Evaluation of Kdo-8-N3 incorporation into lipopolysaccharides of various Escherichia coli strains

Abstract

8-Azido-3,8-dideoxy-α/β-d-manno-oct-2-ulosonic acid (Kdo-8-N₃) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell membrane of Gram-negative bacteria. Several studies have reported successful labeling of LPS using Kdo-8-N₃ and visualization of LPS by a fluorescent reagent through click chemistry on a selection of Gram-negative bacteria such as Escherichia coli strains, Salmonella typhimurium, and Myxococcus xanthus. Motivated by the promise of Kdo-8-N₃ to be useful in the investigation of LPS biosynthesis and cell surface labeling across different strains, we set out to explore the variability in nature and efficiency of LPS labeling using Kdo-8-N₃ in a variety of E. coli strains and serotypes. We optimized the chemical synthesis of Kdo-8-N₃ and subsequently used Kdo-8-N₃ to metabolically label pathogenic E. coli strains from commercial and clinical origin. Interestingly, different extents of labeling were observed in different E. coli strains, which seemed to be dependent also on growth media, and the majority of labeled LPS appears to be of the 'rough' LPS variant, as visualized using SDS-PAGE and fluorescence microscopy. This knowledge is important for future application of Kdo-8-N₃ in the study of LPS biosynthesis and dynamics, especially when working with clinical isolates.

Fig. 1. Overview of the method of fluorescent LPS labeling used in this work.

Fig. 2. Synthesis of Kdo (1) and Kdo-8-N₃ (4). Reagents and conditions: (i) oxaloacetate, NaHCO₃, NaOH, H₂O, pH 12, rt; (ii) NiCl₂, Amberlite-H⁺, pH 5, 50 °C (1 : 36%, 4 : 80%); (iii) 0.18 N HCl in MeOH, 0 °C to rt, 20 h; (iv) I₂, imidazole, PPh₃, THF, 0 to 65 °C, 20 h (56% over two steps); (v) NaN₃, DMF, 80 °C, 20 h; (vi) Amberlite-H⁺, H₂O, pH 4, 60 °C, 20 h (72% over two steps).

Fig. 3. E. coli BW25113 grown in; (A): LB or M9 media with or without 5 mM Kdo. (B): LB or M9 media with or without 5 mM Kdo-8-N₃. OD₆₀₀ values were obtained by growing the bacteria in microtiter 96-well plates and measuring OD₆₀₀ on a microtiter plate reader over 24 h. Values are calculated from triplicates and the average values are displayed. Error bars indicate standard deviations.

Fig. 4. Kdo-8-N₃ is labeled on the circumference of E. coli BW25113 cells. Cells were visualized on a Nikon Ti-E microscope using fluorescence optics (A1, B1: green channel), phase contrast (A2, B2), and co-localization of fluorescent and phase contrast images (A3, B3: merged channels). While E. coli cells supplied with Kdo-8-N₃ show green fluorescence signal localized on the periphery of the cell (A panels), E. coli cells grown without the presence of Kdo-8-N₃ show no peripheral fluorescence (B panels).

Fig. 5. Total LPS was separated on 16% Tris-Tricine SDS-PAGE and the labeled bands were visualized under fluorescence. E. coli BW25113 cells grown with or without Kdo-8-N₃ (1 mM or 5 mM) in M9 minimal medium or the rich LB medium and 'clicked' with Cy3-DBCO; (A): fluorescence image taken in the red channel. (B): Gel imaged with UV-B (300 nm) illumination after staining with the Emerald 300 gel LPS staining kit. (C): Gel scanned after silver staining protocol. (D): Superimposed images of the fluorescent gel (panel A) and after staining with Pro Q Emerald 300 LPS staining kit (panel B). A comparison between the chemically synthesized Kdo-8-N₃ (compound 4) and commercial Kdo-8-N₃ revealed similar incorporation efficiency. (E): Fluorescence image taken in the red channel. (F): Gel scanned after silver staining protocol.

Fig. 6. (I): Fluorescent bands and total LPS visualized on 16% Tris-Tricine SDS-PAGE images: (A) fluorescence image showing FAM-DBCO, Cy3-DBCO, TAMRA-DBCO and FAM-alkyne labeling on LPS. (B) Total LPS visualized by silver staining. (C) Superimposed images of fluorescent and silver-stained gels showing the match/mismatch of labeled LPS structures. (II): Microscopy images showing peripheral labeling by strain-promoted click reactions with FAM-DBCO, Cy3-DBCO, and nonselective labeling of cellular compounds with TAMRA-DBCO.

Fig. 7. Labeled LPS bands and total LPS visualized on 16% Tris-Tricine SDS-PAGE images: (A) fluorescence gel image showing the labeling of the LPS in different E. coli strains grown in either M9 or LB media supplemented with 5 mM Kdo-8-N₃. (B) Image of panel A shown with enhanced fluorescence intensity. (C) Silver-stained gel image showing the total LPS of the different E. coli strains. Each lane is labeled with the name of the E. coli strain and the media used. The plus (+) sign indicates the medium used for cell growth.

Fig. 8. Silver-stained 16% Tris-Tricine SDS-PAGE gels showing the variety of LPS structures between different E. coli strains when grown in LB with 5 mM Kdo-8-N₃ (+N₃), with 5 mM Kdo (−N₃) or without the supplementation of either substrate (−). See Fig. S4 (ESI†) for fluorescence and merged images of these gels.

Fig. 9. Selected pathogenic E. coli cells visualized on a Nikon Ti-E microscope using fluorescence optics (A1–A5: green channel), phase contrast (B1–B5), and co-localization of fluorescence and phase contrast images (C1–C5: merged channels). All fluorescent images were adjusted to the same settings (i.e. brightness, contrast) to allow visualization of different fluorescence intensities of the samples. See Fig. S5 (ESI†) for the control images for LPS labeling using SDS-PAGE.