- Email: [email protected]
Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid
Accepted Manuscript Note Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid Sof’ya N. Senchenkova, Alexander S. Shashkov, Mikhail M. Shneider, Nikolay P. Arbatsky, Anastasiya V. Popova, Konstantin A. Miroshnikov, Nikolay V. Volozhantsev, Yuriy A. Knirel PII: DOI: Reference:
S0008-6215(14)00128-1 http://dx.doi.org/10.1016/j.carres.2014.04.002 CAR 6719
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
11 February 2014 26 March 2014 1 April 2014
Please cite this article as: Senchenkova, S.N., Shashkov, A.S., Shneider, M.M., Arbatsky, N.P., Popova, A.V., Miroshnikov, K.A., Volozhantsev, N.V., Knirel, Y.A., Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid, Carbohydrate Research (2014), doi: http://dx.doi.org/ 10.1016/j.carres.2014.04.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Note
Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid Sof’ya N. Senchenkova,a Alexander S. Shashkov,a Mikhail M. Shneider,b Nikolay P. Arbatsky,a Anastasiya V. Popova,с Konstantin A. Miroshnikov,b Nikolay V. Volozhantsev,с 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 с
State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region,
Russia
Abstract Capsular polysaccharide was isolated by the phenol-water extraction of Acinetobacter baumannii ACICU cells and studied by sugar analysis, partial acid hydrolysis, 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide was found to contain 5,7-diacetamido-3,5,7,9-tetradeoxy-Lglycero-L-manno-non-2-ulosonic or di-N-acetylpseudaminic acid (Pse5Ac7Ac), and the following structure of the branched tetrasaccharide repeating unit was established:
The genes present in the polysaccharide gene cluster of A. baumannii ACICU are appropriate to the structure established.
Keywords: Acinetobacter baumannii; Capsular polysaccharide structure; Pseudaminic acid; Polysaccharide gene cluster
*
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: [email protected] (Yuriy A. Knirel).
Gram-negative bacteria Acinetobacter baumannii are an opportunistic human pathogen affecting immunocompromised people. They are specific to the hospital environment and most commonly encountered in intensive care units. Treatment of A. baumannii infections is complicated by increasing multidrug resistance, and panresistant strains are known. Capsular polysaccharide (CPS) is one of the major virulence factors of A. baumannii1 important for growth and survival of bacteria in the human body environment. CPS structures are highly variable in this species, and a number of them have been determined.2 Recently, two attempts have been made to genotype A. baumannii based on the nucleotide sequences in the CPS biosynthesis genomic locus, one resulting in recognition of 9 types3 and the other being more comprehensive.4 A. baumannii strain ACICU belongs to the highly virulent European epidemic clone II group.5 The ACICU-specific CPS locus type designated Kl23 or PSgc124 is widely distributed in A. baumannii populations.4,6 Our analysis (data not shown) has revealed that this type of CPS is inherent to more than 10% strains with NCBI-deposited genomes. In this work, aiming at integration of the structural and genetic data into a comprehensive classification system of A. baumannii, we established the CPS structure of strain ACICU and found it to be in accord with putative functions of the genes in the CPS locus. A polysaccharide (PS-1) was isolated by the phenol-water extraction of cells of A. baumannii ACICU and purified by GPC. Sugar analysis by GLC of the acetylated alditols and (S)-2-octyl glycosides revealed D-Glc, D-Gal, and D-GalNAc in the ratios ~1:1:0.4 (detector response). Mild acid hydrolysis of PS-1 yielded di-N-acetylpseudaminic acid (Pse5Ac7Ac) (Chart 1) and a modified polysaccharide (PS-2). The 1H and 13C NMR chemical shifts and 3JH,H coupling constants of the isolated Pse5Ac7Ac were identical to the published data.7 Both polysaccharides obtained were studied by 2D NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC and HMBC experiments. As judged by the number of signals in the spectra, including those for anomeric protons and carbons, PS-1 and PS-2 are built up of tetra- and tri-saccharide repeating units, respectively. The 1H and 13C NMR spectra were assigned (Fig. 1, Table 1), and spin systems for one residues each of Glc, Gal, GalNAc (all in both polysaccharides), and Pse5Ac7Ac (in PS-1) were identified. As judged by 3JH,H coupling constants compared with published data,7,8 all monosaccharides were in the pyranosidic form and, except for Pse5Ac7Ac, β-linked (J1,2 7-8 Hz.). Structure of PS-2 was determined by a 1H,13C HMBC experiments, which showed Gal H-1/GalNAc C-3, GalNAc H-1/Gal C-3, and Glc H-1/Gal C-6 cross-peaks at δ 4.46/80.8, 4.68/82.7, and 4.49/70.8, respectively. Positions of substitution of the monosaccharides were confirmed by downfield displacements of the NMR signals for linkage carbons (Table 1), 2
namely C-3 of GalNAc and C-3 and C-6 of Gal, as compared with their positions in the corresponding non-substituted monosaccharides.9 Therefore, PS-2 has a branched structure with a side-chain β-Glcp attached at position 6 of β-Galp at the branching point (Chart 1). The NMR data (Table 1) showed that PS-1 differed from PS-2 in the presence of Pse5Ac7Ac only. The 1H,13C HMBC spectrum of PS-1 showed a cross peak between C-2 of Pse5Ac7Ac and H-6b of Glc at δ 101.3/3.70, and, hence, Pse5Ac7Ac was attached at position 6 of Glc in the side chain. This conclusion was confirmed by a comparison of 13C NMR chemical shifts of PS-2 and PS-1, which revealed displacements of the signals for C-6 and C-5 of Glc from δ 62.0 and 77.3 in PS-2 to δ 63.8 and 75.9 in PS-1, respectively, (positive α-effect and negative β-effect of 6-glycosylation) with no significant shifts of the other signals. The position of the signal for C-6 of Pse5Ac7Ac at δ 72.0 and a relatively small difference (0.52 ppm) between the H-3ax and H-3eq chemical shifts compared with published data7 indicated the α configuration of this monosaccharide. Therefore, based on the data obtained, it was concluded that the CPS of A. baumannii ACICU has the structure shown in Chart 1. A peculiar feature of the polysaccharide is the presence of a higher monosaccharide, di-N-acetylpseudaminic acid. Various N-acyl derivatives of pseudaminic acid, including Pse5Ac7Ac, have been reported as components of a number of bacterial polysaccharides7,10,11 but this is the first time that pseudaminic acid is found in A. baumannii. The CPS biosynthesis locus of A. baumannii ACICU located between the fkpA and lldP genes has been sequenced 3,4 and classified into the PSgc124 or KL2 3 group. The genes that have been annotated based on similarities to genes from available databases3,4 were found to be appropriate to the structure established. Particularly, the locus includes a set of six psa genes for synthesis of nucleotide-activated pseudaminic acid (CMP-Pse5Ac7Ac) (Fig. 2). They are organized in the same manner as the psb genes of Shigella boydii type 7,3,12 whose O-antigen contains another Pse derivative, 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Ac7Hb).13 Most Psa proteins share 57 % to 83 % sequence identity with the corresponding Psb proteins but PsaD and PsaE are only 38 % and 31 % identical to Psb4 and Psb5, respectively. In the predicted CMP-Pse5Ac7Hb biosynthetic pathway,12 PsaE mediates 4-Nacylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-β-L-altrose, and PsaD then cleaves the UDP moiety from the reaction product. A lower homology observed for these enzymes is evidently accounted for by the substrate difference between Psb5 and PsaE, which transfer acetyl or 3-hydroxybutanoyl group, respectively, that in turn makes different the substrates of Psb4 and PsaD, which function downstream in the pathway.
3
Downstream of the CMP-Pse5Ac7Ac synthesis genes, there are the wzx and wzy genes for processing and three glycosyl transferase genes (gtr3-gtr5) for assembly of the tetrasaccharide repeating unit of the CPS. Between wzx and wzy, there is a homologue of the kpsS gene that is required for synthesis/transfer of lysophosphatidylglycerol-poly-(3-deoxy-Dmanno-oct-2-ulosonic acid), the reducing termini of CPSs of Escherichia coli and Neisseria meningitidis.14 The role of kpsS in the CPS locus of A. baumannii ACICU remains unknown. Finally, the presence of a homologue of the itrA2 gene for initiating transferase of the A2 type, whose predicted substrate is UDP-D-GalpNAc,3 fits with the presence of D-GalNAc in the CPS of A. baumannii ACICU and indicates that this monosaccharide is first in the repeating unit. 1. Experimental 1.1. Cultivation of bacteria A. baumannii strain ACICU, a representative of a clone that caused an outbreak in an intensive care unit in Rome in 2005,15 was kindly provided by Dr. R. Zarilli (University of Naples Federico II, Naples, Italy). Bacteria were cultivated in 2TY media overnight; cells were harvested by centrifugation (10,000g, 20 min), washed with distilled water, suspended in aq 70 % acetone, precipitated and dried. 1.2. Isolation of CPS Bacterial cells (dry weight 0.9 g) were extracted with 45% aq phenol as described,16 the mixture was dialyzed, cells were removed by centrifugation, the supernatant was treated with aq 50% CCl3CO2 H at 4 °C to pH 2.5, the supernatant was dialyzed and freeze-dried. The product (130 mg) was dissolved in 25 mM Tris-HCl buffer pH 7.63 containing 2 mM CaCl2 and treated with DNAse and RNAse (24 h) and Proteinase K (48 h) at 20 °C. The solution was dialyzed and freeze-dried to give PS-1 (105 mg). 1.3. Mild acid hydrolysis A CPS sample (55 mg) was hydrolyzed with aq 2 % HOAc (100 °C, 1.5 h), the products were fractionated by GPC on a column of TSK HW-40 (S) to give a modified polysaccharide (PS-2) (25 mg) and Pse5Ac7Ac (9 mg). 1.4. Chemical analyses A PS-1 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 4
temperature program of 160 °C (1 min) to 290 °C at 7 °C min-1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.17 1.5. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9 % D2O and then examined as solutions in 99.95 % D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 45 °C (for the polysaccharides) or 20 °C (for Pse5Ac7Ac). 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 mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively.
Acknowledgement Authors thank Dr. R. Zarilli for providing A. baumannii strain ACICU. This work was supported by the Russian Foundation for Basic Research (project No. 14-04-00657).
5
References 1. 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. 2. 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. 3. Kenyon, J. J.; Hall, R. M. PLoS ONE 2013, 8, e62160. 4. Hu, D.; Liu, B.; Dijkshoorn, L.; Wang, L.; Reeves, P. R. PLoS ONE 2013, 8, e70329. 5. Iacono, M.; Villa, L.; Fortini, D.; Bordoni, R.; Imperi, F.; Bonnal, R. J.; Sicheritz-Ponten, T.; De Bellis, G.; Visca, P.; Cassone, A.; Carattoli, A. Antimicrob. Agents Chemother. 2008, 52, 2616–2625. 6. Wright, M. S.; Haft, D. H.; Harkins, D. M.; Perez, F.; Hujer, K. M.; Bajaksouzian, S.; Benard, M. F.; Jacobs, M. R.; Bonomo, R. A.; Adams, M. D. MBio 2014, 5, e00963-13. 7. Knirel, Y. A.; Shashkov, A. S.; Tsvetkov, Y. E.; Jansson, P.-E.; Zähringer, U. Adv. Carbohydr. Chem. Biochem. 2003, 58, 371-417. 8. Altona, C.; Haasnoot, C. A. G. Org. Magn. Reson. 1980, 13, 417-429. 9. Lipkind, G. M.; Shashkov, A. S.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59-75. 10. Knirel, Y. A.; Shevelev, S. D.; Perepelov, A. V. Mendeleev Commun. 2011, 21, 173-182. 11. Zunk, M.; Kiefel, M. J. RSC Adv. 2014, 4, 3413-3421. 12. Liu, B.; Knirel, Y. A.; Feng, L.; Perepelov, A. V.; Senchenkova, S. N.; Wang, Q.; Reeves, P. R.; Wang, L. FEMS Microbiol. Rev. 2008, 32, 627-653. 13. L'vov, V. L.; Shashkov, A. S.; Dmitriev, B. A. Bioorg. Khim. 1987, 13, 223-233. 14. Willis, L. M.; Stupak, J.; Richards, M. R.; Lowary, T. L.; Li, J.; Whitfield. C. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7868-7873. 15. Iacono, M.; Villa, L.; Fortini, D.; Bordoni, R.; Imperi, F.; Bonnal, R. J.; Sicheritz-Ponten, T.; De Bellis, G.; Visca, P.; Cassone, A.; Carattoli, A. Antimicrob. Agents Chemother. 2008, 52, 2616-2625. 16. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83-91. 17. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87-89.
6
Table 1. 1H and 13C NMR chemical shifts (δ, ppm) Residue
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
H-1
H-2
H-3 (ax, eq) H-4
H-5
H-6 (a, b)
H-7
H-8
H-9
52.7
80.7
69.3
75.9
62.3
4.70
4.03
3.89
4.17
3.67
3.77
105.8
71.2
82.5
69.9
74.4
71.0
4.47
3.59
3.70
4.12
3.84
3.86; 3.97
104.7
74.4
76.8
70.6
75.9
63.8
4.45
3.27
3.45
3.51
3.51
3.54; 3.70
174.7
101.3 36.2
66.0
49.8
72.0
54.8
68.4
17.3
4.24
4.24
3.88
4.18
4.18
1.15
Capsular polysaccharide (PS-1)
→3)-β-GalpNAc-(1→ 104.0 →3,6)-β-Galp-(1→ →6)-β-Glсp-(1 → α-Psep5Ac7Ac-(2→
1.64; 2.16 C-1
C-2
C-3
C-4
C-5
C-6
H-1
H-2
H-3
H-4
H-5
H-6 (a, b)
104.2
52.8
80.8
69.3
76.0
62.4
4.68
4.06
3.92
4.19
3.70
3.78
106.0
71.1
82.7
69.9
74.5
70.8
4.46
3.58
3.74
4.16
3.86
3.88; 4.03
104.3
74.3
77.1
71.3
77.3
62.0
4.49
3.28
3.47
3.38
3.46
3.72; 3.93
Modified polysaccharide (PS-2) →3)-β-GalpNAc-(1→
→3,6)-β-Galp-(1→
β-Glсp-(1→
1
H NMR chemical shifts are shown in italics. Chemical shifts for NAc are δH 1.96-2.04, δC 23.2-
23.6 (CH3) and 174.9-176.3 (CO).
7
Fig. 1. 13C NMR spectrum of the CPS of A. baumannii ACICU (PS-1). Signals for Gal and GalNAc that build up the main chain, are significantly less intense than those of Glc and Pse5Ac7Ac, which are located in the side chain (for the PS-1 structure see Chart 1). Abbreviations: G, Glc; Ga, Gal; Gn, GalN; P, Pse.
Fig. 2. Organization of the CPS biosynthesis locus of A. baumannii ACICU. Specific genes for synthesis of the KL2 (PSgc12) group CPS are located between the conserved gna and galU genes. Adopted from ref. 3.
Chart 1. Structures of the CPS of A. baumannii ACICU (PS-1), the modified Pse5Ac7Aclacking polysaccharide (PS-2), and the monosaccharide Pse5Ac7Ac released by mild acid hydrolysis of PS-1.
Legends to Figures Fig. 1. 13C NMR spectrum of the CPS of A. baumannii ACICU (PS-1). Signals for Gal and GalNAc that build up the main chain, are significantly less intense than those of Glc and Pse5Ac7Ac, which are located in the side chain (for the PS-1 structure see Chart 1). Abbreviations: G, Glc; Ga, Gal; Gn, GalN; P, Pse.
Fig. 2. Organization of the CPS biosynthesis locus of A. baumannii ACICU. Specific genes for synthesis of the KL2 (PSgc12) group CPS are located between the conserved gna and galU genes. Adopted from ref. 3.
Chart 1. Structures of the CPS of A. baumannii ACICU (PS-1), the modified Pse5Ac7Aclacking polysaccharide (PS-2), and the monosaccharide Pse5Ac7Ac released by mild acid hydrolysis of PS-1.
8
Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-Nacetylpseudaminic acid Sofya N. Senchenkova, Alexander S. Shashkov, Mikhail M. Shneider, Nikolay P. Arbatsky, Anastasiya V. Popova, Konstantin A. Miroshnikov, Nikolay V. Volozhantsev, Yuriy A. Knirel CH 3
CH2OH
OH
O
HO HNAc
AcN H
O AcNH
O
O
CH2
HO HO 2C
HO
O HO OH
OH
O
HO
9
O
O
CH2
Capsular polysaccharide was isolated from Acinetobacter baumannii ACICU The polysaccharide contains a higher acidic monosaccharide – pseudaminic acid Structure of the polysaccharide was established by NMR and chemical approaches
10
S0008-6215(14)00128-1 http://dx.doi.org/10.1016/j.carres.2014.04.002 CAR 6719
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
11 February 2014 26 March 2014 1 April 2014
Please cite this article as: Senchenkova, S.N., Shashkov, A.S., Shneider, M.M., Arbatsky, N.P., Popova, A.V., Miroshnikov, K.A., Volozhantsev, N.V., Knirel, Y.A., Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid, Carbohydrate Research (2014), doi: http://dx.doi.org/ 10.1016/j.carres.2014.04.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Note
Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid Sof’ya N. Senchenkova,a Alexander S. Shashkov,a Mikhail M. Shneider,b Nikolay P. Arbatsky,a Anastasiya V. Popova,с Konstantin A. Miroshnikov,b Nikolay V. Volozhantsev,с 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 с
State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region,
Russia
Abstract Capsular polysaccharide was isolated by the phenol-water extraction of Acinetobacter baumannii ACICU cells and studied by sugar analysis, partial acid hydrolysis, 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide was found to contain 5,7-diacetamido-3,5,7,9-tetradeoxy-Lglycero-L-manno-non-2-ulosonic or di-N-acetylpseudaminic acid (Pse5Ac7Ac), and the following structure of the branched tetrasaccharide repeating unit was established:
The genes present in the polysaccharide gene cluster of A. baumannii ACICU are appropriate to the structure established.
Keywords: Acinetobacter baumannii; Capsular polysaccharide structure; Pseudaminic acid; Polysaccharide gene cluster
*
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: [email protected] (Yuriy A. Knirel).
Gram-negative bacteria Acinetobacter baumannii are an opportunistic human pathogen affecting immunocompromised people. They are specific to the hospital environment and most commonly encountered in intensive care units. Treatment of A. baumannii infections is complicated by increasing multidrug resistance, and panresistant strains are known. Capsular polysaccharide (CPS) is one of the major virulence factors of A. baumannii1 important for growth and survival of bacteria in the human body environment. CPS structures are highly variable in this species, and a number of them have been determined.2 Recently, two attempts have been made to genotype A. baumannii based on the nucleotide sequences in the CPS biosynthesis genomic locus, one resulting in recognition of 9 types3 and the other being more comprehensive.4 A. baumannii strain ACICU belongs to the highly virulent European epidemic clone II group.5 The ACICU-specific CPS locus type designated Kl23 or PSgc124 is widely distributed in A. baumannii populations.4,6 Our analysis (data not shown) has revealed that this type of CPS is inherent to more than 10% strains with NCBI-deposited genomes. In this work, aiming at integration of the structural and genetic data into a comprehensive classification system of A. baumannii, we established the CPS structure of strain ACICU and found it to be in accord with putative functions of the genes in the CPS locus. A polysaccharide (PS-1) was isolated by the phenol-water extraction of cells of A. baumannii ACICU and purified by GPC. Sugar analysis by GLC of the acetylated alditols and (S)-2-octyl glycosides revealed D-Glc, D-Gal, and D-GalNAc in the ratios ~1:1:0.4 (detector response). Mild acid hydrolysis of PS-1 yielded di-N-acetylpseudaminic acid (Pse5Ac7Ac) (Chart 1) and a modified polysaccharide (PS-2). The 1H and 13C NMR chemical shifts and 3JH,H coupling constants of the isolated Pse5Ac7Ac were identical to the published data.7 Both polysaccharides obtained were studied by 2D NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC and HMBC experiments. As judged by the number of signals in the spectra, including those for anomeric protons and carbons, PS-1 and PS-2 are built up of tetra- and tri-saccharide repeating units, respectively. The 1H and 13C NMR spectra were assigned (Fig. 1, Table 1), and spin systems for one residues each of Glc, Gal, GalNAc (all in both polysaccharides), and Pse5Ac7Ac (in PS-1) were identified. As judged by 3JH,H coupling constants compared with published data,7,8 all monosaccharides were in the pyranosidic form and, except for Pse5Ac7Ac, β-linked (J1,2 7-8 Hz.). Structure of PS-2 was determined by a 1H,13C HMBC experiments, which showed Gal H-1/GalNAc C-3, GalNAc H-1/Gal C-3, and Glc H-1/Gal C-6 cross-peaks at δ 4.46/80.8, 4.68/82.7, and 4.49/70.8, respectively. Positions of substitution of the monosaccharides were confirmed by downfield displacements of the NMR signals for linkage carbons (Table 1), 2
namely C-3 of GalNAc and C-3 and C-6 of Gal, as compared with their positions in the corresponding non-substituted monosaccharides.9 Therefore, PS-2 has a branched structure with a side-chain β-Glcp attached at position 6 of β-Galp at the branching point (Chart 1). The NMR data (Table 1) showed that PS-1 differed from PS-2 in the presence of Pse5Ac7Ac only. The 1H,13C HMBC spectrum of PS-1 showed a cross peak between C-2 of Pse5Ac7Ac and H-6b of Glc at δ 101.3/3.70, and, hence, Pse5Ac7Ac was attached at position 6 of Glc in the side chain. This conclusion was confirmed by a comparison of 13C NMR chemical shifts of PS-2 and PS-1, which revealed displacements of the signals for C-6 and C-5 of Glc from δ 62.0 and 77.3 in PS-2 to δ 63.8 and 75.9 in PS-1, respectively, (positive α-effect and negative β-effect of 6-glycosylation) with no significant shifts of the other signals. The position of the signal for C-6 of Pse5Ac7Ac at δ 72.0 and a relatively small difference (0.52 ppm) between the H-3ax and H-3eq chemical shifts compared with published data7 indicated the α configuration of this monosaccharide. Therefore, based on the data obtained, it was concluded that the CPS of A. baumannii ACICU has the structure shown in Chart 1. A peculiar feature of the polysaccharide is the presence of a higher monosaccharide, di-N-acetylpseudaminic acid. Various N-acyl derivatives of pseudaminic acid, including Pse5Ac7Ac, have been reported as components of a number of bacterial polysaccharides7,10,11 but this is the first time that pseudaminic acid is found in A. baumannii. The CPS biosynthesis locus of A. baumannii ACICU located between the fkpA and lldP genes has been sequenced 3,4 and classified into the PSgc124 or KL2 3 group. The genes that have been annotated based on similarities to genes from available databases3,4 were found to be appropriate to the structure established. Particularly, the locus includes a set of six psa genes for synthesis of nucleotide-activated pseudaminic acid (CMP-Pse5Ac7Ac) (Fig. 2). They are organized in the same manner as the psb genes of Shigella boydii type 7,3,12 whose O-antigen contains another Pse derivative, 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Ac7Hb).13 Most Psa proteins share 57 % to 83 % sequence identity with the corresponding Psb proteins but PsaD and PsaE are only 38 % and 31 % identical to Psb4 and Psb5, respectively. In the predicted CMP-Pse5Ac7Hb biosynthetic pathway,12 PsaE mediates 4-Nacylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-β-L-altrose, and PsaD then cleaves the UDP moiety from the reaction product. A lower homology observed for these enzymes is evidently accounted for by the substrate difference between Psb5 and PsaE, which transfer acetyl or 3-hydroxybutanoyl group, respectively, that in turn makes different the substrates of Psb4 and PsaD, which function downstream in the pathway.
3
Downstream of the CMP-Pse5Ac7Ac synthesis genes, there are the wzx and wzy genes for processing and three glycosyl transferase genes (gtr3-gtr5) for assembly of the tetrasaccharide repeating unit of the CPS. Between wzx and wzy, there is a homologue of the kpsS gene that is required for synthesis/transfer of lysophosphatidylglycerol-poly-(3-deoxy-Dmanno-oct-2-ulosonic acid), the reducing termini of CPSs of Escherichia coli and Neisseria meningitidis.14 The role of kpsS in the CPS locus of A. baumannii ACICU remains unknown. Finally, the presence of a homologue of the itrA2 gene for initiating transferase of the A2 type, whose predicted substrate is UDP-D-GalpNAc,3 fits with the presence of D-GalNAc in the CPS of A. baumannii ACICU and indicates that this monosaccharide is first in the repeating unit. 1. Experimental 1.1. Cultivation of bacteria A. baumannii strain ACICU, a representative of a clone that caused an outbreak in an intensive care unit in Rome in 2005,15 was kindly provided by Dr. R. Zarilli (University of Naples Federico II, Naples, Italy). Bacteria were cultivated in 2TY media overnight; cells were harvested by centrifugation (10,000g, 20 min), washed with distilled water, suspended in aq 70 % acetone, precipitated and dried. 1.2. Isolation of CPS Bacterial cells (dry weight 0.9 g) were extracted with 45% aq phenol as described,16 the mixture was dialyzed, cells were removed by centrifugation, the supernatant was treated with aq 50% CCl3CO2 H at 4 °C to pH 2.5, the supernatant was dialyzed and freeze-dried. The product (130 mg) was dissolved in 25 mM Tris-HCl buffer pH 7.63 containing 2 mM CaCl2 and treated with DNAse and RNAse (24 h) and Proteinase K (48 h) at 20 °C. The solution was dialyzed and freeze-dried to give PS-1 (105 mg). 1.3. Mild acid hydrolysis A CPS sample (55 mg) was hydrolyzed with aq 2 % HOAc (100 °C, 1.5 h), the products were fractionated by GPC on a column of TSK HW-40 (S) to give a modified polysaccharide (PS-2) (25 mg) and Pse5Ac7Ac (9 mg). 1.4. Chemical analyses A PS-1 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 4
temperature program of 160 °C (1 min) to 290 °C at 7 °C min-1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.17 1.5. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9 % D2O and then examined as solutions in 99.95 % D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 45 °C (for the polysaccharides) or 20 °C (for Pse5Ac7Ac). 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 mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively.
Acknowledgement Authors thank Dr. R. Zarilli for providing A. baumannii strain ACICU. This work was supported by the Russian Foundation for Basic Research (project No. 14-04-00657).
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References 1. 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. 2. 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. 3. Kenyon, J. J.; Hall, R. M. PLoS ONE 2013, 8, e62160. 4. Hu, D.; Liu, B.; Dijkshoorn, L.; Wang, L.; Reeves, P. R. PLoS ONE 2013, 8, e70329. 5. Iacono, M.; Villa, L.; Fortini, D.; Bordoni, R.; Imperi, F.; Bonnal, R. J.; Sicheritz-Ponten, T.; De Bellis, G.; Visca, P.; Cassone, A.; Carattoli, A. Antimicrob. Agents Chemother. 2008, 52, 2616–2625. 6. Wright, M. S.; Haft, D. H.; Harkins, D. M.; Perez, F.; Hujer, K. M.; Bajaksouzian, S.; Benard, M. F.; Jacobs, M. R.; Bonomo, R. A.; Adams, M. D. MBio 2014, 5, e00963-13. 7. Knirel, Y. A.; Shashkov, A. S.; Tsvetkov, Y. E.; Jansson, P.-E.; Zähringer, U. Adv. Carbohydr. Chem. Biochem. 2003, 58, 371-417. 8. Altona, C.; Haasnoot, C. A. G. Org. Magn. Reson. 1980, 13, 417-429. 9. Lipkind, G. M.; Shashkov, A. S.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59-75. 10. Knirel, Y. A.; Shevelev, S. D.; Perepelov, A. V. Mendeleev Commun. 2011, 21, 173-182. 11. Zunk, M.; Kiefel, M. J. RSC Adv. 2014, 4, 3413-3421. 12. Liu, B.; Knirel, Y. A.; Feng, L.; Perepelov, A. V.; Senchenkova, S. N.; Wang, Q.; Reeves, P. R.; Wang, L. FEMS Microbiol. Rev. 2008, 32, 627-653. 13. L'vov, V. L.; Shashkov, A. S.; Dmitriev, B. A. Bioorg. Khim. 1987, 13, 223-233. 14. Willis, L. M.; Stupak, J.; Richards, M. R.; Lowary, T. L.; Li, J.; Whitfield. C. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7868-7873. 15. Iacono, M.; Villa, L.; Fortini, D.; Bordoni, R.; Imperi, F.; Bonnal, R. J.; Sicheritz-Ponten, T.; De Bellis, G.; Visca, P.; Cassone, A.; Carattoli, A. Antimicrob. Agents Chemother. 2008, 52, 2616-2625. 16. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83-91. 17. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87-89.
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Table 1. 1H and 13C NMR chemical shifts (δ, ppm) Residue
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
H-1
H-2
H-3 (ax, eq) H-4
H-5
H-6 (a, b)
H-7
H-8
H-9
52.7
80.7
69.3
75.9
62.3
4.70
4.03
3.89
4.17
3.67
3.77
105.8
71.2
82.5
69.9
74.4
71.0
4.47
3.59
3.70
4.12
3.84
3.86; 3.97
104.7
74.4
76.8
70.6
75.9
63.8
4.45
3.27
3.45
3.51
3.51
3.54; 3.70
174.7
101.3 36.2
66.0
49.8
72.0
54.8
68.4
17.3
4.24
4.24
3.88
4.18
4.18
1.15
Capsular polysaccharide (PS-1)
→3)-β-GalpNAc-(1→ 104.0 →3,6)-β-Galp-(1→ →6)-β-Glсp-(1 → α-Psep5Ac7Ac-(2→
1.64; 2.16 C-1
C-2
C-3
C-4
C-5
C-6
H-1
H-2
H-3
H-4
H-5
H-6 (a, b)
104.2
52.8
80.8
69.3
76.0
62.4
4.68
4.06
3.92
4.19
3.70
3.78
106.0
71.1
82.7
69.9
74.5
70.8
4.46
3.58
3.74
4.16
3.86
3.88; 4.03
104.3
74.3
77.1
71.3
77.3
62.0
4.49
3.28
3.47
3.38
3.46
3.72; 3.93
Modified polysaccharide (PS-2) →3)-β-GalpNAc-(1→
→3,6)-β-Galp-(1→
β-Glсp-(1→
1
H NMR chemical shifts are shown in italics. Chemical shifts for NAc are δH 1.96-2.04, δC 23.2-
23.6 (CH3) and 174.9-176.3 (CO).
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Fig. 1. 13C NMR spectrum of the CPS of A. baumannii ACICU (PS-1). Signals for Gal and GalNAc that build up the main chain, are significantly less intense than those of Glc and Pse5Ac7Ac, which are located in the side chain (for the PS-1 structure see Chart 1). Abbreviations: G, Glc; Ga, Gal; Gn, GalN; P, Pse.
Fig. 2. Organization of the CPS biosynthesis locus of A. baumannii ACICU. Specific genes for synthesis of the KL2 (PSgc12) group CPS are located between the conserved gna and galU genes. Adopted from ref. 3.
Chart 1. Structures of the CPS of A. baumannii ACICU (PS-1), the modified Pse5Ac7Aclacking polysaccharide (PS-2), and the monosaccharide Pse5Ac7Ac released by mild acid hydrolysis of PS-1.
Legends to Figures Fig. 1. 13C NMR spectrum of the CPS of A. baumannii ACICU (PS-1). Signals for Gal and GalNAc that build up the main chain, are significantly less intense than those of Glc and Pse5Ac7Ac, which are located in the side chain (for the PS-1 structure see Chart 1). Abbreviations: G, Glc; Ga, Gal; Gn, GalN; P, Pse.
Fig. 2. Organization of the CPS biosynthesis locus of A. baumannii ACICU. Specific genes for synthesis of the KL2 (PSgc12) group CPS are located between the conserved gna and galU genes. Adopted from ref. 3.
Chart 1. Structures of the CPS of A. baumannii ACICU (PS-1), the modified Pse5Ac7Aclacking polysaccharide (PS-2), and the monosaccharide Pse5Ac7Ac released by mild acid hydrolysis of PS-1.
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Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-Nacetylpseudaminic acid Sofya N. Senchenkova, Alexander S. Shashkov, Mikhail M. Shneider, Nikolay P. Arbatsky, Anastasiya V. Popova, Konstantin A. Miroshnikov, Nikolay V. Volozhantsev, Yuriy A. Knirel CH 3
CH2OH
OH
O
HO HNAc
AcN H
O AcNH
O
O
CH2
HO HO 2C
HO
O HO OH
OH
O
HO
9
O
O
CH2
Capsular polysaccharide was isolated from Acinetobacter baumannii ACICU The polysaccharide contains a higher acidic monosaccharide – pseudaminic acid Structure of the polysaccharide was established by NMR and chemical approaches
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