Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner

Silvie Hansenová Maňásková, Kamram Nazmi, Alex van Belkum, Floris J. Bikker, Willem J.B. van Wamel, Enno C.I. Veerman

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Abstract

The majority of Staphylococcus aureus virulence-and colonization-associated surface proteins contain a pentapeptide recognition motif (LPXTG). This motif can be recognized and cleaved by sortase A (SrtA) which is a membrane-bound transpeptidase. After cleavage these proteins are covalently incorporated into the peptidoglycan. Therefore, SrtA plays a key role in S. aureus virulence. We aimed to generate a substrate mimicking this SrtA recognition motif for several purposes: to incorporate this substrate into the S. aureus cell-wall in a SrtA-dependent manner, to characterize this incorporation and to determine the effect of substrate incorporation on the incorporation of native SrtA-dependent cell-surface-associated proteins. We synthesized substrate containing the specific LPXTG motif, LPETG. As a negative control we used a scrambled version of this substrate, EGTLP and a S. aureus srtA knockout strain. Both substrates contained a fluorescence label for detection by FACScan and fluorescence microscope. A spreading assay and a competitive Luminex assay were used to determine the effect of substrate treatment on native LPXTG containing proteins deposition in the bacterial cell-wall. We demonstrate a SrtA-dependent covalent incorporation of the LPETG-containing substrate in wild type S. aureus strains and several other Gram-positive bacterial species. LPETG-containing substrate incorporation in S. aureus was growth phase-dependent and peaked at the stationary phase. This incorporation negatively correlated with srtA mRNA expression. Exogenous addition of the artificial substrate did not result in a decreased expression of native SrtA substrates (e. g. clumping factor A/B and protein A) nor induced a srtA knockout phenotype.
Original languageEnglish
JournalPLoS One (print)
Volume9
Issue number2
DOIs
Publication statusPublished - 19 Feb 2014

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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