Aggregation causes listeriolysin inactivation

Andrej Bavdek

Biotehniška fakulteta, Oddelek za biologijo, Univerza v Ljubljani

SUMMARY:

Listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagolysosome, which enables its replication in the cytosol of host cells during the infection. LLO belongs to the family of cholesterol dependent citolysins, produced by various Gram-positive bacteria. Its unique feature is low pH optimum, which enables maximal activity in the phagosomal environment. It is believed that PEST sequence leads protein to proteolitical degradation in the cytosol, which diminish the possibility to damage the plasma membrane from the interior of the cell and thus reducing Listeria survival. We and others have found that LLO rapidly aggregates in solution at higher pH values. LLO aggregation in solution was thoroughly examined in this study.

Recombinant His-tagged LLO was produced in an E. coli expression system. According to fluorescence and CD spectroscopy, LLO properties are not dependent on pH. LLO aggregation in solution was followed by tryptophan fluorescence, ANS fluorescence and light scattering. The aggregation is temperature and pH dependent. Aggregates start to form immediately after temperature shift from 20 to 37 oC at physiological pH (7.5), whereas aggregation was not observed at acidic pH (5.5). pH shift from 5.5 to 7.5 at 37 °C also results in rapid aggregate formation. LLO also aggregates when incubated at 37 °C in cytosolic conditions (similar ionic and macromolecular composition and pH). Aggregation is irreversible and agrgegates are not hemolytically active. We suggest that LLO aggregation may provide the major route of rapid LLO inactivation in the cytosol of the host cells.