The global proteome of Streptococcus pneumoniae EF3030 under nutrient-defined in vitro conditions

Streptococcus pneumoniae is a pathobiont that colonises the upper respiratory tract of humans without causing symptoms but can cause a range of life-threatening diseases including pneumonia, sepsis, and meningitis. It also causes less severe, non-invasive infections such as otitis media and sinusitis. This bacterium thrives in the nasopharynx, where nutrient availability is limited, and has adapted to this environment by developing mechanisms to survive host stress and regulate protein abundance. To study the molecular biology of S. pneumoniae under in vitro and infection-related conditions, a suitable cultivation medium is essential for reproducible experiments. In this study, we optimized a chemically-defined minimal medium that mimics the in vivo nutrient-limited condition and used it for proteome analysis. This optimized medium not only shortened the lag phase but also improved the growth of S. pneumoniae clinical isolates and other streptococcal species. We applied this medium to analyse the global proteome of the pneumococcal colonising strain EF3030, focusing on the transition from the early to late log phase. Our proteomic analysis revealed distinct patterns of protein abundance in different functional categories including metabolism, amino acid synthesis, natural competence, RNA synthesis, cell wall synthesis, protein degradation, and stress responses. Notably, choline-binding protein CbpD, competence factors ComGA and ComEA as well as proteins involved in processing internalized single-stranded DNA (ssDNA) such as Dpr and DprA were higher in abundance in the late log phase. This proteomic profiling provides valuable insights into the pathophysiology of strain S. pneumoniae EF3030 under defined nutrient conditions.