Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes

Listeriolysin O (LLO) is a major virulence factor secreted by the pathogeni c Listeria monocytogenes and acts as pore-forming cytolysin. Based on seque nce similarities between LLO and perfringolysin (PFO), the cytolysin from C lostridium perfringens of known crystallographic structure, two truncated L LO proteins were produced: LLO-d123, comprising the first three predicted d omains, and LLO-d4, the last C-terminal domain. The two proteins were effic iently secreted into the culture supernatant of L. monocytogenes and were a ble to bind to cell membranes. Strikingly, when expressed simultaneously, t he two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytical ly active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion create d a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 parti cipates in the interaction between the two portions of the monomer.