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Molecular and Cellular Biology, December 1999, p. 8604-8615, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Kylie A. Browne,1 Elizabeth Blink,2 Vivien R. Sutton,1 Christopher J. Froelich,3 David A. Jans,2 and Joseph A. Trapani1,*

The Austin Research Institute, Heidelberg, Victoria 3084,1 and Nuclear Signalling Laboratory, John Curtin School of Medical Research, Australian National University, Canberra City 2600,2 Australia, and Cell Death Program, ENH Research Institute, Evanston, Illinois 602013

Received 5 April 1999/Returned for modification 3 June 1999/Accepted 31 August 1999

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 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.

* Corresponding author. Mailing address: The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, Australia. Phone: 61-3-9287-0651. Fax: 61-3-9287-0604. E-mail: j.trapani{at}ari.unimelb.edu.au.

Molecular and Cellular Biology, December 1999, p. 8604-8615, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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