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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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