Understanding infectious disease using membrane topography engineering: case studies of membrane remodeling in viral replication and antimicrobial treatment

The membrane topography of a cell alters dramatically in various essential cellular processes, such as cell migration, cell division, and vesicle trafficking. Its regulation is often hijacked during viral infection to generate unique curved membrane compartments for viral replication and is a potential target for the development of new antiviral or therapeutic agents. However, many of such membrane topography changes happen at the scale of tens to hundreds of nanometers, making it not only difficult to resolve optically for characterization but also challenging to manipulate accurately for in-depth study. My thesis here targets to establish a nanofabrication-enabled membrane topography engineering platform to address this issue. Two critical membrane topography remodeling cases are chosen to study: (i) viral replication; and (ii) antimicrobial treatment. To establish the nanofabrication-based membrane topography engineering platform, a nanobar-supported lipid bilayer (SLB) system was first developed, and we successfully identified a novel curvature sensing domain in vitro using it, i.e., an intrinsically disordered region (IDR) outside the reported F-BAR domain of the formin binding protein 17 (FBP17). Moreover, using model proteins of FBP17 and epsin N-terminal homology (ENTH), we also demonstrated the ability of the nanotopography platform to benchmark protein curvature sensing ability measured in vitro with their response to similarly curved membranes captured in live cells. (Chapter II) Furthermore, a novel cell-membrane-mimicking system on nanotopography arrays was established using giant plasma membrane vesicle (GPMV) to evaluate the curvature sensitivity of the membrane proteins with post-translational modifications such as GPI-anchored proteins and palmitoylated proteins. (Chapter III) With the established nanotopography engineering platform, two membrane topography remodeling cases related to infectious diseases were investigated. The first one is the formation of highly curved membrane spherules by viral replication complexes. Positive-sense single-stranded RNA viruses, including the well-known Zika, Dengue, Hepatitis C, and the most recent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are found to generate subcellular membrane compartments for their replication. Taking a simple type of positive-sense single-stranded RNA virus, Chikungunya virus (CHIKV), as a model, we found its non-structural protein 1 (nsP1) was able to sense positively curved membrane through its membrane association loops, and its further binding with RNA template and other nsPs enhances the curvature preference, indicating the curved membrane topography positively associated with the formation and stabilization of the membrane compartments for viral replication. (Chapter IV) Besides the preferential binding of viral proteins to curved membranes during viral replication, I also evaluate the impact of membrane topography on the membrane intercalating of a new class of antimicrobial agent, conjugated oligoelectrolytes (COEs). Long COE species, COE-S6, with reported membrane stabilization ability shows preferential intercalating at the curved membrane. Short COE species, such as COE2-3C-C6 and COE2-3C-C4hexyl, with proved antimicrobial function, rapidly induce dramatic membrane remodeling and selectively generate flexible membrane nanotubes along the nanobar curved membrane sites. These membrane remodeling patterns revealed on engineered membrane topographies provide new insights into the mechanism of COE-induced membrane disruption. (Chapter V) Overall, using the nanostructure-based membrane topography engineering, membrane curvature was shown to serve as a significant modulator of membrane remodeling during viral replication and antimicrobial treatment. I envisioned that the nanopattern-enabled membrane topography engineering platform provides promising and versatile strategies to dissect more fundamental principles of membrane remodeling in infectious diseases and pave the way for novel antimicrobial therapeutics design and evaluation.Doctor of Philosoph