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

Supradipta De¹Larissa Busch²Gerhard Burchhardt³Manuela Gesell-Salazar²Rabea Schlüter⁴Leif Steil²Uwe Völker²Sven Hammerschmidt^1*

• ¹University of Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Molecular Genetics and Infection Biology, Center for Functional Genomics of Microbes, Greifswald, Germany
• ²University Medicine Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, Center for Functional Genomics of Microbes, Greifswald, Germany
• ³Interfaculty Institute of Genetics and Functional Genomics, Department of Molecular Genetics and Infection Biology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
• ⁴University of Greifswald, Imaging Center of the Department of Biology, Greifswald, Germany

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 2 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.