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Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds
Published on: September 8, 2021
An engineered protein-based submicromolar competitive inhibitor of the Staphylococcus aureus virulence factor
Soraia R Mendes1, Ulrich Eckhard1, Arturo Rodríguez-Banqueri1
1Proteolysis Laboratory, Department of Structural Biology, Molecular Biology Institute of Barcelona (CSIC), Barcelona Science Park, Baldiri Reixac 15-21, 08028 Barcelona, Catalonia, Spain.
Abstract:
Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhibitors are currently more common in the clinic, therapeutic proteins and peptides (TPs) are favourable due to their high selectivity, which reduces off-target toxicity and allows dosage tuning. The greater wax moth Galleria mellonella produces a unique defensive protein known as the insect metallopeptidase inhibitor (IMPI), which selectively inhibits some thermolysins from pathogenic bacteria. We determined the ability of IMPI to inhibit aureolysin in vitro and used crystal structures to ascertain its mechanism of action. This revealed that IMPI uses the "standard mechanism", which has been poorly characterised for MPs in general. Accordingly, we designed a cohort of 12 single and multiple point mutants, the best of which (I57F) inhibited aureolysin with an estimated inhibition constant (K i) of 346 nM. Given that animals lack thermolysins, our strategy may facilitate the development of safe TPs against staphylococcal infections, including strains resistant to conventional antibiotics.
Insights
Insect metallopeptidase inhibitor (IMPI) shows promise for combating Staphylococcus aureus infections. A modified IMPI effectively inhibits aureolysin, a key virulence factor, offering a potential new strategy against antibiotic-resistant bacteria.
Area of Science:
- Biochemistry
- Microbiology
- Structural Biology
Background:
- Aureolysin is a metallopeptidase and a critical virulence factor in Staphylococcus aureus.
- No specific aureolysin inhibitors are currently available, highlighting a need for novel antimicrobial drug development against antibiotic-resistant strains.
- Therapeutic proteins and peptides (TPs) offer high selectivity and reduced toxicity compared to small-molecule inhibitors.
Purpose of the Study:
- To investigate the inhibitory potential of insect metallopeptidase inhibitor (IMPI) against aureolysin.
- To elucidate the mechanism of action of IMPI inhibition on aureolysin using structural biology.
- To engineer improved IMPI variants for enhanced aureolysin inhibition.
Main Methods:
- In vitro enzymatic assays to assess IMPI's inhibition of aureolysin.
- X-ray crystallography to determine the structural basis of IMPI-aureolysin interaction.
- Site-directed mutagenesis to create and test IMPI mutants for improved inhibitory activity.
Main Results:
- IMPI was confirmed to inhibit aureolysin in vitro.
- Crystal structures revealed IMPI utilizes a "standard mechanism" for metallopeptidase inhibition.
- A designed IMPI mutant (I57F) demonstrated potent inhibition of aureolysin with an inhibition constant (Ki) of 346 nM.
Conclusions:
- IMPI effectively inhibits aureolysin, a key virulence factor in Staphylococcus aureus.
- The study elucidates the mechanism of metallopeptidase inhibition by IMPI.
- Engineered IMPI variants represent a promising therapeutic strategy for treating staphylococcal infections, including those resistant to conventional antibiotics.
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