Activation of secreted protease virulence factors of Staphylococcus aureus is controlled by a novel cascade pathway.

摘要

Staphylococcus aureus employs a sophisticated proteolytic cascade to control the maturation of three extracellular proteases, the metalloprotease aureolysin, serine protease SspA (V8 protease), and cysteine protease SspB (Staphopain B). These proteases function together to control bacterial adhesion and invasion. SspA and SspB are encoded in the Staphylococcus serine protease (ssp) operon along with a third gene sspC, which forms a stable inhibitory complex with mature SspB. The aim of this thesis is to uncover the activation mechanism of each protease in an attempt to understand how the cascade functions during infection. Aureolysin is the first member of the cascade and undergoes intramolecular autocatalytic activation. Furthermore, activation proceeds through a novel processing intermediate within the Fungalysin/Thermolysin-Propeptide (FTP) domain, which is required to produce a stable active protease. Aureolysin is required for the activation of the SspA serine protease precursor, which is also facilitated by SspA intra- and inter-molecular cleavage intermediates. Intermolecular processing of proSspA results in the production of an isoform of mature protease with a three amino acid extension, HAN↓1VILP. Molecular modeling suggested that the histidine residue is essential to maintain the protease as an inactive zymogen by forming a bifurcated hydrogen bond with the active site serine. SspA efficiently removes the propeptide of the SspB cysteine protease by a single cleavage at Glu183 to release an activate protease. SspB accumulates in the cell as a result of slow secretion kinetics, and the signal peptide was found to have two functional signal peptidase cleavage sites. In addition, SspB production was not affected by the loss of the dedicated intracellular inhibitor SspC. In conclusion, the Staphylococcal proteases have evolved necessary steps in their maturation that allow efficient and timely activation.