title: Synthetic LPETG-containing peptide incorporation in the Staphylococcus aureus cell-wall in a sortase A- and growth phase-dependent manner.
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.
authors.affiliation: Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, 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 Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Kamran
authors.initials: K
authors.lastname: Nazmi
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 Oral Biochemistry, Academic Centre for Dentistry Amsterdam, 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 Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Enno C I
authors.initials: EC
authors.lastname: Veerman
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005434:Flow Cytometry; D055786:Gene Knockout Techniques; D008565:Membrane Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000737:chemistry*; D018709:Statistics, Nonparametric
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases