title: Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation.
abstract: Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogenListeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified proteinin vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain ofL. monocytogenesexpressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild typein vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection modelin vivoThis study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.
authors.affiliation: Graduate Group in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Portman
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Qiongying
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Reniere
authors.affiliation: The California Institute for Quantitative Biosciences and the QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Iavarone
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA portnoy@berkeley.edu.|School of Public Health, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis; D008089:Listeria monocytogenes / Q000472:pathogenicity / Q000502:physiology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D037521:Virulence Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; C038705:hlyA protein, Listeria monocytogenes
keywords: Firmicutes; Gram-positive bacteria; Listeria monocytogenes; bacterial toxins; cholesterol-dependent cytolysins; glutathione; pore-forming toxins; posttranslational modification; thiol-dependent cytolysins; virulence