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Cited by 2Escherichia coli research studiesCarbohydrate Chemistry and SynthesisGlycosylation and Glycoproteins Research

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Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes

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Furevi, Axel, Ståhle, Jonas, Muheim, Claudio, Gkotzis, Spyridon, Daley, Daniel O, Udekwu, Klas I, Widmalm, Göran·2023·Glycobiology
DOI 10.1093/glycob/cwac069PMC PMC10191223

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Abstract

The structure of the O-antigen from the international reference strain Escherichia coli O93:−:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

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Figures

Fig. 1 SDS-PAGE analysis of LPS from different E. coli strains. Lane 1: LPS from E. coli CWG303 ΔwaaG, Lane 2: LPS from E. coli W3110, and Lane 3: LPS from E. coli O93. A 14% Tricine SDS-PAGE gel was used to separate LPS samples, which subsequently were stained with AgNO3. A pre-stained protein marker (Thermo Fisher Scientific) was utilized for calibration of molecular weights.
Fig. 1 SDS-PAGE analysis of LPS from different E. coli strains. Lane 1: LPS from E. coli CWG303 ΔwaaG, Lane 2: LPS from E. coli W3110, and Lane 3: LPS from E. coli O93. A 14% Tricine SDS-PAGE gel was used to separate LPS samples, which subsequently were stained with AgNO3. A pre-stained protein marker (Thermo Fisher Scientific) was utilized for calibration of molecular weights.CC BY · PubMed Central
Fig. 2 The O-antigen gene cluster between galF and hisI in E. coli O93 (Iguchi et al. 2015; DebRoy et al. 2016; Liu et al. 2020).
Fig. 2 The O-antigen gene cluster between galF and hisI in E. coli O93 (Iguchi et al. 2015; DebRoy et al. 2016; Liu et al. 2020).CC BY · PubMed Central
Fig. 3 The anomeric region of O-deacetylated E. coli O93 O-antigen polysaccharide (PS–OH) from a 1H NMR spectrum (a) and a 1H,1H-NOESY spectrum with a mixing time of 150 ms at 700 MHz (b). Transglycosidic cross-peaks are annotated by red color.
Fig. 3 The anomeric region of O-deacetylated E. coli O93 O-antigen polysaccharide (PS–OH) from a 1H NMR spectrum (a) and a 1H,1H-NOESY spectrum with a mixing time of 150 ms at 700 MHz (b). Transglycosidic cross-peaks are annotated by red color.CC BY · PubMed Central
Fig. 4 NMR spectra of selected regions from the O-antigen polysaccharides (PS and PS–OH) of E. coli O93. (a) An overlay of 1H,13C-meHSQC NMR spectra of the anomeric region from PS (black) and PS–OH (red); (b) 1H,13C-meHSQC spectrum of PS–OH plotted in red color with linkage positions annotated and hydroxymethyl correlations plotted in blue color; (c, d) selected regions from the 1H,13C-meHSQC spectrum of the PS sample showing the O-acetylated positions in residues C′/C″ with hydroxymethyl correlations plotted in green color; (e, f) 1H,1H-TOCSY spectrum with a mixing time of 120 ms displaying the cross-peaks from H2 resonances in residues C2′ and C2″; (g) selected region from the coupling-enhanced (2×) 1H,13C-CT-CE-HSQC NMR spectrum resulting in 1JC1,H1 for B′/B″ (158 Hz) and C′/C″ (160 Hz).
Fig. 4 NMR spectra of selected regions from the O-antigen polysaccharides (PS and PS–OH) of E. coli O93. (a) An overlay of 1H,13C-meHSQC NMR spectra of the anomeric region from PS (black) and PS–OH (red); (b) 1H,13C-meHSQC spectrum of PS–OH plotted in red color with linkage positions annotated and hydroxymethyl correlations plotted in blue color; (c, d) selected regions from the 1H,13C-meHSQC spectrum of the PS sample showing the O-acetylated positions in residues C′/C″ with hydroxymethyl correlations plotted in green color; (e, f) 1H,1H-TOCSY spectrum with a mixing time of 120 ms displaying the cross-peaks from H2 resonances in residues C2′ and C2″; (g) selected region from the coupling-enhanced (2×) 1H,13C-CT-CE-HSQC NMR spectrum resulting in 1JC1,H1 for B′/B″ (158 Hz) and C′/C″ (160 Hz).CC BY · PubMed Central
Fig. 5 Top 3 structures using the “Determine Glycan Structure” entry in CASPER for E. coli O93 PS–OH with NMR data obtained from 1H,13C-HSQC and 1H,1H-TOCSY experiments and magnitudes of coupling constants for input to CASPER, viz., those from 1JC1,H1 and 3JH1,H2 obtained from coupled 1H,13C-HSQC and 1H NMR spectra, respectively.
Fig. 5 Top 3 structures using the “Determine Glycan Structure” entry in CASPER for E. coli O93 PS–OH with NMR data obtained from 1H,13C-HSQC and 1H,1H-TOCSY experiments and magnitudes of coupling constants for input to CASPER, viz., those from 1JC1,H1 and 3JH1,H2 obtained from coupled 1H,13C-HSQC and 1H NMR spectra, respectively.CC BY · PubMed Central
Fig. 6 Comparison of 1H (left) and 13C (right) NMR chemical shifts predicted by CASPER versus assigned from NMR experiments for the O-deacetylated (PS–OH) and the 2,6-di-O-acetylated population of the polysaccharide (PS) from the E. coli O93 O-antigen.
Fig. 6 Comparison of 1H (left) and 13C (right) NMR chemical shifts predicted by CASPER versus assigned from NMR experiments for the O-deacetylated (PS–OH) and the 2,6-di-O-acetylated population of the polysaccharide (PS) from the E. coli O93 O-antigen.CC BY · PubMed Central
Fig. 7 (top) A “Sequence plot” summarizing the connectivities observed in a 1H,1H-NOESY spectrum of E. coli O93 PS–OH (upper triangle) and a 1H,13C-HMBC spectrum of E. coli O93 PS (lower triangle) where the connectivities emanate “from” the anomeric position “to” the linkage position, (middle) E. coli O93 PS repeating unit in SNFG-representation (Neelamegham et al. 2019) and (bottom) standard nomenclature with residues annotated by A–D.
Fig. 7 (top) A “Sequence plot” summarizing the connectivities observed in a 1H,1H-NOESY spectrum of E. coli O93 PS–OH (upper triangle) and a 1H,13C-HMBC spectrum of E. coli O93 PS (lower triangle) where the connectivities emanate “from” the anomeric position “to” the linkage position, (middle) E. coli O93 PS repeating unit in SNFG-representation (Neelamegham et al. 2019) and (bottom) standard nomenclature with residues annotated by A–D.CC BY · PubMed Central
Fig. 8 (left) 3D representation of the trisaccharide α-d-GlcNAc-(1→2)-β-d-Man-(1→3)-β-d-Man2Ac6Ac from the E. coli O93 PS in a conformation consistent with the 1D 1H,1H-NOESY spectrum (right) with an irradiation at 2.22 ppm of the methyl group of the O-acetyl group at C2 recorded with a 600 ms mixing time at 700 MHz showing inter-residue correlations. The yellow conical shape represents the proposed anisotropic shielding effect by the carbonyl group of the O-acetyl group at position C2 affecting H2 in residue B by an upfield chemical shift displacement, in comparison to the O-deacetylated polysaccharide. Primes used in the text to differentiate O-acetylated populations in the polysaccharide are not included in the figure for clarity.
Fig. 8 (left) 3D representation of the trisaccharide α-d-GlcNAc-(1→2)-β-d-Man-(1→3)-β-d-Man2Ac6Ac from the E. coli O93 PS in a conformation consistent with the 1D 1H,1H-NOESY spectrum (right) with an irradiation at 2.22 ppm of the methyl group of the O-acetyl group at C2 recorded with a 600 ms mixing time at 700 MHz showing inter-residue correlations. The yellow conical shape represents the proposed anisotropic shielding effect by the carbonyl group of the O-acetyl group at position C2 affecting H2 in residue B by an upfield chemical shift displacement, in comparison to the O-deacetylated polysaccharide. Primes used in the text to differentiate O-acetylated populations in the polysaccharide are not included in the figure for clarity.CC BY · PubMed Central
Fig. 9 Molecular models of 10 repeating units of the O-deacetylated (PS–OH), 2-O-acetylated (PS'), and 2,6-di-O-acetylated (PS″) O-antigens built using CarbBuilder (Kuttel et al. 2016) and visualized with VMD (Humphrey et al. 1996). Sugar-residues are colored with d-GlcNAc in blue, d-Man in green, and d-GlcA in cyan. The O-acetyl groups present in (PS′) and (PS″) are depicted in red color.
Fig. 9 Molecular models of 10 repeating units of the O-deacetylated (PS–OH), 2-O-acetylated (PS'), and 2,6-di-O-acetylated (PS″) O-antigens built using CarbBuilder (Kuttel et al. 2016) and visualized with VMD (Humphrey et al. 1996). Sugar-residues are colored with d-GlcNAc in blue, d-Man in green, and d-GlcA in cyan. The O-acetyl groups present in (PS′) and (PS″) are depicted in red color.CC BY · PubMed Central
Fig. 10 Protein structure obtained from the AlphaFold Protein Structure Database of a putative glycosyltransferase (orf5 in the O-antigen gene cluster) from E. coli O93 carrying out the last glycosylation reaction in formation of the oligosaccharyl-PP-undecaprenyl entity. The molecular model was made using UCSF Chimera (Pettersen et al. 2004).
Fig. 10 Protein structure obtained from the AlphaFold Protein Structure Database of a putative glycosyltransferase (orf5 in the O-antigen gene cluster) from E. coli O93 carrying out the last glycosylation reaction in formation of the oligosaccharyl-PP-undecaprenyl entity. The molecular model was made using UCSF Chimera (Pettersen et al. 2004).CC BY · PubMed Central

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Metadata

Authors
Furevi, A., Ståhle, J., Muheim, C., Gkotzis, S., Daley, D. O., Udekwu, K. I., & Widmalm, G.
Affiliations
Stockholm University (SE)
Corresponding author
Widmalm, Göran
ORCID
Venue
Glycobiology
Publisher
Oxford University Press (OUP)
Published
2022-10-14
Volume
33
Issue
4
Pages
289-300
ISSN
  • 0959-6658 (ISSN-L)
  • 1460-2423
DOI
10.1093/glycob/cwac069
PMC
PMC10191223
OpenAlex
W4306174605
Cited by
2
References
68
Citations by year
2024: 2
FWCI
0.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Glycobiology (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Epsilon Open Archive (Sveriges lantbruksuniversitet biblioteket (Swedish University of Agricultural Sciences)) (submittedVersion)
Funders
  • Swedish Research Council (2017-03703)
  • Knut and Alice Wallenberg Foundation
Topics
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
Keywords
lipopolysaccharide, O-antigen, tetrasaccharide repeating unit, O-acetylation, glycosyltransferases, biosynthetic gene clusters, chain length distribution, capsular polysaccharide, NMR structure elucidation, CASPER
MeSH terms
Escherichia coli, Multigene Family, Lipopolysaccharides, Magnetic Resonance Spectroscopy, O Antigens

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