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Infection and Immunity, November 2007, p. 5135-5147, Vol. 75, No. 11
0019-9567/07/$08.00+0     doi:10.1128/IAI.00164-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Pamela Schnupf,1,{dagger} Jianmin Zhou,4 Alexander Varshavsky,4 and Daniel A. Portnoy1,2,3*

Graduate Group in Microbiology,1 Department of Molecular and Cellular Biology,2 School of Public Health, University of California, Berkeley, California 94720-3202,3 Division of Biology, California Institute of Technology, Pasadena, California 911254

Received 31 January 2007/ Returned for modification 6 March 2007/ Accepted 17 July 2007

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.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202. Phone: (510) 643-3925. Fax: (510) 643-6334. E-mail: portnoy{at}berkeley.edu

{triangledown} Published ahead of print on 6 August 2007.

Editor: J. B. Bliska

{dagger} Present address: Unité de Pathogenie Microbienne Moléculaire, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France.

Infection and Immunity, November 2007, p. 5135-5147, Vol. 75, No. 11
0019-9567/07/$08.00+0     doi:10.1128/IAI.00164-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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