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Cytosolic Delivery of Granzyme B by Bacterial Toxins: Evidence that Endosomal Disruption, in Addition to Transmembrane Pore Formation, Is an Important Function of Perforin

Browne, Kylie; Blink, Elizabeth; Sutton, Vivian; Froelich, Christopher; Jans, David A; Trapani, Joseph A

Description

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and...[Show more]

dc.contributor.authorBrowne, Kylie
dc.contributor.authorBlink, Elizabeth
dc.contributor.authorSutton, Vivian
dc.contributor.authorFroelich, Christopher
dc.contributor.authorJans, David A
dc.contributor.authorTrapani, Joseph A
dc.date.accessioned2015-12-13T23:24:54Z
dc.identifier.issn0270-7306
dc.identifier.urihttp://hdl.handle.net/1885/92441
dc.description.abstractGranule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% 51Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal 51Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.
dc.publisherAmerican Society for Microbiology
dc.sourceMolecular and Cellular Biology
dc.subjectKeywords: bacterial toxin; granzyme B; listeriolysin; nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase; perforin; pneumolysin; Staphylococcus alpha toxin; streptolysin O; animal cell; apoptosis; article; channel gating; endosome; enzyme lo
dc.titleCytosolic Delivery of Granzyme B by Bacterial Toxins: Evidence that Endosomal Disruption, in Addition to Transmembrane Pore Formation, Is an Important Function of Perforin
dc.typeJournal article
local.description.notesImported from ARIES
local.description.refereedYes
local.identifier.citationvolume19
dc.date.issued1999
local.identifier.absfor111502 - Clinical Pharmacology and Therapeutics
local.identifier.ariespublicationMigratedxPub23551
local.type.statusPublished Version
local.contributor.affiliationBrowne, Kylie, Peter MacCallum Cancer Centre
local.contributor.affiliationBlink, Elizabeth, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationSutton, Vivian, Austin Research Institute
local.contributor.affiliationFroelich, Christopher, ENH Research Institute
local.contributor.affiliationJans, David A, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationTrapani, Joseph A, Peter MacCallum Cancer Centre
local.description.embargo2037-12-31
local.bibliographicCitation.issue12
local.bibliographicCitation.startpage8604
local.bibliographicCitation.lastpage8615
dc.date.updated2015-12-12T09:22:28Z
local.identifier.scopusID2-s2.0-0033499729
CollectionsANU Research Publications

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