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* Academic Unit of Respiratory Medicine,
Academic Unit of Biochemical and Musculoskeletal Medicine, and
Academic Unit of Infectious Diseases, University of Sheffield, Sheffield, United Kingdom; and
Department of Medicine, Hampstead Campus, Royal Free and University College School of Medicine, London, United Kingdom
Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramatically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. In this study, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunction, shown by altered lysosomal pH, within 15 min of exposure. Lysosomal disruption was followed by mitochondrial membrane permeabilization, caspase activation, and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin D (CTSD), abrogated pyocyanin-induced apoptosis, and translocation of CTSD to the cytosol followed pyocyanin treatment and lysosomal disruption. A stable analog of cAMP (dibutyryl cAMP) impeded the translocation of CTSD and prevented the destabilization of Mcl-1 by pyocyanin. Thus, pyocyanin activated a coordinated series of events dependent upon lysosomal dysfunction and protease release, the first description of a bacterial toxin using a lysosomal cell death pathway. This may be a pathological pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses.
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 a Wellcome Clinical Research Fellowship (064997) (to S.M.B.). I.S. is a Medical Research Council Senior Clinical Fellow (G116/170), and D.H.D. is a Wellcome Trust Senior Clinical Fellow (076945).
2 L.R.P. and S.M.B. contributed equally to this work and are joint first authors.
3 Address correspondence and reprint requests to Dr. Moira K. B. Whyte, Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, L Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, U.K. E-mail address: m.k.whyte{at}sheffield.ac.uk
4 Abbreviations used in this paper: ROI, reactive oxygen intermediates; 
m, mitochondrial membrane potential; Boc-D-fmk, Boc-Asp(OMe)-fmk; BODIPY FL, boron dipyrromethane difluoride; CTSD, cathepsin D; CTSG, cathepsin G; DAME, diazoacetyl-DL-2-aminohexanoic acid-methyl ester; dbcAMP, dibutyryl cAMP; DHR, dihydrorhodamine; MFI, mean fluorescence intensity; z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone; DEVD-AMC, 7-amino-4-methylcoumarin, N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide.
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