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 approximately 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.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Pamela
P
Schnupf
Jianmin
J
Zhou
Alexander
A
Varshavsky
Daniel A
DA
Portnoy
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D015169:Colony Count, Microbial; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D008088:Listeriosis; D008099:Liver / Q000382:microbiology; D008239:Lysine / Q000235:genetics / Q000502:physiology; D053858:Metabolic Networks and Pathways; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D013154:Spleen / Q000382:microbiology; D014774:Virulence
D001427:Bacterial Toxins; D002457:Cell Extracts; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008239:Lysine; C038705:hlyA protein, Listeria monocytogenes