A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger

• Published in: Scientific reports 2023-03, Vol.13 (1), p.3926-3926, Article 3926
• Main Authors: Kotb, Essam, El-Nogoumy, Baher A., Alqahtani, Haifa A., Ahmed, Asmaa A., Al-shwyeh, Hussah A., Algarudi, Sakina M., Almahasheer, Hanan
• Format: Article
• Language: English
• Subjects: 631/326; 631/326/41/1969; Antimicrobial agents; Cytotoxicity; Dithiothreitol; Electron microscopy; Gel electrophoresis; Humanities and Social Sciences; Hydrogen-Ion Concentration; Hydrophobicity; Isoelectric Point; Molecular Weight; multidisciplinary; o-Phenanthroline; Proteolysis; Salmonella; Salmonella typhimurium - metabolism; Science; Science (multidisciplinary); Serine Endopeptidases - metabolism; Serine Proteases - metabolism; Serine proteinase; Serum proteins; Sodium lauryl sulfate; Substrate Specificity; Temperature; Virulence
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-29847-8

A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving K m of 0.16 mM, K cat / K m of 3.01 × 10 5  S −1  M −1 , and amidolytic activity of 28.9 µmol min −1  L −1 . It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, β-mercaptoethanol, 2,2′-bipyridine, o -phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.