Assessing Aggregation and Membrane Binding of LLO Domains

Abstract

Listeriolysin O (LLO), as one of the major virulence factors of intracellular pathogen Listera monocytogenes, assists in the escape of the bacterium from the phagosome by specifically forming oligomeric transmembrane β‐barrels on the target membrane. Its unique acidic pH optimum lessens the toxicity towards the host cell. Cholesterol is claimed to be the membrane receptor of LLO with the C‐terminal domain 4 (D4) involved in the binding and recognition. In contrast, domains 1 to 3 (D1‐3) are critical for monomer aggregation on the membrane. To better investigate these two distinct interactions with the membrane, we have separately expressed domains D4 and D1‐3 and compared their behavior to full‐length LLO using a variety of biophysical approaches (TEM, FCS, NMR). Full‐length LLO is lytic only towards cholesterol‐containing membranes and at acidic pH. D4 binds to membranes regardless of cholesterol or pH, presumably due to the hydrophobicity of its tip (ECTGLAWEWWR). However, this domain can be stabilized in a well‐folded form in DPC and SDS micelles; these have sufficient resolution in 15N‐1H HSQC experiments for some structural work. D1‐3 is nonlytic and soluble, but it aggregates significantly with the aggregation enhanced at acidic pH. These results are used to propose a modified model for the action of LLO on target membranes. These studies were supported by the NIH (GM60418).