Introduction Pneumococci colonize asymptomatically the upper nasopharyngeal cavity, but upon an external trigger, pneumococci disseminate and cause severe infections. S. pneumoniae can express up to four different extracellular serine proteases called PrtA, HtrA, SFP and CbpG. Strain EF3030, a colonizing strain was chosen for profiling of the extracellular and cytosolic proteome during early and late log phase. Furthermore, mutants lacking these serine proteases will be studied to monitor their impact on the proteome composition.

Goals Monitoring of the abundance of intracellular and extracellular pneumococcal proteins, especially extracellular serine proteases, using global proteomic techniques.

Methods An optimized minimal medium was established for cultivation of pneumococci. Bacterial pellet and supernatant samples were taken in mid log phase and late log phase. Supernatant proteins were obtained by TCA precipitation, while pellet proteins were obtained by cell disruption using a bead mill. Both sample types were subjected to data independent LC-ESI MS/MS analysis (directDIA).

Results Around 1600 proteins, identified with more than 2 peptides, were profiled in the supernatant samples. A significant proportion of these proteins exhibited higher levels in the late log phase compared to the mid log phase. Proteins included extracellular proteases HtrA and PrtA, several choline-binding proteins, and the competence factor ComG. Proteome analysis of the pellet samples identified approximately 1600 proteins with more than 2 peptides, and the relative amount of the vast majority of these proteins remained unchanged upon entering the late log phase. Significantly reduced relative amounts were observed for proteins involved in energy metabolism, such as alpha amylase Amy, 1-phospho-fructokinase FruB, and galactokinase GalK in the late log phase. The relative amounts of HtrA and PrtA remained unchanged.

Summary We were able to monitor the abundance of intracellular and extracellular pneumococcal proteins, in particular the extracellular serine proteases, using proteomic profiling.