title: Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation.
abstract: The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.
authors.affiliation: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.|Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Silvie
authors.initials: S
authors.lastname: Hansenová Maňásková
authors.affiliation: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Kamran
authors.initials: K
authors.lastname: Nazmi
authors.affiliation: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Wim
authors.initials: W
authors.lastname: van 't Hof
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: van Belkum
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Nathaniel I
authors.initials: NI
authors.lastname: Martin
authors.affiliation: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Floris J
authors.initials: FJ
authors.lastname: Bikker
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Willem J B
authors.initials: WJ
authors.lastname: van Wamel
authors.affiliation: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Enno C I
authors.initials: EC
authors.lastname: Veerman
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism / Q000502:physiology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D005434:Flow Cytometry; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000378:metabolism*; D000067776:RNA Recognition Motif Proteins / Q000378:metabolism / Q000502:physiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010455:Peptides; D000067776:RNA Recognition Motif Proteins; D014640:Vancomycin; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases