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

Streptococcus pneumoniae 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 S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo. Using a transgenic autophagy reporter line, we found the autophagy marker Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy genes (atg5 and atg16l1) 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 cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae-infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that production of pneumococcal pore-forming toxin - pneumolysin induces LAP-independent Lc3 lipidation, which could be abolished by knockdown of tecpr1a indicating the involvement of the sphingomyelin-TECPR1-induced LC3 lipidation (STIL) pathway. Collectively, our observations shed new light on the host immune response against S. pneumoniae, demonstrating that two distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.