Understanding the principles of biomolecular self-assembly is vital in the context of infectious diseases, as it plays a crucial role in the assembly of pore-forming toxins and viruses, mechanisms central to pathogen virulence and infection propagation. This knowledge facilitates the development of targeted interventions and therapeutics that can disrupt these processes, thereby mitigating infection and disease spread. Moreover, it sheds light on the molecular basis of host-pathogen interactions, enabling the design of innovative strategies to bolster host defenses and prevent disease onset.

A key challenge in studying self-assembly of (cytotoxic) macro-assemblies lies in tracking the dynamics of their formation via pathways comprising of a large number of diffraction-limited heterogeneous intermediates. We addressed this problem in the study of 2D pore assembly of Cytolysin A, a bacterial pore forming toxin implicated in bacterial virulence, on phospholipid membranes. Using a custom-built total-internal-reflection fluorescence (TIRF) microscope combined with single particle tracking and photobleaching analysis, we demonstrated, that the conformations and oligomeric states of the membrane bound toxin protein can be accurately extracted (Sathyanarayana, et al. PNAS 2018). This allowed us to show how cholesterol, a key component of mammalian cell membranes, allowed the toxin to selectively target the mammalian cells. Due to broad structural similarities between pore-forming toxins, these mechanisms to impart membrane selectivity could be common.  New ways to prepare suspended membranes that remove surface effects are allowing us to extend such studies to other membrane phenomena like viral membrane fusion (Sannigrahi, et al. Membranes 2022)

Furthermore, using artificially designed polymer supported bilayers that are better mimics of cell membranes, we are currently exploring the effect of molecular crowding on the diffusional properties and kinetics of molecular assembly on the membrane (Maurya et al. JoVE 2022). 

1. P Sathyanarayana, S Maurya, A Behera, M Ravichandran, SS Visweswariah, KG Ayappa, and R Roy, "Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation" PNAS (USA), 115 (31) E7323-E7330, doi:10.1073/pnas.1721228115 (2018) 

2. A Sannigrahi, et al. "A Versatile Suspended Lipid Membrane System for Probing Membrane Remodeling and Disruption" Membranes, www.mdpi.com/2077-0375/12/12/1190 (2022)

3. S Maurya, et al., "Single-molecule Diffusion and Assembly on Polymer-crowded Lipid Membranes" JoVE,  https://dx.doi.org/10.3791/64243 (2022)