Minimalist Tetrazine N‑Acetyl
Muramic Acid Probes for Rapid and Efficient Labeling of Commensal
and Pathogenic Peptidoglycans in Living Bacterial Culture and During
Macrophage Invasion
posted on 2024-03-01, 16:05authored byAshlyn
S. Hillman, Stephen N. Hyland, Kimberly A. Wodzanowski, DeVonte L. Moore, Sushanta Ratna, Andrew Jemas, Liam-Michael D. Sandles, Timothy Chaya, Arit Ghosh, Joseph M. Fox, Catherine L. Grimes
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems.