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

Abstract

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.

Figure 1

Evolution of NAM Probes. NAM derivatives bearing bioorthogonal handles are metabolically incorporated through recycling enzymes, AmgK and MurU, to achieve the corresponding UDP-NAM precursor necessary to shuttle into PG biosynthesis that includes MurC-F, MraY, MurG, and MurJ. Action by transpeptidases and transglycosylases display the bioorthogonal handle in mature PG. The first-generation system involved CuAAC chemistry between Alk-NAM and azide-conjugate fluorophores. A second-generation system utilized the SPAAC reaction of Az-NAM with a cyclooctyne–fluorophore. A third system applied MeTzPh-NAM through a Tz-TCO ligation but was limited by low incorporation and abnormal morphology for probe-incorporated cells. Introduced here is the new probe, HTz-NAM, which can successfully incorporate and undergo Tz-TCO ligation at rapid rates.

Figure 2

(A) Synthesis of HTz-Pfp-ester (5) and HTz-NAM, as well as small molecule fluorescent probes NBD-NAM and 4-DMAPth-NAM. See Supporting Information Figures 1–3 for DSC safety testing for tetrazine precursors 3, 4, and 5. (B) In vitro chemoenzymatic studies for NBD-NAM, 4-DMAPth NAM, and HTz-NAM. (C) Workflow for analyzing HTz-NAM incorporation into EQKU cells. (D) Growth curve for EQKU with varying concentrations of HTz-NAM to determine minimum concentration needed for growth recovery in the presence of a lethal dose of fosfomycin. (E) Lysozyme digestion to confirm HTz-NAM incorporation into mature PG via mass spectrometry analysis of disaccharide fragments.

Figure 3

(A) Labeling workflow for PG visualization in EQKU. (B) EQKU PG visualization using either NAM (left) or HTz-NAM (right) with aTCO-TAMRA or aTCO-SiR under fixed (top) or live conditions (bottom), respectively. (C) Tz-TCO kinetics analysis through real-time addition of aTCO-SiR to fixed EQKU cells that had been remodeled to display HTz-NAM in the PG. The kinetics were analyzed starting at 1 s following the 5.6 μM aTCO-SiR addition and represents an average across eight different cell regions across one cell population. See SI Videos 1 and 2, as well as SI Figures 10–13, for experimental details and biological replicate analysis.

Figure 4

Versatility of HTz-NAM remodeling across a variety of bacterial species. (A) Confocal fluorescent images of (left to right) B. subtilus (BSKU), Gram-positive; E. coli strains (DH5α-Tf-KU and RFM795-Tf-KU), Gram-negative; wild-type Pseudomonas species (P. putida and P. aeruginosa). Scale bars, 10 μm. (B) Observation of cell wall detail throughout bacterial growth and division stages. EQKU and DH5α-Tf-KU: aTCO-TAMRA (red) imaged on Zeiss LSM 800. BSKU and RFM795-Tf-KU: aTCO-SiR (pink) imaged on Zeiss LSM 800. P. putida and P. aeruginosa: aTCO-SiR (purple) imaged on Andor Dragonfly 600. Scale bars, 2 μm. (C) P. aeruginosa high-resolution images of no-wash, live labeling with aTCO-SiR. Scale bars, 2 μm. Deconvolution achieved through Imaris software. All images were taken of samples that were performed in biological and technical replicates (see SI Figures 38–92).

Figure 5

Imaging of bacteria engulfment by macrophages. (A) Fixed labeling of EQKU cells inside macrophages with workflow visualization. EQKU cells were remodeled with HTz-NAM prior to macrophage engulfment. The cells were fixed and then reacted with either aTCO-TAMRA or aTCO-SiR. (B) Live imaging of prelabeled bacteria by macrophages. Macrophages were stained using CellMask Orange (white), and bacteria were labeled with aTCO-SiR (purple) (see SI Figures 93–95 and SI Videos 5 and 6).

Figure 6

Live visualization of Tz-TCO ligation of HTz-NAM-remodeled bacteria engulfed by a macrophage. (A) Workflow for rapid, real-time, live-cell, no-wash labeling of HTz-NAM remodeled EQKU inside of live macrophages. (B) Prior to aTCO-SiR addition, EQKU cells were remodeled with HTz-NAM and engulfed by macrophages that were stained using CellMask Orange (white). (C) Labeling with aTCO-SiR was complete within 5 min. Orthogonal slices of bacterial cell confirming presence inside macrophage (see SI Video 7).