Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.
Bottom Line: 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).The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP.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.
Affiliation: Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom.Show MeSH
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Mentions: PLY contributed to LMP independently of phagocytosis. In contrast, the execution phase of macrophage apoptosis required phagocytosis of bacteria, suggesting a role for intracellular microbial factors. Since PLY translocated to the cytosol, we addressed whether intracellular PLY was also required to execute apoptosis or was required solely to induce LMP. We reconstituted comparable levels of LMP in macrophages containing the PLY-deficient strain and determined whether apoptosis occurred to an extent similar to that observed with wild-type bacteria. The lysomotrophic detergent l-Leucyl-l-leucine methyl ester (LeuLeuOMe) is a dipeptide which, in lysosomes, is converted by the lysosomal enzyme dipeptidyl peptidase into a detergent-like compound which induces LMP (41, 42). The dose of LeuLeuOMe was selected as the dose that provided levels of LLA and kinetics comparable to those observed following pneumococcal challenge (see Fig. S7A in the supplemental material). It was also confirmed that it reconstituted LLA and LMP in cells challenged with Stop to an extent comparable to that observed in D39-exposed cells (see Fig. S7B and C). The lysomotrophic detergent did not induce significant apoptosis in the absence of bacteria but enhanced apoptosis following phagocytosis of PLY-deficient bacteria, though not to the extent seen with D39 bacteria (see Fig. 6A; see also Fig. S8A). In contrast, addition of PLY to cells exposed to the lysomotrophic detergent in the presence of Stop increased levels of apoptosis over those seen in the absence of PLY (see Fig. 6A; see also Fig. S8A), whereas PLY had no additional effect on LMP (see Fig. S7C). This that suggested PLY contributed to induction of apoptosis in this system downstream of its induction of LMP.
Affiliation: Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom.