Attaching small molecules to the Staphylococcus aureus cell wall

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

James W. Nelson, Alexander G. Chamessian, Patrick J. McEnaney, Ryan P. Murelli, Barbara I. Kazmiercak, David A. Spiegel*

Departments of Chemistry and Medicine, Yale University, New Haven, CT

ACS Chem. Bio. 2010, 5, 1147-55.

Over the past few decades, many hospital Staphylococcus aureus infections have become resistant to available antibiotic treatments, resulting in a major public health threat. S. aureus, a Gram-positive bacterium–meaning it has a thick peptidoglycan cell well–is believed to have developed its virulence in part by the proteins embedded in its cell wall.  These proteins interact with the hosts’ cells and escape the immune response. A transpeptidase, sortase A (Srt A), is known for incorporating proteins into the bacterial cell wall.

The authors of this article postulated that re-engineering the S. aureus cell wall to incorporate small molecules would have both fundamental and therapeutic applications. As demonstrated in the figure below, the mechanism of action for Srt A involves a pentapeptide motif, LPXTG, and specifically LPETG in S. aureus, of secreted proteins. Once this peptide is recognized by the Srt A, it is cleaved between the threonine and glycine residues, forming an acyl-enzyme intermediate. The intermediate is then attacked by a pentaglycine motif within the peptidoglycan cell wall, resulting in a covalent attachment to the cell wall.

Non-native derivatives of the LPETG substrate were prepared containing fluorescein, biotin, and azide for fluorescence, affinity chromatography, and bioorthogonal chemical reactions at the cell surface of S. aureus, respectively.  Additionally, scrambled peptide controls (i.e. EGTLP) were synthesized containing the same derivatives for comparison of experimental data.

Substrate incorporation was investigated using fluorescence of a fluorescein-LPETG substrate on wild type S. aureus. The authors found that the bacteria incubated with the fluorescein-LPETG were highly fluorescent in comparison to the scrambled control, indicating that the substrate was incorporated into the cell wall.

To confirm that it was indeed incorporated into the cell wall, the biotin derivative was synthesized and incorporated into the cell wall, using the same method as the previous experiment. Upon modification, the cell wall was digested to break down the peptidoglycan, and then the digests were subjected to streptavidin affinity chromatography to pull down biotinylated molecules. The molecules that were pulled down by the streptavidin were then analyzed by mass spectrometry. The authors found that their biotinylated peptide was indeed covalently linked to a LPETGGG sequence. The tri-glycine motif was postulated to have originated from the peptidoglycan cell wall. As a result, this provided strong evidence that Srt A can incorporate a derivatized LPETG substrate into the S. aureus cell wall.

Finally, given the success of their fluorescein and biotin LPETG derivatives, the authors chose to make a bioorthogonal derivative for the addition of different types of reactive groups onto the cell wall of S. aureus. Utilizing the [3+2] cycloaddition (“click”) reaction, the authors synthesized an azide-LPETG substrate to be used with alkyne-containing reactants. This strategy is highly selective and has potential for developing new therapeutic approaches for treating antibiotic-resistant bacteria.

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