The authors have declared that no competing interests exist.
Conceived and designed the experiments: QS RL JX YAK. Performed the experiments: QS YAK JW SNS DJ ASS SX AVP QC YW HW. Analyzed the data: RL QS YAK JX. Wrote the paper: QS YAK RL JX.
In 2001, a novel
Since serotype Xv can agglutinate with both group antigen-specific monoclonal antibodies MASF IV-1 and 7,8, it was initially named as serotype 4X and then as 4c
In this study, we analyzed the O-antigen structure of serotype Xv strains and found a phosphoethanolamine (PEtN) residue attached at position 3 of RhaII, which is absent from the typical serotype X O-antigen. This modification was shown to confer the MASF IV-1 positive phenotype in serotype Xv strains. The gene named as
This study was reviewed and approved by the ethics committee of National Institute for Communicable Disease Control and Prevention, the Chinese CDC.
Serotype Xv strains 2002017
LPS were isolated from dried bacterial cells by the phenol-water method
Samples were deuterium-exchanged by freeze-drying twice from 99.9% D2O and then examined as solutions in 99.95% D2O at 40°C. NMR spectra were recorded on an Avance II 600 spectrometer (Bruker, Germany) using internal sodium 3-(trimethylsilyl) propanoate-2,2,3,3-d4 (δH 0) and acetone (δC 31.45) as references. Two-dimensional NMR spectra were obtained using standard Bruker software and parameters set as described
The serotypes of
Chromosomal DNA was isolated from
PCR products using primers
A plasmid purification kit (Qiagen, Germany) was used to isolate the plasmids in accordance with the manufacturer’s recommendations. Plasmid DNA was separated by electrophoresis with a Chef Mapper system (Bio-Rad) on a 1% SeaKem Gold agarose gel and visualized by ethidium bromide staining. Plasmids isolated from strain 2002017 were used as the positive control. Lambda DNA cleaved with
The plasmid DNA separated on Gold agarose gel were further transferred onto a nylon membrane (Amersham) using a Vacuum Blotter (Bio-Rad). Southern hybridization was performed using an ECL™ Direct Nucleic Acid Labelling and Detection System (Amersham) as recommended by the manufacturer. DNA product amplified from strain 2002017 using primer pair
To identify the MASF IV-1 determinant in serotype Xv strains, the isolated O-polysaccharide of serotype Xv strain 2002017 was analyzed by NMR techniques. The 1H NMR and 13C NMR spectra of the O-polysaccharide demonstrated a non-O-acetylated pentasaccharide O-unit containing one residue each of glucose (Glc) and
Numbers refer to protons in sugar residues denoted as follows: G, Glc; GN, GlcNAc; RI, RhaI; RII, RhaII; RIII, RhaIII. In the spectrum of the transformant 51580_Xv O-polysaccharide, the annotations for the signals of the minor bisphosphorylated O-unit are italicized. The most important difference between X and Xv is the presence in the latter of peaks for ethanolamine, which are annotated as EtN 1 and EtN 2. The different positions of the signals for C3 of RhaII (non-phosphorylated in X and phosphorylated in Xv) are shown by a dotted line.
The NMR spectra of the serotype Xv O-polysaccharide were assigned and analyzed using two-dimensional 1H,1H and 1H,13C NMR techniques (
Residue | Monosaccharide | EtN/NAc | ||||||
C1 | C2 | C3 | C4 | C5 | C6 | C1 | C2 | |
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Serotype X | ||||||||
→2,3)-α-L-Rha |
102.2 | 75.6 | 74.9 | 72.1 | 70.7 | 18.0 | ||
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→2)-α-L-Rha |
101.9 | 79.6 | 71.1 | 73.3 | 70.4 | 17.9 | ||
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→3)-α-L-Rha |
102.2 | 71.8 | 78.4 | 72.9 | 70.2 | 17.6 | ||
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→3)-β-D-Glc |
102.5 | 56.7 | 82.8 | 69.5 | 77.3 | 62.0 | 175.2 | 23.8 |
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2.10 | ||
α-D-Glc |
95.7 | 72.6 | 74.4 | 70.7 | 72.7 | 61.5 | ||
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Serotype Xv | ||||||||
→2,3)-α-L-Rha |
101.8 | 75.4 | 75.0 | 72.2 | 70.7 | 18.0 | ||
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→2)-α-L-Rha |
101.9 | 77.5 | 76.5 | 72.3 | 70.5 | 17.9 | 63.2 | 41.4 |
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→3)-α-L-Rha |
102.4 | 71.9 | 78.6 | 73.0 | 70.2 | 17.6 | ||
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→3)-β-D-Glc |
102.7 | 56.8 | 82.9 | 69.7 | 77.4 | 62.0 | 175.2 | 23.8 |
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2.10 | ||
α-D-Glc |
95.8 | 72.6 | 74.4 | 70.7 | 72.6 | 61.6 | ||
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The corresponding parts of the 1H NMR and 31P NMR spectra are displayed along the horizontal and vertical axes, respectively. RII and RIII indicate RhaII and RhaIII, respectively. The annotations for the cross-peaks of the minor bisphosphorylated O-unit are italicized.
The O-antigen of another serotype Xv strain 2003055 was also analyzed and found to have the same structure as that of 2002017, suggesting that the PEtN modification is common among serotype Xv strains and responsible for the MASF IV-1 positive phenotype in serotype Xv.
Addition of PEtN to the inner core lipopolysaccharide (LPS) or lipooligosaccharide (LOS) has been reported in a number of bacterial species
To confirm this hypothesis, we first performed PCR assay with 9 more serotype Xv strains (2008129, 2008131, 05AH016, 05AH022, 05AH027, 05AH030, 06HN005, 06HN019 and 2008164 ) and 5 serotype X strains (06HN400, 05BJ002, 03HL001, 03HL020 and 2003055) to determine whether the
Plasmid pSFXv_2 is a double-stranded circular plasmid of 6,850 bp in length. BLAST search revealed no homology with any plasmids or bacterial genomes in the NCBI database, except for 2 regions (4186–5525 and 6058–6847) showing high similarity (96% and 78% identity, respectively) to those of pJHCMW1, a multiresistance plasmid from
To determine whether the
To elucidate the function of the
Strains | Serotype | Reaction with monoclonal antibody of MASF | Reaction with monovalent antisera of Seiken | ||||||||||||||||||||
Typing antibodies | Grouping antibodies | 1C | Typing antisera | Grouping antisera | |||||||||||||||||||
I | II | IV-2 | V | VI | Y-5 | 6 | 7,8 | IV-1 | I | II | III | IV | V | VI | 3,4 | 6 | 7,8 | ||||||
51580 | X | − | − | − | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − | + | |||
51580_Xv | Xv | − | − | − | − | − | − | − | + | + | − | − | − | − | + | − | − | − | − | + | |||
2002017 | Xv | − | − | − | − | − | − | − | + | + | − | − | − | − | + | − | − | − | − | + | |||
NCTC 9725 | 4a | − | − | + | − | − | + | − | − | - | − | − | − | − | + | − | − | + | − | − | |||
NCTC 9725_4av | 4av | − | − | + | − | − | + | − | − | + | − | − | − | − | + | − | − | + | − | − | |||
G1668 | 4av | − | − | + | − | − | + | − | − | + | − | − | − | − | + | − | − | + | − | − |
A previous study has identified that serotype 4a strain G1668 contains a PEtN residue on RhaIII at position 3
Motif analysis revealed that the
To confirm that the gain of the
NMR spectroscopic analysis showed that the major O-unit of the NCTC 9725_4 av transformant (∼50% of the total O-units) has the same structure as that of wild-type 4 av strains reported earlier
To determine whether the
Serotypes | Number of strains tested | Number of serological reactive strains with |
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MASF IV-1 | IV | |||
1a | 25 | – | – | – |
1b | 17 | – | – | – |
1c | 2 | – | – | – |
1d | 5 | – | – | – |
2a | 24 | – | – | – |
2b | 27 | – | – | – |
3a | 8 | – | – | – |
3b | 2 | – | – | – |
4a | 3 | – | 3 | – |
4av |
3 | 3 | 3 | +(3) |
4b | 5 | – | – | −/+(1) |
5a | 4 | – | – | – |
X | 70 | – | – | −/+(4) |
Xv | 59 | 59 | 59 | +(59) |
Y | 15 | – | – | – |
Yv |
19 | 19 | 19 | +(19) |
6 | 23 | – | – | – |
Reactivity with monoclonal antibody MASF IV-1 (Reagensia AB) and monovalent antisera IV (Denka Seiken). Numbers of
+ and −/+ indicate the presence of the functional and inactivated
Serotype 4 av strains (2002091, NCTC 8296 and G1668) were serologically different from typical serotype 4a strains in the MASF IV-1 reactivity.
Serotype Yv strains were serologically different from typical serotype Y strains in the MASF IV-1 and monovalent antisera IV reactivity.
The
The O-antigen structure and its diversity of
The presence and location of non-carbohydrate substituents in LPS, such as PEtN, are critical for the immune recognition of LPS by antibodies in some bacterial species
In this study, the
The known O-antigen modifications through glucosylation or O-acetylation in
In conclusion, the chemistry and genetics of a new O-antigen modification in