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Structure of the K128 capsular polysaccharide produced by Acinetobacter baumannii KZ-1093 from Kazakhstan
Carbohydrate Research 485 (2019) 107814
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Structure of the K128 capsular polysaccharide produced by Acinetobacter baumannii KZ-1093 from Kazakhstan
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Nikolay P. Arbatskya, Anastasiya A. Kasimovaa,b, Alexander S. Shashkova, Mikhail M. Shneiderc,d, Anastasiya V. Popovad,e,f, Dmitry A. Shaging, Andrey A. Shelenkovg, Yuliya V. Mikhailovag, Yurii G. Yanushevichg, Ilya S. Azizovd, Mikhail V. Edelsteind, Ruth M. Hallh, Johanna J. Kenyoni,∗,1, Yuriy A. Knirela,1 a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Higher Chemical College of the Russian Academy of Sciences, D. I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia M. M. Shemyakin & Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia d Institute of Antimicrobial Chemotherapy, Smolensk State Medical University, Smolensk, Russia e Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia f State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russia g Central Scientific Research Institute of Epidemiology, Moscow, Russia h School of Life and Environmental Sciences, University of Sydney, Sydney, Australia i Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia b c
ARTICLE INFO
ABSTRACT
Keywords: Acinetobacter baumannii Capsular polysaccharide K locus Glycosyltransferase Wzy polymerase
The structure of the K128 capsular polysaccharide (CPS) produced by Acinetobacter baumannii isolate KZ-1093 from Kazakhstan was established by sugar analysis and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The CPS was found to consist of branched pentasaccharide repeating units containing only neutral sugars, and its composition and topology are closely related to those of the A. baumannii K116 CPS. The K128 and K116 oligosaccharide units differ in the linkage between the disaccharide side chain and the main chain, with a β-(1 → 6) linkage in K128 replacing a β-(1 → 4) linkage in K116. The linkages between the repeating units in the K128 and K116 CPSs are also different, with K128 units linked by β-D-GalpNAc-(1 → 4)-DGalp, and β-D-GalpNAc-(1 → 3)-D-Galp linkages between K116 units. The KZ-1093 genome was sequenced and the CPS biosynthesis gene cluster at the chromosomal K locus was designated KL128. Consistent with the CPS structures, KL128 differs from KL116 in one glycosyltransferase gene and the gene for the Wzy polymerase. In KL128, the gtr200 glycosyltransferase gene replaces gtr76 in KL116, and Gtr200 was therefore assigned to the different β-D-GalpNAc-(1 → 6)-D-Galp linkage in K128. Similarly, the WzyK128 polymerase could be assigned to the β-D-GalpNAc-(1 → 4)-D-Galp linkage between the K128 units.
1. Introduction Capsular polysaccharides (CPSs) form a hydrated layer on the cell surface of many bacterial species. For the clinically important Gramnegative pathogen, Acinetobacter baumannii, CPS is a major virulence determinant that also provides protection against desiccation [1,2]. The CPS is made up of repeating ‘K unit’ oligosaccharides that can vary between different isolates in the species in sugar composition and linkages between sugars, and the linkages between K units can also vary (e.g. Refs. [3–6] and references cited therein). The genes driving CPS
biosynthesis in A. baumannii usually include those for the initiation of capsule synthesis (itr), glycosyltransfer (gtr), translocation (wzx), polymerisation (wzy), and the addition of non-carbohydrate groups. Most of these genes are clustered together at the K locus (KL) in the A. baumannii chromosome [7], and variation generally reflects replacement of CPS genes [8,9] or recombination between KL gene clusters giving rise to a hybrid KL [10]. Some CPS gene clusters in A. baumannii genomes represent closely related pairs or groups that differ only in one or two genes in the region specific for the construction of the K-unit oligosaccharide [11–15]. In
Corresponding author. E-mail address: [email protected] (J.J. Kenyon). 1 Authors contributed equally. ∗
https://doi.org/10.1016/j.carres.2019.107814 Received 22 July 2019; Received in revised form 20 August 2019; Accepted 8 September 2019 Available online 09 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The A. baumannii KL128 gene cluster and the closely related KL116 gene cluster. Genes are colour coded according to the functions of their encoded products and the colour scheme is shown below. Dark grey shading indicates regions that share > 90% nucleotide sequence identity, whereas light grey shading is 75–90% nucleotide sequence identity. Figure is drawn to scale from GenBank accession numbers MK399428.1 (KL128) and MK399425.1 (KL116). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
includes genes for capsule export (wza-wzb-wzc) and for simple sugar synthesis (galU-ugd-gpi-gne1-pgm). The absence of further sugar synthesis genes indicated that the CPS would include only one or more of the following sugars, Glcp, Galp, GlcpNAc and GalpNAc [7]. The KL128 gene cluster also includes genes for four glycosyltransferases (gtr75, gtr201, gtr25, and gtr5), and an initiating transferase (itrA2) known to link D-GalpNAc-1-P to the undecaprenyl phosphate (UndP) lipid carrier in the inner membrane and thus initiate synthesis of the K-unit synthesis from its component sugars [18], as well as K-unit processing genes (wzx and wzy). The arrangement of the KL128 gene cluster is closely related to the previously reported A. baumannii KL116 gene cluster (GenBank accession number MK399425.1). The two gene clusters share gtr75, gtr25, and gtr5 glycosyltransferase genes but differ in a short sequence segment that includes the fourth glycosyltransferase gene (gtr200 in KL128 replaces gtr76 in KL116) and the gene for the Wzy polymerase (Fig. 1). The structure of the K116 CPS has been elucidated, and the linkages formed by the glycosyltransferases encoded by KL116 have been deduced [10]. Gtr25K116 and Gtr5K116 form α-D-Galp-(1 → 6)-D-Galp and β-D-Galp-(1 → 3)-D-GalpNAc linkages, respectively, in the K116 main chain and also in the K units of several other A. baumannii CPSs, including K14 and K93 [19,20]. Gtr75K116 was found to form a β-D-Glcp(1 → 6)-D-GalpNAc side-branch linkage, and this side-branch is joined by the fourth glycosyltransferase (Gtr76) to a D-Galp residue in the main chain via a β-(1 → 4) linkage (Fig. 2A). Thus, it is expected that the K128 structure will include the same linkages formed by Gtr75, Gtr25 and Gtr5 but differ from K116 in the remaining linkage of the disaccharide side-chain to the main chain, and also in the linkage between the K units in the CPS.
Fig. 2. A. The K116 CPS structure elucidated in a previous study [10]. B. Structure of the K128 CPS determined in this study. Glycosyltransferases, Wzy polymerase and ItrA2 initiating transferase are shown near the linkage that each are predicted to form. Grey shading indicates differences between the K116 and K128 CPS structures.
these cases, the differences in the resulting CPS structures are limited and these pairs can be used to unambiguously determine the linkages formed by enzymes encoded by the genes that are the same or differ between the gene clusters. This approach has been successfully used to determine the linkages formed by Gtr proteins and the specific Wzy polymerases responsible for linking K units together to generate highmolecular mass CPS [11–13,15–17]. In this work, we established the structure of the CPS of a multiply antibiotic resistant A. baumannii isolate, KZ-1093, from Kazakhstan that carries a novel KL gene cluster related to the previously reported A. baumannii KL116 gene cluster [10], and deduce the linkages formed by the encoded Gtr and Wzy proteins.
2.2. Elucidation of the K128 CPS structure A CPS preparation was isolated from cells of A. baumannii KZ-1093. Analysis of monosaccharide components using a sugar analyzer after full acid hydrolysis of the CPS showed the presence of Glc, Gal, and GalNAc in the ratio ~1.0 : 0.9: 1.7, respectively. The CPS structure was established by NMR spectroscopy, including 2D 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY (Fig. 3), and 1H,13C HSQC (Fig. 4) experiments. The assigned 1H and 13C NMR chemical shifts of the CPS are tabulated in Table 1. NMR analysis revealed spin systems for five monosaccharide residues (units A-E), all being in the pyranose form (Table 1). In the 1H,1H TOCSY spectra, there were correlations for H-1 with H-2,3,4 for sugars having the galacto configuration (Gal and GalNAc) and with H-2,3,4,5 for Glc. The signals within each spin system were assigned using the
2. Results 2.1. The KL128 capsule biosynthesis gene cluster The draft genome sequence of A. baumannii KZ-1093 was obtained by Illumina MiSeq sequencing, and the chromosomal K locus was found to contain a novel CPS gene cluster, designated KL128 (GenBank accession number MK399428.1). As for most KL, this gene cluster (Fig. 1) 2
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Fig. 3. Part of a 2D 1H,1H ROESY spectrum of the K128 CPS of A. baumannii KZ-1093. The corresponding parts of the 1H NMR spectrum are shown along the axes. Numbers refer protons in sugar residues denoted by letters as indicated in Table 1. 1
H,1H COSY spectra, and those for H-5 and H-6 of Gal and GalNAc were found by H-4/H-5 correlations in the 1H,1H ROESY spectrum and H-5/ H-6 correlations in the 1H,1H COSY spectrum. Relatively large J1,2 coupling constants of 7–8 Hz indicated that all monosaccharide residues are β-linked, except for one Gal residue that is α-linked as judged by a relatively low 3J1,2 coupling constant of < 3 Hz. Downfield displacements by 6–10 ppm of the NMR signals for the linkage carbons (Fig. 4), as compared with their positions in the spectra of the corresponding non-substituted monosaccharides [21], showed that the CPS is branched and defined the glycosylation pattern in the K unit. The 1H,1H ROESY spectrum (Fig. 3) showed the following correlations between the anomeric proton and protons at the linkage carbons of the neighbouring sugar residues: E H-1/D H-6a and H-6b, D H-1/C H6a, C H-1/B H-6a, B H-1/A H-3, and A H-1/C H-4 at δ 4.50/3.93 and 4.07, 4.49/3.82, 4.94/3.70, 4.49/3.87, and 4.69/4.09, respectively. An alternative assignment, D H-1/A H-3 and B H-1/C H-6a, for correlations of units B and D having the same H-1 chemical shift of δ 4.49 could be excluded based on genetic data, particular by the presence of Gtr5 glycosyltransferase responsible for the formation of the β-D-Galp-(1 → 3)-D-GalpNAc (B→A) linkage (see below section 2.3). These data confirmed the positions of substitution and defined the sequence of the monosaccharides in the K unit. Based on the data obtained, it was concluded that the K128 CPS has the structure shown in Fig. 2B. The K128 unit consists of a β-D-Glcp(1 → 6)-D-GalpNAc disaccharide side chain β-(1 → 6)-linked to a D-Galp residue in a trisaccharide main chain. Given that only one D-GalpNAc residue is present in the main chain, this is the first sugar of the K unit due to the known function of the ItrA2 initiating transferase encoded by KL128.
monosaccharides, D-Glcp, D-Galp and D-GlcpNAc (Fig. 2A and B). The linkages predicted to be formed by Gtr75, Gtr25, and Gtr5 are all present in the K128 unit, and the Gtrs were assigned to these linkages accordingly (Fig. 2B). As the first sugar of the K128 unit is known, the linkage between units in the K128 CPS chain is unambiguous. This linkage, β-D-GalpNAc-(1 → 4)-D-Galp, would therefore be formed by WzyK128. Consistent with this assignment, WzyK128 shares 54% identity with WzyK27 from A. baumannii KL27 (GenBank accession number KT266827.1), which is known to link K units in the K27 CPS via the same β-D-GalpNAc-(1 → 4)-D-Galp linkage [11]. The remaining linkage, β-D-GalpNAc-(1 → 6)-D-Galp, that joins the disaccharide side-chain to the main chain, would therefore be formed by the Gtr200K128 glycosyltransferase. Gtr200K128 shares 33% identity with Gtr75, which forms the similar β-D-Glcp-(1 → 6)-D-GalpNAc linkage in K116 and K128. 3. Discussion The K128 CPS joins a growing number of A. baumannii CPS structures made up of only neutral sugars [10,15,17,19,22]. The α-D-Galp(1 → 6)-β-D-Galp-(1 → 3)-D-GalpNAc trisaccharide formed by Gtr5 and Gtr25 coupled with ItrA2 is found in several of these structures, including K128, K116, K14 [19] and K93 [20], and the corresponding gene clusters share the gtr25/gtr5/itrA2 sequence segment. This suggests that the gtr25/gtr5/itrA2 module may be maintained together or transferred as a gene block in the assembly of new KL gene clusters via recombination, conserving the availability of specific substrates for this set of transferases. The coupling of Gtrs for the construction of specific polysaccharides has also been observed in other species [23,24]. Determination of CPS structures of A. baumannii gene cluster pairs have enabled unambiguous identification of the specific linkages formed by the Wzy polymerases (e.g. Refs. [11–13]). This approach has proven invaluable for characterising Wzy proteins in the species, as Wzy polymerases are notoriously difficult to classify due to the enormous amount of amino acid sequence diversity observed in nature.
2.3. Assignment of glycosyltransferases and the Wzy polymerase The K128 and K116 units both consist of a trisaccharide mainchain with a disaccharide sidechain and include only simple, neutral 3
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Fig. 4. Parts of a 2D 1H,13C HSQC spectrum of the K128 CPS of A. baumannii KZ-1093. The corresponding parts of the 1H and 13C NMR spectra are shown along the horizontal and vertical axes, respectively. Numbers refer to H/C pairs in sugar residues denoted by letters as indicated in Table 1.
different clonal lineages [10], suggesting a more widespread distribution.
Table 1 1 H and13C NMR chemical shifts (δ, ppm) of the K128 CPS of A. baumannii KZ1093. Sugar residue
C-1 H-1
C-2 H-2
C-3 H-3
C-4 H-4
C-5 H-5
C-6 H-6 (6a, 6b)
→3)-β-D-GalpNAc-(1→ A →6)-β-D-Galp-(1→ B →4,6)-α-D-Galp-(1→ C →6)-β-D-GalpNAc-(1→ D β-D-Glcp-(1→ E
103.5 4.69 106.2 4.49 100.1 4.94 103.9 4.49 104.3 4.50
53.8 3.99 72.3 3.55 69.5 3.72 53.2 3.92 74.7 3.32
81.5 3.87 74.1 3.64 71.2 3.90 75.1 3.86 77.4 3.51
69.4 4.15 70.1 3.98 78.5 4.09 70.1 3.99 71.3 3.41
76.1 3.63 73.8 3.83 71.3 4.00 73.8 3.72 77.5 3.47
62.7 3.78, 67.8 3.70, 72.2 3.82, 70.4 3.93, 62.5 3.75,
4. Materials and methods 4.1. Bacterial strain, cultivation, and isolation of CPS A. baumannii clinical isolate KZ-1093 was obtained from a bronchoalveolar lavage specimen of a 2-year-old male patient with nosocomial pneumonia in Nur-Sultan (Astana), Kazakhstan, in August 2016, and was deposited in the collection of the Institute of Antimicrobial Chemotherapy, Smolensk State Medical University (Smolensk, Russia). Bacteria were cultivated in 2 × TY media overnight; cells were harvested by centrifugation (10,000×g, 15 min), and suspended in phosphate-buffered saline. The suspension was cooled down to 4 °C, 0.2 volume of CCl3CO2H was added, cells were precipitated by centrifugation (15,000×g, 20 min), and two volumes of acetone were added to the supernatant. After intense shaking, the precipitate (CPS) was separated by centrifugation (8000×g, 20 min), dissolved in water, the pH value was adjusted to pH 8 by adding 1 M NaOH, the CPS was precipitated with acetone and separated by centrifugation as above, dissolved in distilled water and applied to a column (53 × 3.5 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden). Elution was performed with 0.1% HOAc and monitored using a UV-detector (Uvicord, Sweden) at 206 nm to give a purified CPS sample (21 mg).
3.78 3.84 4.07 4.07 3.92
1
H NMR chemical shifts are italicized. Chemical shifts for the N-acetyl groups are δH 2.03–2.07, δC 23.7–24.1 (CH3) and 175.7–176.3 (CO).
We have successfully used the correlation of CPS structural data with KL sequence to establish the specific linkage formed by more than 35 different Wzy proteins in A. baumannii, and here we used the same approach to demonstrate that WzyK128 forms the β-D-GalpNAc-(1 → 4)D-Galp linkage in the K128 CPS. The KL128 gene cluster appears to be sporadic, found to date only in the genomes of KZ-1093 and one further A. baumannii isolate, 4300STDY7045753 (WGS accession number UFKI01000022.1), available in the NCBI non-redundant and WGS databases that include more than 3000 sequences. In comparison, the related KL116 gene cluster has previously been identified in a wide range of A. baumannii isolates from
4.2. Chemical analyses A CPS sample (1 mg) was hydrolyzed with 3 M CF3CO2H (120 °C, 2 h). Monosaccharides were analyzed using a Biotronik LC-200 sugar analyzer. Neutral sugars were identified on a column (15 × 0.4 cm) of 4
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Dionex A × 8 anion-exchange resin in 0.5 M sodium borate buffer pH 8 at 70 °C. Amino sugars were determined on a column (22 × 0.4 cm) of Ostion LC AN B cation-exchange resin in 0.2 M borate buffer pH 5 at 70 °C.
[7] [8]
4.3. NMR spectroscopy [9]
A CPS sample was deuterium-exchanged by freeze-drying from 99.9% D2O and then examined as a solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 60 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (δH 0, δC –1.6) was used as internal reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A 60-ms MLEV-17 spin-lock time and a 150-ms mixing time were used in 1 H,1H TOCSY and ROESY experiments, respectively.
[10]
[11]
[12]
4.4. Sequencing and bioinformatic analysis The draft genome sequence of A. baumannii KZ-1093 was obtained by MiSeq sequencing using a Nextera DNA library preparation kit (Illumina, San Diego, CA), and assembly of reads using SPAdes v. 3.10 [25]. The capsule biosynthesis gene cluster was located in the chromosome, and the genes were annotated and characterised as described previously [7]. The sequence was deposited into GenBank under accession number MK399428.1.
[13]
[14]
Funding [15]
Identification, cultivation, and genome sequencing of the bacterial strain KZ-1093 was supported by the Russian Science Foundation (project No. 18-15-00403). Monosaccharide analysis and CPS structure determination was supported by the Russian Science Foundation (project No. 19-14-00273). Bioinformatics was supported by an Australian Research Council (ARC) DECRA Fellowship 180101563 to JJK.
[16]
[17]
Acknowledgements The authors thank Sarah Cahill (Queensland University of Technology, Australia) for bioinformatics assistance.
[18]
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Structure of the K128 capsular polysaccharide produced by Acinetobacter baumannii KZ-1093 from Kazakhstan
T
Nikolay P. Arbatskya, Anastasiya A. Kasimovaa,b, Alexander S. Shashkova, Mikhail M. Shneiderc,d, Anastasiya V. Popovad,e,f, Dmitry A. Shaging, Andrey A. Shelenkovg, Yuliya V. Mikhailovag, Yurii G. Yanushevichg, Ilya S. Azizovd, Mikhail V. Edelsteind, Ruth M. Hallh, Johanna J. Kenyoni,∗,1, Yuriy A. Knirela,1 a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Higher Chemical College of the Russian Academy of Sciences, D. I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia M. M. Shemyakin & Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia d Institute of Antimicrobial Chemotherapy, Smolensk State Medical University, Smolensk, Russia e Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia f State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russia g Central Scientific Research Institute of Epidemiology, Moscow, Russia h School of Life and Environmental Sciences, University of Sydney, Sydney, Australia i Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia b c
ARTICLE INFO
ABSTRACT
Keywords: Acinetobacter baumannii Capsular polysaccharide K locus Glycosyltransferase Wzy polymerase
The structure of the K128 capsular polysaccharide (CPS) produced by Acinetobacter baumannii isolate KZ-1093 from Kazakhstan was established by sugar analysis and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The CPS was found to consist of branched pentasaccharide repeating units containing only neutral sugars, and its composition and topology are closely related to those of the A. baumannii K116 CPS. The K128 and K116 oligosaccharide units differ in the linkage between the disaccharide side chain and the main chain, with a β-(1 → 6) linkage in K128 replacing a β-(1 → 4) linkage in K116. The linkages between the repeating units in the K128 and K116 CPSs are also different, with K128 units linked by β-D-GalpNAc-(1 → 4)-DGalp, and β-D-GalpNAc-(1 → 3)-D-Galp linkages between K116 units. The KZ-1093 genome was sequenced and the CPS biosynthesis gene cluster at the chromosomal K locus was designated KL128. Consistent with the CPS structures, KL128 differs from KL116 in one glycosyltransferase gene and the gene for the Wzy polymerase. In KL128, the gtr200 glycosyltransferase gene replaces gtr76 in KL116, and Gtr200 was therefore assigned to the different β-D-GalpNAc-(1 → 6)-D-Galp linkage in K128. Similarly, the WzyK128 polymerase could be assigned to the β-D-GalpNAc-(1 → 4)-D-Galp linkage between the K128 units.
1. Introduction Capsular polysaccharides (CPSs) form a hydrated layer on the cell surface of many bacterial species. For the clinically important Gramnegative pathogen, Acinetobacter baumannii, CPS is a major virulence determinant that also provides protection against desiccation [1,2]. The CPS is made up of repeating ‘K unit’ oligosaccharides that can vary between different isolates in the species in sugar composition and linkages between sugars, and the linkages between K units can also vary (e.g. Refs. [3–6] and references cited therein). The genes driving CPS
biosynthesis in A. baumannii usually include those for the initiation of capsule synthesis (itr), glycosyltransfer (gtr), translocation (wzx), polymerisation (wzy), and the addition of non-carbohydrate groups. Most of these genes are clustered together at the K locus (KL) in the A. baumannii chromosome [7], and variation generally reflects replacement of CPS genes [8,9] or recombination between KL gene clusters giving rise to a hybrid KL [10]. Some CPS gene clusters in A. baumannii genomes represent closely related pairs or groups that differ only in one or two genes in the region specific for the construction of the K-unit oligosaccharide [11–15]. In
Corresponding author. E-mail address: [email protected] (J.J. Kenyon). 1 Authors contributed equally. ∗
https://doi.org/10.1016/j.carres.2019.107814 Received 22 July 2019; Received in revised form 20 August 2019; Accepted 8 September 2019 Available online 09 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The A. baumannii KL128 gene cluster and the closely related KL116 gene cluster. Genes are colour coded according to the functions of their encoded products and the colour scheme is shown below. Dark grey shading indicates regions that share > 90% nucleotide sequence identity, whereas light grey shading is 75–90% nucleotide sequence identity. Figure is drawn to scale from GenBank accession numbers MK399428.1 (KL128) and MK399425.1 (KL116). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
includes genes for capsule export (wza-wzb-wzc) and for simple sugar synthesis (galU-ugd-gpi-gne1-pgm). The absence of further sugar synthesis genes indicated that the CPS would include only one or more of the following sugars, Glcp, Galp, GlcpNAc and GalpNAc [7]. The KL128 gene cluster also includes genes for four glycosyltransferases (gtr75, gtr201, gtr25, and gtr5), and an initiating transferase (itrA2) known to link D-GalpNAc-1-P to the undecaprenyl phosphate (UndP) lipid carrier in the inner membrane and thus initiate synthesis of the K-unit synthesis from its component sugars [18], as well as K-unit processing genes (wzx and wzy). The arrangement of the KL128 gene cluster is closely related to the previously reported A. baumannii KL116 gene cluster (GenBank accession number MK399425.1). The two gene clusters share gtr75, gtr25, and gtr5 glycosyltransferase genes but differ in a short sequence segment that includes the fourth glycosyltransferase gene (gtr200 in KL128 replaces gtr76 in KL116) and the gene for the Wzy polymerase (Fig. 1). The structure of the K116 CPS has been elucidated, and the linkages formed by the glycosyltransferases encoded by KL116 have been deduced [10]. Gtr25K116 and Gtr5K116 form α-D-Galp-(1 → 6)-D-Galp and β-D-Galp-(1 → 3)-D-GalpNAc linkages, respectively, in the K116 main chain and also in the K units of several other A. baumannii CPSs, including K14 and K93 [19,20]. Gtr75K116 was found to form a β-D-Glcp(1 → 6)-D-GalpNAc side-branch linkage, and this side-branch is joined by the fourth glycosyltransferase (Gtr76) to a D-Galp residue in the main chain via a β-(1 → 4) linkage (Fig. 2A). Thus, it is expected that the K128 structure will include the same linkages formed by Gtr75, Gtr25 and Gtr5 but differ from K116 in the remaining linkage of the disaccharide side-chain to the main chain, and also in the linkage between the K units in the CPS.
Fig. 2. A. The K116 CPS structure elucidated in a previous study [10]. B. Structure of the K128 CPS determined in this study. Glycosyltransferases, Wzy polymerase and ItrA2 initiating transferase are shown near the linkage that each are predicted to form. Grey shading indicates differences between the K116 and K128 CPS structures.
these cases, the differences in the resulting CPS structures are limited and these pairs can be used to unambiguously determine the linkages formed by enzymes encoded by the genes that are the same or differ between the gene clusters. This approach has been successfully used to determine the linkages formed by Gtr proteins and the specific Wzy polymerases responsible for linking K units together to generate highmolecular mass CPS [11–13,15–17]. In this work, we established the structure of the CPS of a multiply antibiotic resistant A. baumannii isolate, KZ-1093, from Kazakhstan that carries a novel KL gene cluster related to the previously reported A. baumannii KL116 gene cluster [10], and deduce the linkages formed by the encoded Gtr and Wzy proteins.
2.2. Elucidation of the K128 CPS structure A CPS preparation was isolated from cells of A. baumannii KZ-1093. Analysis of monosaccharide components using a sugar analyzer after full acid hydrolysis of the CPS showed the presence of Glc, Gal, and GalNAc in the ratio ~1.0 : 0.9: 1.7, respectively. The CPS structure was established by NMR spectroscopy, including 2D 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY (Fig. 3), and 1H,13C HSQC (Fig. 4) experiments. The assigned 1H and 13C NMR chemical shifts of the CPS are tabulated in Table 1. NMR analysis revealed spin systems for five monosaccharide residues (units A-E), all being in the pyranose form (Table 1). In the 1H,1H TOCSY spectra, there were correlations for H-1 with H-2,3,4 for sugars having the galacto configuration (Gal and GalNAc) and with H-2,3,4,5 for Glc. The signals within each spin system were assigned using the
2. Results 2.1. The KL128 capsule biosynthesis gene cluster The draft genome sequence of A. baumannii KZ-1093 was obtained by Illumina MiSeq sequencing, and the chromosomal K locus was found to contain a novel CPS gene cluster, designated KL128 (GenBank accession number MK399428.1). As for most KL, this gene cluster (Fig. 1) 2
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Fig. 3. Part of a 2D 1H,1H ROESY spectrum of the K128 CPS of A. baumannii KZ-1093. The corresponding parts of the 1H NMR spectrum are shown along the axes. Numbers refer protons in sugar residues denoted by letters as indicated in Table 1. 1
H,1H COSY spectra, and those for H-5 and H-6 of Gal and GalNAc were found by H-4/H-5 correlations in the 1H,1H ROESY spectrum and H-5/ H-6 correlations in the 1H,1H COSY spectrum. Relatively large J1,2 coupling constants of 7–8 Hz indicated that all monosaccharide residues are β-linked, except for one Gal residue that is α-linked as judged by a relatively low 3J1,2 coupling constant of < 3 Hz. Downfield displacements by 6–10 ppm of the NMR signals for the linkage carbons (Fig. 4), as compared with their positions in the spectra of the corresponding non-substituted monosaccharides [21], showed that the CPS is branched and defined the glycosylation pattern in the K unit. The 1H,1H ROESY spectrum (Fig. 3) showed the following correlations between the anomeric proton and protons at the linkage carbons of the neighbouring sugar residues: E H-1/D H-6a and H-6b, D H-1/C H6a, C H-1/B H-6a, B H-1/A H-3, and A H-1/C H-4 at δ 4.50/3.93 and 4.07, 4.49/3.82, 4.94/3.70, 4.49/3.87, and 4.69/4.09, respectively. An alternative assignment, D H-1/A H-3 and B H-1/C H-6a, for correlations of units B and D having the same H-1 chemical shift of δ 4.49 could be excluded based on genetic data, particular by the presence of Gtr5 glycosyltransferase responsible for the formation of the β-D-Galp-(1 → 3)-D-GalpNAc (B→A) linkage (see below section 2.3). These data confirmed the positions of substitution and defined the sequence of the monosaccharides in the K unit. Based on the data obtained, it was concluded that the K128 CPS has the structure shown in Fig. 2B. The K128 unit consists of a β-D-Glcp(1 → 6)-D-GalpNAc disaccharide side chain β-(1 → 6)-linked to a D-Galp residue in a trisaccharide main chain. Given that only one D-GalpNAc residue is present in the main chain, this is the first sugar of the K unit due to the known function of the ItrA2 initiating transferase encoded by KL128.
monosaccharides, D-Glcp, D-Galp and D-GlcpNAc (Fig. 2A and B). The linkages predicted to be formed by Gtr75, Gtr25, and Gtr5 are all present in the K128 unit, and the Gtrs were assigned to these linkages accordingly (Fig. 2B). As the first sugar of the K128 unit is known, the linkage between units in the K128 CPS chain is unambiguous. This linkage, β-D-GalpNAc-(1 → 4)-D-Galp, would therefore be formed by WzyK128. Consistent with this assignment, WzyK128 shares 54% identity with WzyK27 from A. baumannii KL27 (GenBank accession number KT266827.1), which is known to link K units in the K27 CPS via the same β-D-GalpNAc-(1 → 4)-D-Galp linkage [11]. The remaining linkage, β-D-GalpNAc-(1 → 6)-D-Galp, that joins the disaccharide side-chain to the main chain, would therefore be formed by the Gtr200K128 glycosyltransferase. Gtr200K128 shares 33% identity with Gtr75, which forms the similar β-D-Glcp-(1 → 6)-D-GalpNAc linkage in K116 and K128. 3. Discussion The K128 CPS joins a growing number of A. baumannii CPS structures made up of only neutral sugars [10,15,17,19,22]. The α-D-Galp(1 → 6)-β-D-Galp-(1 → 3)-D-GalpNAc trisaccharide formed by Gtr5 and Gtr25 coupled with ItrA2 is found in several of these structures, including K128, K116, K14 [19] and K93 [20], and the corresponding gene clusters share the gtr25/gtr5/itrA2 sequence segment. This suggests that the gtr25/gtr5/itrA2 module may be maintained together or transferred as a gene block in the assembly of new KL gene clusters via recombination, conserving the availability of specific substrates for this set of transferases. The coupling of Gtrs for the construction of specific polysaccharides has also been observed in other species [23,24]. Determination of CPS structures of A. baumannii gene cluster pairs have enabled unambiguous identification of the specific linkages formed by the Wzy polymerases (e.g. Refs. [11–13]). This approach has proven invaluable for characterising Wzy proteins in the species, as Wzy polymerases are notoriously difficult to classify due to the enormous amount of amino acid sequence diversity observed in nature.
2.3. Assignment of glycosyltransferases and the Wzy polymerase The K128 and K116 units both consist of a trisaccharide mainchain with a disaccharide sidechain and include only simple, neutral 3
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Fig. 4. Parts of a 2D 1H,13C HSQC spectrum of the K128 CPS of A. baumannii KZ-1093. The corresponding parts of the 1H and 13C NMR spectra are shown along the horizontal and vertical axes, respectively. Numbers refer to H/C pairs in sugar residues denoted by letters as indicated in Table 1.
different clonal lineages [10], suggesting a more widespread distribution.
Table 1 1 H and13C NMR chemical shifts (δ, ppm) of the K128 CPS of A. baumannii KZ1093. Sugar residue
C-1 H-1
C-2 H-2
C-3 H-3
C-4 H-4
C-5 H-5
C-6 H-6 (6a, 6b)
→3)-β-D-GalpNAc-(1→ A →6)-β-D-Galp-(1→ B →4,6)-α-D-Galp-(1→ C →6)-β-D-GalpNAc-(1→ D β-D-Glcp-(1→ E
103.5 4.69 106.2 4.49 100.1 4.94 103.9 4.49 104.3 4.50
53.8 3.99 72.3 3.55 69.5 3.72 53.2 3.92 74.7 3.32
81.5 3.87 74.1 3.64 71.2 3.90 75.1 3.86 77.4 3.51
69.4 4.15 70.1 3.98 78.5 4.09 70.1 3.99 71.3 3.41
76.1 3.63 73.8 3.83 71.3 4.00 73.8 3.72 77.5 3.47
62.7 3.78, 67.8 3.70, 72.2 3.82, 70.4 3.93, 62.5 3.75,
4. Materials and methods 4.1. Bacterial strain, cultivation, and isolation of CPS A. baumannii clinical isolate KZ-1093 was obtained from a bronchoalveolar lavage specimen of a 2-year-old male patient with nosocomial pneumonia in Nur-Sultan (Astana), Kazakhstan, in August 2016, and was deposited in the collection of the Institute of Antimicrobial Chemotherapy, Smolensk State Medical University (Smolensk, Russia). Bacteria were cultivated in 2 × TY media overnight; cells were harvested by centrifugation (10,000×g, 15 min), and suspended in phosphate-buffered saline. The suspension was cooled down to 4 °C, 0.2 volume of CCl3CO2H was added, cells were precipitated by centrifugation (15,000×g, 20 min), and two volumes of acetone were added to the supernatant. After intense shaking, the precipitate (CPS) was separated by centrifugation (8000×g, 20 min), dissolved in water, the pH value was adjusted to pH 8 by adding 1 M NaOH, the CPS was precipitated with acetone and separated by centrifugation as above, dissolved in distilled water and applied to a column (53 × 3.5 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden). Elution was performed with 0.1% HOAc and monitored using a UV-detector (Uvicord, Sweden) at 206 nm to give a purified CPS sample (21 mg).
3.78 3.84 4.07 4.07 3.92
1
H NMR chemical shifts are italicized. Chemical shifts for the N-acetyl groups are δH 2.03–2.07, δC 23.7–24.1 (CH3) and 175.7–176.3 (CO).
We have successfully used the correlation of CPS structural data with KL sequence to establish the specific linkage formed by more than 35 different Wzy proteins in A. baumannii, and here we used the same approach to demonstrate that WzyK128 forms the β-D-GalpNAc-(1 → 4)D-Galp linkage in the K128 CPS. The KL128 gene cluster appears to be sporadic, found to date only in the genomes of KZ-1093 and one further A. baumannii isolate, 4300STDY7045753 (WGS accession number UFKI01000022.1), available in the NCBI non-redundant and WGS databases that include more than 3000 sequences. In comparison, the related KL116 gene cluster has previously been identified in a wide range of A. baumannii isolates from
4.2. Chemical analyses A CPS sample (1 mg) was hydrolyzed with 3 M CF3CO2H (120 °C, 2 h). Monosaccharides were analyzed using a Biotronik LC-200 sugar analyzer. Neutral sugars were identified on a column (15 × 0.4 cm) of 4
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Dionex A × 8 anion-exchange resin in 0.5 M sodium borate buffer pH 8 at 70 °C. Amino sugars were determined on a column (22 × 0.4 cm) of Ostion LC AN B cation-exchange resin in 0.2 M borate buffer pH 5 at 70 °C.
[7] [8]
4.3. NMR spectroscopy [9]
A CPS sample was deuterium-exchanged by freeze-drying from 99.9% D2O and then examined as a solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 60 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (δH 0, δC –1.6) was used as internal reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A 60-ms MLEV-17 spin-lock time and a 150-ms mixing time were used in 1 H,1H TOCSY and ROESY experiments, respectively.
[10]
[11]
[12]
4.4. Sequencing and bioinformatic analysis The draft genome sequence of A. baumannii KZ-1093 was obtained by MiSeq sequencing using a Nextera DNA library preparation kit (Illumina, San Diego, CA), and assembly of reads using SPAdes v. 3.10 [25]. The capsule biosynthesis gene cluster was located in the chromosome, and the genes were annotated and characterised as described previously [7]. The sequence was deposited into GenBank under accession number MK399428.1.
[13]
[14]
Funding [15]
Identification, cultivation, and genome sequencing of the bacterial strain KZ-1093 was supported by the Russian Science Foundation (project No. 18-15-00403). Monosaccharide analysis and CPS structure determination was supported by the Russian Science Foundation (project No. 19-14-00273). Bioinformatics was supported by an Australian Research Council (ARC) DECRA Fellowship 180101563 to JJK.
[16]
[17]
Acknowledgements The authors thank Sarah Cahill (Queensland University of Technology, Australia) for bioinformatics assistance.
[18]
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