Editors' ChoiceImmunology

Dying on the Inside

Science Signaling  17 Jun 2014:
Vol. 7, Issue 330, pp. ec163
DOI: 10.1126/scisignal.2005592

Cytotoxic lymphocytes, including natural killer cells and CD8+ T cells (CTLs), respond to bacterial infection by killing the host cell to limit the spread of infection. These lymphocytes secrete granules containing the pore-forming protein perforin and serine proteases called granzymes, which enter the host cell through the perforated plasma membrane. These proteases cleave host proteins, including members of complex I of the mitochondrial electron transport chain, to produce reactive oxygen species (ROS) and induce apoptosis (see Commentary by Narni-Mancinelli and Vivier). Granulysin is a saposin-like, lipid-degrading protein secreted by human, but not mouse, killer lymphocytes that disrupts bacterial and other parasitic cell membranes. Walch et al. found that incubation of extracellular bacteria with sublytic concentrations of granulysin enabled fluorescently tagged granzymes (A or B) to enter the bacterial cytoplasm. Moreover, incubation with granzymes, but not mutant granzymes that are proteolytically inactive, killed multiple species of bacteria in a granulysin-dependent manner. Incubation of multiple species of bacteria with granulysin and granzyme B, but not either protein alone, induced the production of ROS and the expression of ROS-dependent genes. In Escherichia coli, overexpression of enzymes or exposure to small molecules that detoxify ROS inhibited bacterial death induced by granulysin and granzyme B. Granzymes (A or B) cleaved multiple bacterial ROS-detoxifying enzymes and several subunits of complex I of the electron transport chain both in vitro and in bacteria. E. coli deficient in complex I or expressing a cleavage-resistant mutant component of complex I were resistant to ROS production and death induced by granulysin and granzyme B. In cultured human cancer (HeLa) cells infected with Listeria monocytogenes (Lm), incubation with perforin and granzyme B killed the host cell and this effect was not enhanced by granulysin. In contrast, bacterial cell viability in infected cells was not reduced by perforin and granzyme B, but was somewhat reduced by granulysin and either perforin or granzyme B, whereas it was most reduced by all three proteins. Inhibition of host cell death by overexpression of the anti-apoptotic protein BCL2 did not inhibit bacterial death induced by these three proteins. In monocyte-derived dendritic cells infected with Lm, coculture with human Lm-specific CTLs killed bacteria and host cells; however, bacterial death was faster, required fewer CTLs, and was not affected by caspase inhibition, unlike that of host cells. Transgenic mice expressing human granulysin cleared primary and secondary Lm infections more efficiently than did wild-type mice. Together, these data suggest that human cytotoxic lymphocytes use perforin and granulysin as part of a two-step process to kill intracellular bacteria without killing the infected cell.

M. Walch, F. Dotiwala, S. Mulik, J. Thiery, T. Kirchhausen, C. Clayberger, A. M. Krensky, D. Martinvalet, J. Lieberman, Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell 157, 1309–1323 (2014). [PubMed]

E. Narni-Mancinelli, E. Vivier, Delivering three punches to knockout intracellular bacteria. Cell 157, 1251–1252 (2014). [PubMed]

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