Pneumolysin-dependent and independent non-canonical autophagy processes mediate host defense against pneumococcal infection

<i>Streptococcus pneumoniae</i> is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against <i>S. pneumoniae</i> remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci <i>in vivo</i>. Using a transgenic macroautophagy/autophagy reporter line, we found the autophagy marker Map1lc3/Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy gene <i>atg5</i> led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of <i>cyba</i> and <i>rubcn</i> moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in <i>S. pneumoniae</i>-infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that the pneumococcal pore-forming toxin pneumolysin induces ROS-independent CASM pathways, one of which is abolished by knockdown of <i>tecpr1a</i> indicating the involvement of sphingomyelin-Tecpr1-induced LC3 lipidation (STIL). Collectively, our observations shed new light on the host immune response against <i>S. pneumoniae</i>, demonstrating that several distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.<b>Abbreviations</b>: ATG: autophagy related; BMDM: bone marrow-derived macrophage; CASM: conjugation of ATG8 to single membranes; CFU: colony-forming units; Cyba: cytochrome b-245, alpha polypeptide; DPI: diphenyleneiodonium, GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LAP: LC3-associated phagocytosis; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NADPH: nicotinamide adenine dinucleotide phosphate; Optn: optineurin; PINCA: pore-forming toxin-induced non-canonical autophagy; Ply: pneumolysin; ROS: reactive oxygen species; SLR: sequestosome-like receptors; Sqstm1: sequestosome 1; STIL: sphingomyelin-TECPR1-induced LC3 lipidation; Tecpr1: tectonin beta-propeller repeat containing 1.