberculosis and macrophages

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.

Grant Number: 5DP2AI154429-05
NIH Institute/Center: NIH
Principal Investigator: Samantha Bell