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.
Schnupf
Pamela
P
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Zhou
Jianmin
J
Varshavsky
Alexander
A
Portnoy
Daniel A
DA
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