Degradation Products of the Extracellular Pathogen Streptococcus pneumoniae Access the Cytosol via Its Pore-Forming Toxin

Abstract

Streptococcus pneumoniae is a leading pathogen with an extracellular lifestyle; however, it is detected by cytosolic surveillance systems of macrophages. The innate immune response that follows cytosolic sensing of cell wall components results in recruitment of additional macrophages, which subsequently clear colonizing organisms from host airways. In this study, we monitored cytosolic access by following the transit of the abundant bacterial surface component capsular polysaccharide, which is linked to the cell wall. Confocal and electron microscopy visually characterized the location of cell wall components in murine macrophages outside membrane-bound organelles. Quantification of capsular polysaccharide through cellular fractionation demonstrated that cytosolic access of bacterial cell wall components is dependent on phagocytosis, bacterial sensitivity to the host’s degradative enzyme lysozyme, and release of the pore-forming toxin pneumolysin. Activation of p38 mitogen-activated protein kinase (MAPK) signaling is important for limiting access to the cytosol; however, ultimately, these are catastrophic events for both the bacteria and the macrophage, which undergoes cell death. Our results show how expression of a pore-forming toxin ensures the death of phagocytes that take up the organism, although cytosolic sensing results in innate immune detection that eventually allows for successful host defense. These findings provide an example of how cytosolic access applies to an extracellular microbe and contributes to its pathogenesis.Importance Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen that is a leading cause of pneumonia. Pneumococcal disease is preceded by colonization of the nasopharynx, which lasts several weeks before being cleared by the host’s immune system. Although S. pneumoniae is an extracellular microbe, intracellular detection of pneumococcal components is critical for bacterial clearance. In this study, we show that following bacterial uptake and degradation by phagocytes, pneumococcal products access the host cell cytosol via its pore-forming toxin. This phenomenon of cytosolic access results in phagocyte death and may serve to combat the host cells responsible for clearing the organism. Our results provide an example of how intracellular access and subsequent immune detection occurs during infection with an extracellular pathogen.

Sensing of Interleukin-1 Cytokines during Streptococcus pneumoniae Colonization Contributes to Macrophage Recruitment and Bacterial Clearance

“…We have previously reported that infection of macrophages with a type 23F strain of S. pneumoniae results in host cell death, subsequent to bacterial uptake and pneumolysin-dependent cytosolic access of pneumococcal fragments (12). To determine whether pneumolysin-dependent cytosolic access causes proinflammatory cytokine release upon in vitro infection, we incubated bone marrow-derived macrophages (BMMs) with the wild-type (WT) 23F isolate and 24 h later assayed for the presence of IL-1␤ in the cell culture supernatants by Western blotting.…”

“…Although S. pneumoniae is an extracellular pathogen, this macrophage influx is the result of intracellular innate immune recognition by the cytosolic Nod-like receptor 2 (Nod2) (9). Nod2 detects peptidoglycan (10,11) that accesses the macrophage cytosol via the pneumococcal poreforming toxin pneumolysin following phagocytosis and bacterial degradation (12). Nod2 activation results in nuclear factor B (NF-B) activation (13) and drives proinflammatory cytokine production, including the C-C motif chemokine ligand 2 (CCL2), which contributes to monocyte/macrophage-dependent pneumococcal clearance (9).…”

“…Nod2 activation results in nuclear factor B (NF-B) activation (13) and drives proinflammatory cytokine production, including the C-C motif chemokine ligand 2 (CCL2), which contributes to monocyte/macrophage-dependent pneumococcal clearance (9). Cytosolic access, however, is a fatal event for the host cell following bacterial uptake (12), although the type of cell death that occurs and its contribution to the host immune response remain unclear.…”

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

“…Last, we examined whether the minimal PG turnover was altered by ⌬pgdA or ⌬adr mutation, each of which alters PG glycan chain modification and, thus, could potentially serve as a turnover signal. PgdA deacetylates GlcNAc in the PG glycan chains of wild-type cells, thereby imparting resistance to host lysozyme produced by the innate immune response (23,39,64). Adr O-acetylates the 6 position of MurNAc in PG glycan chains, which imparts lysozyme and penicillin resistance to strains of some serotypes (38,64).…”

“…For some phagocytosed extracellular pathogens, such as Streptococcus pneumoniae (pneumococcus), lysozyme digestion concomitantly produces PG fragments and releases a pore-forming toxin that damages the phagosome membrane (23). This damage enables the release of PG fragments into the cytosol, where they can interact with Nod2 receptors that induce proinflammatory signaling, leading to the recruitment of additional phagocytic cells to infection sites (23).…”