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Published November 2007 | Published
Journal Article Open

Listeriolysin O Secreted by Listeria monocytogenes into the Host Cell Cytosol Is Degraded by the N-End Rule Pathway


The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an ~100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.

Additional Information

© 2007, American Society for Microbiology. Received 31 January 2007/ Returned for modification 6 March 2007/ Accepted 17 July 2007. Published ahead of print on 6 August 2007. We thank Partho Ghosh (University of California, San Diego) for advice about LLO structure and Meredith L. Leong, Dirk G. Brockstedt, and Tom W. Dubensky (Cerus Corporation) for the LD50 determination for BALB/c mice and helpful discussions. This research was supported by the National Institute of Health grants AI27655 (to D.A. Portnoy) and DK39520 (to A. Varshavsky) and by a PGSB award from the National Science and Engineering Research Council of Canada (to P. Schnupf). D.A. Portnoy consults with and has a financial interest in Cerus Corporation, a company that may stand to benefit from the results of this research.

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