Michno, B. J. | Search bioRxiv

bioRxiv2025

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 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 atg5 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 the pneumococcal pore-forming toxin pneumolysin induces ROS-independent CASM pathways, one of which is abolished by knockdown of tecpr1a indicating the involvement of sphingomyelin-Tecpr1-induced LC3 lipidation (STIL). Collectively, our observations shed new light on the host immune response against S. pneumoniae, 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.

immunology↗

bioRxiv2024

Enterococcal cell wall remodelling underpins pathogenesis via the release of the Enteroccocal Polysaccharide Antigen (EPA)

Enterococci are opportunistic pathogens displaying a characteristic ovoid shape, typically forming pairs of cells (diplococci) and short chains. Control of cell chain length in Enterococcus faecalis relies on the activity of the major N-acetylglucosaminidase AtlA. The formation of short chains and diplococci is critical during pathogenesis for dissemination in the host and to limit recognition by innate immune effectors such as complement molecules and phagocytes. Here, we identify AtlE, an N-acetylmuramidase that contributes to septum cleavage during stationary phase in the absence of AtlA. AtlE is encoded by the locus required to produce the decoration subunits of the Enterococcal Polysaccharide Antigen (EPA), which mediate evasion of phagocytosis. We show that peptidoglycan hydrolysis by AtlE is essential for pathogenesis and demonstrate that soluble cell wall fragments containing EPA decorations increase the virulence of E. faecalis, suggesting that EPA plays a role as a decoy molecule to evade host defences. This research sheds light on the complex interplay between bacterial cell division, cell wall remodelling, and the host immune system, providing valuable insights into a novel mechanism underlying the virulence of E. faecalis. Author summaryThe major component of the bacterial cell envelope (peptidoglycan) undergoes partial hydrolysis during growth. This process, referred to as remodelling, is required for the incorporation of novel peptidoglycan building blocks, and cell separation at the end of division. In Enterococcus faecalis, only one ubiquitous peptidoglycan hydrolase, named AtlA, has been described so far. AtlA plays a prominent role in septum cleavage and is responsible for the characteristic formation of diplococci and short cell chains. The minimization of cell chain length by AtlA is critical for innate immune evasion and underpins pathogenesis. Here, we identify another ubiquitous peptidoglycan hydrolase named AtlE encoded by the Enterococcal Polysaccharide Antigen (EPA) biosynthetic locus. We show that AtlE displays N-acetylmuramidase activity and requires strain-specific EPA decorations to be active. Whilst AtlE only plays a marginal role in septum cleavage during growth, AtlE is essential for virulence in the zebrafish model of infection. We demonstrate that AtlE activity contributes to release cell wall fragments and promotes phagocyte evasion, indicating that EPA plays a role as a decoy molecule produced by enterococci to counteract host immune defenses.

microbiology↗