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bioRxiv · 10.1101/2024.12.18.629304

Novel approaches to label the surface of S. aureus with DBCO for click chemistry-mediated deposition of sensitive cargo

Baryakova, T.·Segatori, L.·McHugh, K.
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Abstract

The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications, including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for in situ conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity for some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of Staphylococcus aureus and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes - a sortase peptide substrate (LPETG), two D-alanine derivatives, and vancomycin - can selectively label the S. aureus surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-D-alanine variants do not adversely affect the growth of S. aureus or lead to off-target labeling or toxicity in HEK293T cells, even at high concentrations. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified S. aureus. We believe the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of S. aureus and other gram-positive bacteria, opening the door for new applications using azido-modified cargo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/629304v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16c7d1corg.highwire.dtl.DTLVardef@189bd12org.highwire.dtl.DTLVardef@1dc1bcborg.highwire.dtl.DTLVardef@1a4e1b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Baryakova, T., Segatori, L., McHugh, K.. 2024-12-21. Novel approaches to label the surface of S. aureus with DBCO for click chemistry-mediated deposition of sensitive cargo. https://doi.org/10.1101/2024.12.18.629304

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