Identification and Co-complex Structure of a New <i>S. pyogenes</i> SpeB Small Molecule Inhibitor

The secreted <i>Streptococcus pyogenes</i> cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB <b>2477</b> identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. <b>2477</b> inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of <b>2477</b> bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of <b>2477</b> necessary for binding. An assessment against a panel of <b>2477</b> derivatives confirms our structural findings and shows that a carbamate and nitrile on <b>2477</b> are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite <b>2477</b> having a reduced inhibitory potential against papain, the majority of <b>2477</b>-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to <b>2477</b> for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics

Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram^– and Gram⁺ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble postlysis fractions differ significantly. Additionally, in the case of Gram^– Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture

Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram^– and Gram⁺ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble postlysis fractions differ significantly. Additionally, in the case of Gram^– Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture