A Biosynthetic Strategy for Re-engineering the Staphylococcus aureus Cell Wall with Non-native Small Molecules

Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
ACS Chemical Biology (Impact Factor: 5.33). 10/2010; 5(12):1147-55. DOI: 10.1021/cb100195d
Source: PubMed


Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has emerged as a major public health threat. Here we report that the cell wall of S. aureus can be covalently re-engineered to contain non-native small molecules. This process makes use of endogenous levels of the bacterial enzyme sortase A (SrtA), which ordinarily functions to incorporate proteins into the bacterial cell wall. Thus, incubation of wild-type bacteria with rationally designed SrtA substrates results in covalent incorporation of functional molecular handles (fluorescein, biotin, and azide) into cell wall peptidoglycan. These conclusions are supported by data obtained through a variety of experimental techniques (epifluorescence and electron microscopy, biochemical extraction, and mass spectrometry), and cell-wall-incorporated azide was exploited as a chemical handle to perform an azide-alkyne cycloaddition reaction on the bacterial cell surface. This report represents the first example of cell wall engineering of S. aureus or any other pathogenic Gram-positive bacteria and has the potential for widespread utility.

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Available from: Alexander Chamessian, Feb 19, 2015
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    • "FITC-labelling was achieved by incorporating a fluorescein-containing lysine at the N-terminus of the LPXTG-amide. More detailed description of the peptide synthesis procedure and the chemical structure has been described elsewhere [17]. The substrates used in this study are shown in Table 1. "
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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.
    PLoS ONE 02/2014; 9(2):e89260. DOI:10.1371/journal.pone.0089260 · 3.23 Impact Factor
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    ABSTRACT: Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.
    Angewandte Chemie International Edition 05/2011; 50(22):5024-32. DOI:10.1002/anie.201008267 · 11.26 Impact Factor
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    ABSTRACT: Sortasen gehören zu einer Klasse bakterieller Enzyme, die Transpeptidaseaktivität aufweisen. Die Fähigkeit, Peptidbindungen ortsspezifisch zu brechen und neue Bindungen mit einem angreifenden Nukleophil zu bilden, macht Sortase zu einem hervorragenden Hilfsmittel für das Protein-Engineering. Diese Methode wurde für eine Reihe von Anwendungen angepasst, die von der Chemie über die Zellbiologie bis hin zu technischen Gebieten reichen. Wir wollen hier einen kurzen Überblick über die Biologie der Sortaseenzyme und ihre Anwendungen im Protein-Engineering geben, auf Gebiete für zukünftige Innovationen hinweisen und neue Anwendungen vorschlagen.
    Angewandte Chemie 05/2011; 123(22). DOI:10.1002/ange.201008267
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