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

Abstract

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-1β). 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.

Streptococcus pneumoniae disrupts pulmonary immune defence via elastase release following pneumolysin-dependent neutrophil lysis

“…However, RAW264.7 cells were resistant to PLY-induced cell lysis. Although RAW264.7 cell may not accurately reflect characteristics associated with human macrophages, human monocyte-derived macrophages isolated from whole blood are also resistant to PLY-induced cell lysis33, which was consistent with our experimental findings. While no studies have so far reported PLY-induced cytolytic activity on alveolar macrophages, our findings suggested that infiltrated neutrophils are the primary target cells of PLY.…”

Lysosomal membrane permeabilization and cell death

“…Other physiological examples of LDCD include the homeostatic responses of immune cells to infection. In Legionella and Streptococcus pneumoniae infections, the termination of productive infection can give rise to macrophage cell death triggered by cathepsin‐dependent cell death . Viruses have also evolved mechanisms to hijack cellular defenses to their own benefit.…”

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol-dependent cytolysins

“…pneumoniae was also shown to trigger LMP and apoptosis of host macrophages (Bewley et al, ). The involved mechanism is complex and partially requires the pneumolysin toxin but not its pore‐forming activity, as well as additional bacterial factors (Bewley et al, ). This mechanism differs from the one observed in our study in epithelial cells, which strictly requires PLY pore‐forming activity (Figure b).…”