Distinct Mechanisms without Membrane Pore Formation Membrane Permeabilization and Executes Apoptosis by Pneumolysin Activates Macrophage Lysosomal 2014

Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY’s ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1 ). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. IMPORTANCE Streptococcus pneumoniae, the commonest cause of bacterial pneumonia, expresses the toxin pneumolysin, which can make holes in cell surfaces, causing tissue damage. Macrophages, resident immune cells essential for responses to bacteria in tissues, activate a program of cell suicide called apoptosis, maximizing bacterial clearance and limiting harmful inflammation. We examined pneumolysin’s role in activating this response. We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule. Pneumolysin and other bacterial factors released by bacteria that have not been eaten by macrophages activate macrophages to release inflammatory factors but also make the cell compartment containing ingested bacteria leaky. Once inside the cell, pneumolysin ensures that the bacteria activate macrophage apoptosis, rather than necrosis, enhancing bacterial killing and limiting inflammation. This dual response to pneumolysin is critical for an effective immune response to S. pneumoniae. Received 31 July 2014 Accepted 15 September 2014 Published 7 October 2014 Citation Bewley MA, Naughton M, Preston J, Mitchell A, Holmes A, Marriott HM, Read RC, Mitchell TJ, Whyte MKB, Dockrell DH. 2014. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation. mBio 5(5):e01710-14. doi:10.1128/mBio.01710-14. Editor Liise-anne Pirofski, Albert Einstein College of Medicine Copyright © 2014 Bewley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to David H. Dockrell, d.h.dockrell@sheffield.ac.uk. Streptococcus pneumoniae, the pneumococcus, is a significant cause of morbidity and mortality worldwide, contributing to an estimated 2 million deaths per annum. It is the most frequent cause of community-acquired pneumonia (1). The toxin pneumolysin (PLY) is a major virulence factor of S. pneumoniae and is expressed by almost all clinical isolates (2–4). PLY is a member of the cholesteroldependent cytolysin (CDC) family of proteins expressed by a range of bacteria (5, 6). Monomeric soluble PLY binds to cholesterolcontaining membranes and, through adjustment of its four domains, oligomerizes to form a ring-shaped pore (7), which can cause cytolysis in mammalian cells (8). PLY’s role in S. pneumoniae pathogenicity is well documented, with several studies showing that mice infected with PLY-deficient strains of S. pneumoniae display increased survival time (2), lower bacterial numbers in the nasopharynx (8), RESEARCH ARTICLE crossmark September/October 2014 Volume 5 Issue 5 e01710-14 ® mbio.asm.org 1 m b.asm .rg on O cber 1, 2014 P ubished by m b.asm .rg D ow nladed fom and decreased bacteremia and less lung damage (9, 10). PLY can also subvert complement-mediated opsonization (10–12). However, PLY also activates immune responses to pneumococci which contribute to host defense (13). PLY activates T cells (14), induces prostaglandin production in neutrophils (15), and promotes tumor necrosis factor alpha (TNF) production in mononuclear phagocytes (16). Pneumolysin is capable of activating inflammasomes, including members of the nucleotide-binding oligomerization domain (Nod)-like receptor family, pyrin domain-containing protein 3 (NLRP3), and absent in melanoma 2 (AIM 2), in both dendritic cells and macrophages. This leads to caspase 1 activation and secretion of interleukin-1 beta (IL-1 ), a process which requires the cytolytic activity of the molecule and leads to protective immunity to S. pneumoniae (17, 18). Macrophages are an essential contributor to the host response to pneumococcal infection, being responsible for the initial detection of bacteria and subsequent modulation of the inflammatory response (19). S. pneumoniae-infected macrophages undergo host-mediated programmed cell death (PCD), which mediates bactericidal activity in the later stages of the macrophage’s interaction when other killing mechanisms are exhausted and which is a prerequisite for bacterial clearance and resolution of inflammation (20–22). This form of PCD, which is significantly reduced during infections using PLY-deficient strains (21, 23), proceeds along a lysosome-mitochondrion axis involving initial lysosomal/ phagolysosomal membrane permeabilization (LMP), which precedes the sequential loss of the inner mitochondrial transmembrane potential ( m), mitochondrial outer membrane permeabilization (MOMP), caspase 3 activation, and, finally, nuclear condensation and fragmentation reminiscent of “classical” apoptosis (20). Moreover, the cell membrane remains intact until late in the cell death process and permeabilization is a prominent feature only when key regulators of the death program are inhibited (23). In this form of PCD, loss of lysosomal acidification (LLA) is a marker of LMP, which is an apical event in the initiation of PCD, linking activation of the lysosomal protease cathepsin D with regulation of cytosolic proteins that control the execution of the cell death program in the macrophage (20). PLY contributes to both LLA and cathepsin D activation (20). However, although PLY contributes to PCD, it is not sufficient, with internalization of live bacteria being required to unmask the role of PLY (23, 24). This contrasts with PLY-induced PCD in neurons (25, 26), cochlear hair cells (27), lymphocytes (28), and peritoneal macrophages (29) in which exogenous PLY alone can induce PCD directly and implies that host-mediated macrophage PCD in response to PLY proceeds through an alternative mechanism in differentiated macrophages. In this study, we explored how PLY activates host-mediated macrophage PCD. We show that PLY contributes to PCD induction through two distinct processes that are independent of its pore-forming capacity. PLY enhances LMP, contributing to a phagocytosis-independent process involving Toll-like receptor (TLR) signaling, but is also required to ensure that, after bacterial internalization, LMP activates a form of PCD with features of apoptosis rather than a form of cell death with prominent plasma membrane permeabilization and cytolysis.