The Gram-positive human pathogen Streptococcus pneumoniae adapts its metabolism to the environment during colonization and host invasion. Extracellular vesicles (EVs) are produced by S. pneumoniae in the process of infection but the exact interplay between metabolic adaptation and vesicle formation remains elusive. This study investigates the role of environmental cues in modulating pneumococcal EVs biogenesis and function.

Here, we demonstrate that exposure to normal human serum induced rearrangement of the pneumococcal cell wall and considerably increased Sp-EVs production. Temperature and pH were critical factors for Sp-EVs formation: 37 °C supported optimal EV production, while bacterial exposure to either basic or acidic environments slowed down pneumococcal EV biogenesis and led to a heterogeneous subpopulation profile. Proteomic analysis revealed that Sp-EVs are enriched in carbon metabolism-related proteins, specifically those associated with glycolysis (e.g. Eno, GapA, GapN, GpmA, PfkA, PykF, and Tpi). Moderate glucose availability enhanced Sp-EVs production and intracellular ATP level, underlying a relation between metabolic status and EV biogenesis. Functionally, Sp-EVs promoted biofilm formation in both S. pneumoniae and Streptococcus pyogenes. Sp-EVs isolated under glucose-rich conditions enhanced S. pneumoniae biofilms, whereas Sp-EVs from glucose-poor conditions strongly stimulated S. pyogenes biofilm formation.

These findings underscore the role of host and environmental cues in shaping pneumococcal EV production, composition, and function, highlighting their potential involvement in metabolic adaptation and interspecies interactions.