Environmental cues shape extracellular vesicles biogenesis and function in Streptococcus pneumoniae

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. Here, we demonstrate that exposure to normal human serum induced rearrangement of the pneumococcal cell wall and significantly increased Sp-EVs production. Temperature and pH were critical factors for Sp-EVs formation: 37{degrees}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, specially those associated with glycolysis (e.g. eno, gapA, gapN, gpmA, pfkA, pykF, and tpi). Low 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. ImportanceStreptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide, responsible for diseases ranging from community-acquired pneumonia to meningitis and sepsis. It is of great concern in children under the age of five and in low-income countries, despite the existence of vaccination. The success of this pathogen relies on its capacity to adapt to diverse host niches, including the blood, nasopharynx and the lungs. Bacterial extracellular vesicles (EVs) have emerged as mediators of virulence and communication, yet their regulation by environmental and metabolic factors remains poorly understood. By exploring how environmental conditions and nutrient availability shape pneumococcal EV production and how these vesicles contribute to biofilm formation and interspecies interactions, our study contributes to the understanding of how S. pneumoniae applies complex strategies to persist and spread, with potential future applications in infection control and therapeutic strategies.