Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

S Hansenová Maňásková, Kamran Nazmi, Wim van't Hof, Alex Belkum, Nathaniel I. Martin, Floris J. Bikker, Willem van Wamel, Enno C.I. Veerman

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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 pM 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 pM, 100 pM and 250 pM; (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.
Original languageUndefined/Unknown
JournalPLoS One (print)
Volume11
Issue number1
DOIs
Publication statusPublished - 22 Jan 2016

Bibliographical note

This work was supported by ZonMW; grant title: Exploiting Staphylococcus aureus sortase for anti-infective purposes with the project number: 50-51700-98-055.

Research programs

  • EMC MM-04-28-01

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