Release of ActA into a medium via membrane anchor cleavage is required for Listeria monocytogenes host cell invasion

Abstract

The ActA protein, which is involved in intra- and intercellular Listeria monocytogenes movement, is anchored in the bacterial membrane by a hydrophobic C-tail, and it is a membrane-bound form of ActA that is indispensable for intracellular movement. However, soluble ActA is detected in the culture supernatant of L. monocytogenes as a major protein. In this study, the mechanisms of ActA release into the culture supernatant and the role that soluble ActA plays in intracellular virulence were investigated. Our results indicated that the cleavage of the C-terminal membrane anchor is required for ActA release. The MALDI-TOF MS analysis of trypsin-cut soluble ActA suggested that the cleavage occurred between His582 and Thr583. The proposed cleavage site was confirmed by introduction of the His582Pro substitution preventing ActA release, although this did not disturb its surface presentation. The virulence-associated metalloprotease Mpl, which is responsible for pH-dependent ActA degradation, is not involved in membrane anchor cleavage, which requires another still unidentified protease. The mutant strain had a lower invasive effectiveness into HeLa epithelial cells, whereas it did not differ from the wild strain in intracellular proliferation and the ability to form plaques of lysis in the HeLa confluent monolayer. The obtained results suggested that ActA release is important for the invasion of L. monocytogenes into epithelial cells but is not required for intracellular motility and cell-to-cell spread.