Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavag e of other exoproteins released from this organism. To define the target ex oproteins and their sites of cleavage by proteases, high-resolution two-dim ensional polyaerylamide gel electrophoresis followed by N-terminal amino ac id sequencing of exoprotein spots was performed. Two to three hundred exopr otein spots were detected at the early-stationary phase of cultures of S. a ureus NCTC8325, and then at the late-stationary stage most of these high mo lecular protein spots became invisible due to further proteolytic degradati on. As the result of N-terminal analysis, lipase, triacylglycerol lipase, o rf619 protein and orf388 protein were detected as multiple spots at the ear ly-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with ca lculated values in addition to their N-terminal sequences, we showed that t he positions of putative peptides resulted from proteolytic cleavage of the se proteins.