hsdSA regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes

Streptococcus pneumoniae is a notorious human opportunistic pathogen which undergoes a spontaneous and reversible phenotypic change in response to the host environment. We demonstrated that the regulatory gene hsdSA of DNA methylation in the type I restriction modification system altered colony transparency and substantially contributed to S. pneumoniae virulence. Most importantly, hsdSA regulated the production of extracellular vesicles (EVs) which package cytosolic, surface, and secreted proteins, including pneumolysin (PLY). Interestingly, we confirmed that EV-associated PLY utilized internalization into macrophages to prolong the survival of intracellular bacteria as a major immune evasion strategy; that is, EV-associated PLY produced by the D39 strain (EVs-D39) could induce the formation of LC3-associated monolayer vacuoles [LC3-associated phagocytosis (LAP)] and co-localize with the NADPH oxidase 2 (NOX2) complex but not ULK1 when macrophages were infected with the D39Δply strain. In addition, EV-associated PLY derived from the EVs-D39 promoted macrophages to release more reactive oxygen species (ROS) and expression of p-p70s6k than EV-associated PLY derived from the D39ΔhsdSA strain (EVs-D39ΔhsdSA), whereas the expression of p-ULK1 was reversed, indicating that EVs-D39ΔhsdSA was more likely to induce conventional xenophagy. Furthermore, we identified the β1 integrin receptor as a crucial inducer of ROS to mediate LAP activation. Bacterial evasion of host clearance is closely related to insufficient acidification after the fusion of autophagosomes or LAPosomes with lysosomes. Of note, we found EV-associated PLY damaged the integrity of the lysosome membrane and changed the pH gradient, resulting in lysosomes being unable to remove intracellular bacteria and ultimately prolonging the survival of S. pneumoniae in macrophages. Finally, the extracted mouse alveolar macrophages and mouse intranasal infection models were employed to further verify the above findings.

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