Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus

Accepted Manuscript Note Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus Alexander S. Shashkov, Mikhail M. Shneider, Sof’ya N. Senchenkova, Anastasiya V. Popova, Anastasia S. Nikitina, Vladislav V. Babenko, Elena S. Kostryukova, Konstantin A. Miroshnikov, Nikolay V. Volozhantsev, Yuriy A. Knirel PII: DOI: Reference:

S0008-6215(14)00429-7 http://dx.doi.org/10.1016/j.carres.2014.11.013 CAR 6895

To appear in:

Carbohydrate Research

Received Date: Revised Date: Accepted Date:

14 September 2014 12 November 2014 17 November 2014

Please cite this article as: Shashkov, A.S., Shneider, M.M., Senchenkova, S.N., Popova, A.V., Nikitina, A.S., Babenko, V.V., Kostryukova, E.S., Miroshnikov, K.A., Volozhantsev, N.V., Knirel, Y.A., Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.11.013

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Note

Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus Alexander S. Shashkov,a Mikhail M. Shneider,b Sof’ya N. Senchenkova,a Anastasiya V. Popova,c Anastasia S. Nikitina,d,e Vladislav V. Babenko,d Elena S. Kostryukova,d,f Konstantin A. Miroshnikov,b Nikolay V. Volozhantsev,c Yuriy A. Knirela,* a

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

b

M. M. Shemyakin & Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of

Sciences, Moscow, Russia c

State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region,

Russia d

Research Institute of Physical−Chemical Medicine, Moscow, Russia

e

Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia

f

Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University,

Kazan, Russia Abstract − Acinetobacter baumannii 1053 is the type strain for the maintenance of specific bacteriophage AP22, which infects a fairly broad range of A. baumannii strains circulating in Russian clinics and hospitals. A capsular polysaccharide (CPS) was isolated from cells of strain 1053 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established : →4)-β-D-ManpNAcA-(1→4)-β-D-ManpNAcA-(1→3)-α-D-FucpNAc-(1→ where ManNAcA and FucNAc indicate 2-acetamido-2-deoxymannuronic acid and 2-acetamido2,6-dideoxygalactose, respectively. A polysaccharide having the same repeating unit but a shorter chain was isolated by the phenol-water extraction of bacterial cells. Sequencing of the CPS biosynthesis gene locus showed that A. baumannii 1053 belongs to a new group designated KL91. The gene functions assigned putatively by a comparison with available databases were in agreement with the CPS structure established. *

Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.

E-mail address: [email protected] (Yuriy A. Knirel).

Keywords: Acinetobacter baumannii; Capsular polysaccharide structure; 2-Acetamido-2-deoxyD-mannuronic

acid; 2-Acetamido-2,6-dideoxy-D-galactose; Polysaccharide biosynthesis gene

locus

Recently, A. baumannii has become one of the most widespread agents causing health-care associated infections. Treatment of these infections is complicated owing to the ability of the bacteria to acquire and to accumulate various antibiotic resistance mechanisms.1 A. baumannii lacks a long-chain lipopolysaccharide with an O-polysaccharide chain2 but has a capsular polysaccharide (CPS) that forms a thick layer around the bacterial cell. The CPS protects A. baumannii from the action of immune system components and is considered as an important virulence factor.3 A number of A. baumannii CPS structures have been established to date (some under the wrong name of O-antigen or O-specific polysaccharide) (refs.4-9 and refs. cited in ref.4) but more remain to be elucidated. Bacteriophages that are able to infect A. baumannii represent a promising alternative to antibiotics to control this pathogen. From 130 A. baumannii isolates collected by us from clinics and hospitals in Russia in 2005-2010, 89 isolates (~68%) were sensitive to Myoviridae bacteriophage AP22,10 making it suitable for development of anti-acinetobacter preparations. To infect the host bacteriophages have to disrupt the CPS layer, and CPS is the primary receptor for the phage AP22 structural depolymerase/adsorption protein (data will be published elsewhere). In this work, we studied structure and genetics of the CPS of A. baumannii 1053, which is the type strain for the phage AP22 maintenance. CPS was isolated from cells of A. baumannii 1053 by extraction with phosphate-buffered saline containing EDTA. Sugar analysis of CPS by GLC of the acetylated alditols revealed 2amino-2,6-dideoxygalactose (fucosamine, FucN). GLC analysis of the acetylated (S)-2-octyl glycosides showed that FucN has the D configuration. The 13C NMR spectrum of the CPS (Fig. 1) showed signals for three anomeric carbons at δ 98.9-100.9, CH3−C groups of one 6-deoxyhexose (C-6 of FucN) at δ 16.6 and three N-acetyl groups at δ 23.2-23.3, three nitrogen-bearing carbons at δ 48.8-54.7, other sugar-ring carbons at δ 68.1-79.1, and CO groups at δ 175.2-176.6 (Table 1). The 1H NMR spectrum contained inter alia signals for three anomeric protons at δ 4.75-4.89, CH3−C groups of one 6-deoxyhexose (H6 of FucN) at δ 1.17 and three N-acetyl groups at δ 1.99-2.08. The NMR spectra were assigned using a set of shift-correlated 2D NMR experiments, and spin-systems for three sugar residues were revealed, which were designated units A, B, and C (Table 1). Unit C was identified as FucNAc as its spin system included the CH3 group (H-6). 2

Units A and B were identified as manno-configurated monosaccharides by relatively small J1,2 and J2,3 and relatively large J3,4 and J4,5 coupling constants estimated from the 2D NMR spectra. Correlations of protons at the nitrogen-linked carbons (H-2) with the corresponding carbons (C2) at δ 48.8-54.7 in the 1 H,13C HSQC spectrum confirmed FucNAc and demonstrated that units A and B also are 2-amino sugars. As there were no signals for HOCH2-C groups but for CO groups, it was suggested that units A and B are hexuronic acid derivatives. These data together indicated that the repeating unit of the CPS consists of one FucNAc residue (C) and two residues of 2-acetamido-2-deoxymannuronic acid (ManNAcA) (A and B). In the ROESY spectrum, H-1 of unit C showed a strong intraresidue correlation with H-2 and, hence, FucNAc is α-linked. Correlations between H-1 and H-5 indicated that units A and B are β-linked. Interresidue correlations between anomeric protons and protons at the linkage carbons, including A H-1/B H-4, B H-1/C H-3, and C H-1/A H-4, defined the sequence and linkage pattern of the monosaccharides. The sites of glycosylation were confirmed by significant low-field displacements of the 13C NMR signals for the linkage carbons, namely, C-3 of unit C and C-4 of units A and B, as compared with their positions in the non-substituted FucNAc and ManNAc.11 Analysis of the displacements of the signals for the linkage and neighboring carbons using known regularities12 showed that all constituent monosaccharides had the same absolute configuration, and, hence, ManNAcA had the D configuration. Therefore, CPS is built up of trisaccharide repeating units having the structure shown in Chart 1. To the best of our knowledge, the structure established is unique among known bacterial polysaccharide structures. Another polysaccharide (PS) was obtained by phenol-water extraction of cells of A. baumannii 1053 followed by cleavage of the accompanying lipopolysaccharide by mild acid hydrolysis of the isolated material and fractionation of the water-soluble carbohydrate portion by GPC on Sephadex G-50. Studies of the PS by NMR spectroscopy as described above for the CPS showed that it consisted of the same oligosaccharide repeats but had a shorter chain (Chart 1). As a result, signals for the terminal non-reducing monosaccharide were clearly seen in the NMR spectra and were assigned to the terminal unit A (ManNAcA) designated A* (Table 1). With unit A being the last, unit C (FucNAc) is the first monosaccharide of the repeating unit, whose transfer to a lipid carrier initiate the polysaccharide biosynthesis (see below). As judged by the ~3:1 ratio of the signals for the terminal and interior oligosaccharide units, the PS consists on the average of four trisaccharide repeats. The genome of A. baumannii 1053 was sequenced and the complete sequence of the CPS gene locus (26616 bp) between the conserved fkpA and lldP genes was obtained (Fig. 2), which was submitted to GenBank and received accession number KM402814. The 1053 CPS sequence 3

was essentially identical to that of A. baumannii ANC 4097 isolated in Czech Republic (full genome sequence GenBank accession number APRF00000000), except that the former lacked gne1 gene for UDP-D-glucose/UDP-N-acetyl-D-glucosamine 4-epimerase located between gpi and pgm genes in the latter. Therefore, it is suggested to classify strain 1053 into a new molecular group designated KL91, which is close to the KL40 group that has been proposed for strain ANC 4097 (Kenyon J. J. & Hall, R. M., personal communication). As in other A. baumannii strains studied,2,13 the KL91 CPS gene locus contained the wza, wzb, and wzc genes for transport of the CPS from the periplasm to the cell surface. There were also sugar pathway genes that are always present in the A. baumannii gene loci, including gna gene located near wza and a set of genes (galU, ugd, gpi, and pgm) at the other side of the locus. galU, ugd, gpi, and pgm that are not usually found in polysaccharide gene loci have functions both in central metabolism and synthesis of CPS components of A. baumannii.13 The remaining genes between gna and galU were evidently involved with the synthesis of the KL91 group CPS, and their annotations shown in Fig. 2 matched with the CPS structure of strain 1053. There were homologues of mnaA and mnaB that encode 2-epimerase for synthesis of the D-ManNAc precursor from UDP-D-GlcpNAc and dehydrogenase that converts UDP-DManpNAc to UDP-D-ManpNAcA, respectively. These genes were 93%-94% identical at the amino acid level to mnaA and mnaB of A. baumannii AB5075 (full genome sequence GenBank accession number AHAH00000000), which had been classified into the KL25 group (Kenyon J. J. & Hall, R. M., personal communication) and whose CPS also contained ManNAcA (authors' unpublished data). In addition, there were two putative genes for synthesis of D-FucNAc, including gdr for UDP-D-GlcpNAc 4,6-dehydratase and fnr for NAD-dependent 4-reductase. gdr of strain 1053 was 98% identical to the corresponding gene of the 2,4-diamino-2,4,6-dideoxy-Dglucose synthesis pathway in A. baumannii AB5075. fnr had a high level homology (64-100% identity) to many polysaccharide biosynthesis family proteins of A. baumannii and is 50% identical to wbjF, which encodes putative UDP-6-deoxy-4-keto-D-GlcNAc 4-reductase involved in the D-FucNAc biosynthesis pathway in Pseudomonas aeruginosa PA103 (O11).14 Just downstream of fnr, the itrB1 gene was located, which had been predicted to encode initiating transferase to add the first sugar to the lipid carrier. ItrB1 of A. baumannii 1053 shared 60-100% identity to a number of glycosyl transferase 4 family proteins of A. baumannii and 51% identity to the O-antigen initiating glycosyl transferase wbpL of P. aeruginosa PA103 (O11),14 whose substrate is D-FucNAc. That D-FucNAc was the first monosaccharide of the A. baumannii 1053 repeating unit too was confirmed by elucidation of the PS structure (see above). The presence of two glycosyltransferase genes (gtr85 and gtr86), as well as the flippase wzx and polymerase wzy genes, also was appropriate to the trisaccharide repeating unit of the 4

1053 CPS. No function for the atr3 gene predicted to encode an acyltransferase could be assigned based on the CPS structure. Therefore, except for atr3, the genes in the CPS biosynthesis locus of A. baumannii 1053 were appropriate to the CPS structure established. As the phage AP22 depolymerase is highly specific to A. baumannii CPS, all AP22 sensitive strains must have the same CPS type as strain 1053, and this type is prevalent among Russian clinical isolates. 1. Experimental 1.1. Bacterial strain and cultivation A. baumannii 1053 was isolated from a burn patient in Chelyabinsk, Russia, in 2008. Bacteria were cultivated in 2TY media overnight; cells were harvested by centrifugation (10,000×g, 20 min), washed with phosphate-buffered saline, suspended in aq 70 % acetone, precipitated, and dried. 1.2. Isolation of CPS and PS Freshly harvested bacterial cells were suspended in phosphate-buffered saline containing 10 mM EDTA and stored at 37 ºC for 30 min. Cells were removed by centrifugation (10,000×g, 20 min), and CPS was precipitated from the supernatant by adding acetone to the final concentration 70%. PS was isolated by phenol-water extraction15 of bacterial mass, the extract was dialyzed without layer separation and freed from insoluble contaminations by centrifugation. The resultant solution was treated with cold aq 50 % CCl3CO2 H at 4 °C; after centrifugation the supernatant was dialyzed against distilled water and freeze-dried. To cleave the accompanying O-antigen-lacking lipopolysaccharide,2 the crude PS preparation (85 mg) was heated with 2% HOAc (100 °C, 3 h), the lipid precipitate was removed by centrifugation, and the supernatant was fractionated by GPC on a column (56 × 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 4.5 monitored using a differential refractometer (Knauer, Germany) to give a purified PS sample (33 mg). 1.3. Sugar analysis A CPS sample (1 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were analyzed by GLC of the alditol acetates on a Maestro (Agilent 7820) chromatograph (Interlab, Russia) equipped with an HP-5 column (0.32 mm × 30 m) using a temperature program of 160 °C (1 min) to 290 °C at 7 °C min-1. The absolute configuration of FucN was determined by GLC of the acetylated (S)-2-octyl glycosides as described.16 5

1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9 % D2O and then examined as solution in 99.95 % D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 20 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (δH 0) and acetone (δC 31.45) were used as internal references 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. Mixing times of 100 and 150 ms were used in TOCSY and ROESY experiments, respectively.

1.5. Genome analysis The genome of strain 1053 was sequenced by 454 pyrosequencing technology with 19fold coverage and median length of 910 bp using Roche 454 Life Science Genome Sequencer. FLX+. Library was constructed using Rapid Library Preparation Kit and Rapid Library MID Adaptors Kit. Emulsion PCR and sequencing were performed using GS FLX Titanium LV emPCR Kit (Lib-L), GS FLX Titanium emPCR Breaking Kits LV/MV, GS FLX Titanium Sequencing Kit XL+ and GS FLX Titanium PicoTiterPlate Kit 70 x 75. All procedures were carried out according to the protocols of the manufacturer (Roche 454 Life Science, USA). The raw reads dataset was assembled with Newbler version 2.9, resulting in 127 contigs (>2000 bp in size) and estimated genome size of 4.1 MB. The CPS gene locus was found within contigs 31 (42296 bp) and 50 (28413 bp). In order to fill the gap between the contigs and to acquire the full CPS gene locus sequence specific primers were designed and the corresponding amplicons were generated. The sequences of amplicons were obtained by the Sanger dideoxy sequencing method using Big DyeTM Terminator v.3.1 Cycle Sequencing Kit and 3ABI Prism Genetic Analyzer 3730XL following the manufacturer’s instructions (Applied Biosystem, USA). Further assembly was conducted with Vector NTI 9.0 software.

Acknowledgements This work was supported by the Russian Foundation for Basic Research (project No. 14-04-00657).

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References 1.

Roca, I.; Espinal, P.; Vila-Farrés, X.; Vila, J. Front. Microbiol. 2012, 3, Article 148.

2.

Kenyon, J. J.; Hall, R. M. PLoS ONE 2013, 8, e62160.

3.

Russo, T. A.; Luke, N. R.; Beanan, J. M.; Olson, R.; Sauberan, S. L.; MacDonald, U.; Schultz, L. W.; Umland, T. C.; Campagnari, A. A. Infect. Immun. 2010, 78, 3993-4000.

4.

Knirel, Y. A. In: Bacterial lipopolysaccharides − Structure, chemical synthesis, biogenesis and interaction with host cells. Knirel, Y. A.; Valvano, M. A., Eds.; Springer: Wien–New York, 2011; pp. 42–108.

5.

Senchenkova, S. N.; Shashkov, A. S.; Shneider, M. M.; Arbatsky, N. P.; Popova, A. V.; Miroshnikov, K. A.; Volozhantsev, N. V.; Knirel, Y. A. Carbohydr. Res. 2014, 391, 89-92.

6.

Kenyon, J. J.; Marzaioli, A.; M. Hall, R. M.; De Castro, C. Glycobiology 2014, 24, 554-563.

7.

Vinogradov, E.; MacLean, L.; Xu, H. H.; Chen, W. Carbohydr. Res. 2014, 390, 42-45.

8.

Russo, T. A.; Beanan, J. M.; Olson, R.; Macdonald, U.; Cox, A. D.; St Michael, F.; Vinogradov, E. V.; Spellberg, B.; Luke-Marshall, N. R.; Campagnari, A. A. Infect. Immun. 2013, 81, 915-922.

9.

Fregolino, E.; Gargiulo, V.; Lanzetta, R.; Parrilli, M.; Holst, O.; De Castro, C. Carbohydr. Res. 2011, 346, 973-977.

10. Popova, A. V.; Zhilenkov, E. L.; Myakinina, V. P.; Krasilnikova, V. M.; Volozhantsev, N. V. FEMS Microbiol. Lett. 2012, 332, 40-46. 11. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59-75. 12. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26, 735-747. 13. Hu, D.; Liu, B.; Dijkshoorn, L.; Wang, L.; Reeves, P. R. PLoS ONE 2013, 8, e70329. 14. King, J. D.; Kocincova, D.; Westman, E. L.; Lam, J. S. Innate Immun. 2009, 15, 261-312. 15. Westphal, O.; Jann, K. Methods Carbohydr Chem. 1965, 5, 83-91. 16. Leontein, K.; Lönngren, J. Methods Carbohydr Chem. 1993, 9, 87-89.

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Table 1. 1H and 13C NMR chemical shifts of CPS (δ, ppm) Residue

β-D-ManpNAcA-(1→

→4)-β-D-ManpNAcA-(1→

→4)-β-D-ManpNAcA-(1→

→3)-α-D-FucpNAc-(1→

1

A*

A

B

C

C-1

C-2

C-3

C-4

C-5

C-6

H-1

H-2

H-3

H-4

H-5

H-6

100.1

54.4

74.4

70.1

77.6

175.5

4.75

4.44

3.79

3.59

3.80

100.1

54.7

73.8

74.8

77.4

4.75

4.39

3.99

3.77

3.86

100.9

53.9

71.5

78.9

76.4

4.89

4.43

3.87

3.76

3.83

98.9

48.8

79.1

71.3

68.1

16.6

5.20

4.20

3.93

3.96

4.02

1.17

175.2

175.2

H NMR chemical shifts are italicized. A* indicates the terminal non-reducing residue in the PS.

Chemical shifts for N-acetyl groups are δH 1.99-2.08, δC 23.2-23.3 (CH3) and 175.5-176.6 (CO).

8

Legends to Figures

Chart 1. Structures of the CPS and PS of A. baumannii 1053. In the PS, on the average n = 3. Figure 1. 13C NMR spectrum of the CPS of A. baumannii 1053. Signal of acetone is marked by asterisk. Figure 2. Organization of the CPS biosynthesis gene locus of A. baumannii 1053 (GenBank accession number KM402814). Annotations of the specific genes for synthesis of the KL91 group CPS located between gna and galU were made according to the proposed system.2

9

10

11

Graphical abstract Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis locus Alexander S. Shashkov, Mikhail M. Shneider, Sofya N. Senchenkova, Anastasiya V. Popova, Anastasia S. Nikitina, Vladislav V. Babenko, Elena S. Kostryukova, Konstantin A. Miroshnikov, Nikolay V. Volozhantsev, Yuriy A. Knirel CO2H

O AcNH

O HO

CO2H O HO

HO CH 3 O

O

AcNH

O AcNH

→4)-β-D-ManpNAcA-(1→4)-β-D-ManpNAcA-(1→3)-α-D-FucpNAc-(1→

12


Capsular polysaccharide was isolated from Acinetobacter baumannii 1053
Structure of the polysaccharide was established by NMR and chemical approaches
Capsule biosynthesis locus was analyzed and found to match the structure established

13