title: Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence.
abstract: Listeria monocytogenes is a bacterial pathogen that grows within the cytosol of infected host cells. Entry into the cytosol is largely mediated by a secreted bacterial cytolysin, listeriolysin O (LLO). In order to prevent host cell damage, the pore-forming activity of LLO is restricted to the phagosome. Compartmentalization of LLO requires a PEST-like sequence; PEST sequences can direct eukaryotic proteins for proteasomal degradation. Here we test the hypothesis that LLO's PEST-like sequence compartmentalizes pore-forming activity by targeting this bacterial protein for degradation in the host cytosol. We show that intracellular LLO was degraded in a proteasome-dependent manner, and that, prior to degradation, LLO was ubiquitinated and was phosphorylated within the PEST-like sequence. However, wild-type LLO and PEST region mutants had similarly short intracellular half-lives and both the wild-type and mutant proteins were stabilized by inhibitors of host proteasomes. Additionally, blocking host proteasomes did not cause toxicity in a wild-type infection, but enhanced the cytotoxicity of PEST region mutants. Together with the observation that PEST region mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like region does not mediate proteasomal degradation by the host, but controls LLO production in the cytosol.
authors.affiliation: Graduate Group in Microbiology, University of California, Berkeley, CA 94720-3202, USA.
authors.forename: Pamela
authors.initials: P
authors.lastname: Schnupf
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Portnoy
authors.forename: Amy L
authors.initials: AL
authors.lastname: Decatur
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism / Q000382:microbiology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000254:growth & development / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D009154:Mutation; D010588:Phagosomes / Q000378:metabolism* / Q000382:microbiology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology*; D051381:Rats; D012694:Serine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D021382:Protein Sorting Signals; D025801:Ubiquitin; D012694:Serine; D046988:Proteasome Endopeptidase Complex; C038705:hlyA protein, Listeria monocytogenes