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Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling¹

Jeremy C. D. Wiseman^*, Ling Ling Ma, Kaleb J. Marr^*, Gareth J. Jones and Christopher H. Mody^2,,

Departments of ^* Medical Science, Internal Medicine, and Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada

Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the -regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from Canadian Institutes of Health Research (to C.H.M.), the Canadian Foundation for AIDS Research (to C.H.M.), and the Jessie Bowden Lloyd Professorship in Immunology (to C.H.M.). C.H.M. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.

² Address correspondence and reprint requests to Dr. Christopher H. Mody, Room 273, Heritage Medical Research Building, University of Calgary, Calgary, Alberta, Canada. E-mail address: cmody{at}ucalgary.ca

³ Abbreviations used in this paper: Rac-1, Ras-related C3 botulinum toxin substrate-1; Akt, v-akt murine thymoma viral oncogene homolog; DAPI, 4',6'-diamidino-2-phenylindole; PAK, p21/Cdc42/Rac1-activated kinase; TRITC, tetramethyl rhodamine isothiocyanate; siRNA, small interfering RNA.

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