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Alpha-1-antitrypsin as novel substrate for S. aureus' Spl proteases - implications for virulence
Franziska Scherr1, Murthy N Darisipudi2, Friedemann R Börner1
1Institute of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, Jena, Germany.
Insights
Staphylococcus aureus SplD, E, and F proteases cleave alpha-1-antitrypsin (AAT), releasing a peptide that inhibits NETosis. This reveals a new S. aureus virulence mechanism targeting host defenses.
Area of Science:
- Microbiology
- Immunology
- Protease biochemistry
Background:
- Staphylococcus aureus serine protease like (Spl) proteases are key virulence factors with poorly understood functions.
- Alpha-1-antitrypsin (AAT) is an immunomodulatory serine protease inhibitor crucial for preventing tissue damage during infection.
Purpose of the Study:
- To investigate the interaction between S. aureus Spl proteases and AAT.
- To elucidate the functional consequences of this interaction on host immune responses, specifically NETosis.
Main Methods:
- MALDI-TOF-MS and LC-MS/MS were used to identify AAT cleavage sites by Spl proteases.
- Western Blots confirmed proteolytic cleavage and complex formation.
- In vitro digestions and synthetic peptide assays assessed NETosis inhibition.
Main Results:
- SplD, E, and F proteases cleave AAT within its reactive center loop, releasing a C-terminal 36-amino acid peptide (C36).
- Both synthetic C36 and AAT-Spl digestions inhibited NETosis.
- AAT partially inhibited SplE, forming a covalent complex.
Conclusions:
- S. aureus utilizes SplD/E/F to cleave and inactivate AAT, a novel virulence mechanism.
- The resulting C36 peptide actively inhibits host NETosis, contributing to bacterial pathogenesis.
Background:
The serine protease like (Spl) proteases of Staphylococcus aureus are a family of six proteases whose function and impact on virulence are poorly understood. Here we propose alpha-1-antitrypsin (AAT), an important immunomodulatory serine protease inhibitor as target of SplD, E and F. AAT is an acute phase protein, interacting with many proteases and crucial for prevention of excess tissue damage by neutrophil elastase during the innate immune response to infections.
Methods:
We used MALDI-TOF-MS to identify the cleavage site of Spl proteases within AAT's reactive center loop (RCL) and LC-MS/MS to quantify the resulting peptide cleavage product in in vitro digestions of AAT and heterologous expressed proteases or culture supernatants from different S. aureus strains. We further confirmed proteolytic cleavage and formation of a covalent complex with Western Blots, investigated AAT's inhibitory potential against Spls and examined the NETosis inhibitory activity of AAT-Spl-digestions.
Results:
SplD, E and F, but not A or B, cleave AAT in its RCL, resulting in the release of a peptide consisting of AAT's C-terminal 36 amino acids (C36). Synthetic C36, as well as AAT-SplD/E/F-digestions exhibit NETosis inhibition. Only SplE, but not D or F, was partly inhibited by AAT, forming a covalent complex.
Conclusion:
We unraveled a new virulence trait of S. aureus, where SplD/E/F cleave and inactivate AAT while the cleavage product C36 inhibits NETosis.
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