Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we i dentify a putative PEST sequence close to the N-terminus of LLO and focus o n the role of this motif in the biological activities of LLO. Two LLO varia nts were constructed: a deletion mutant protein, lacking the 19 residues co mprising this sequence (residues 32-50), and a recombinant protein of wild- type size, in which all the P, E, S or T residues within this motif have be en substituted. The two mutant proteins were fully haemolytic and were secr eted in culture supernatants of L. monocytogenes in quantities comparable w ith that of the wild-type protein. Strikingly, both mutants failed to resto re virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired i n its ability to escape from the phagosomal vacuole and, subsequently, to d ivide in the cytosol of infected cells. This work reveals for the first tim e that the N-terminal portion of LLO plays an important role in the develop ment of the infectious process of L. monocytogenes.