Structure of a new pseudaminic acid-containing capsular polysaccharide of Acinetobacter baumannii LUH5550 having the KL42 capsule biosynthesis locus

Article abstract of DOI:10.1016/j.carres.2015.02.006

The capsular polysaccharide from Acinetobacter baumannii LUH5550 was studied by 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the branched trisaccharide repeating unit was established: (Formula presented.) where Pse5Ac7RHb indicates 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutanoylamino]-l-glycero-l-manno-non-2-ulosonic acid. The genes in the capsule biosynthesis locus designated KL42 are consistent with the structure established.

Full text of DOI:10.1016/j.carres.2015.02.006

Accepted Manuscript
Structure of a new pseudaminic acid-containing capsular polysaccharide of
Acinetobacter baumannii LUH5550 having the KL42 capsule biosynthesis locus
Sof’ya N. Senchenkova, Anastasiya V. Popova, Alexander S. Shashkov, Mikhail M.
Shneider, Zhu Mei, Nikolay P. Arbatsky, Bin Liu, Konstantin A. Miroshnikov, Nikolay
V. Volozhantsev, Yuriy A. Knirel
PII:
S0008-6215(15)00068-3
10.1016/j.carres.2015.02.006
CAR 6946
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 22 January 2015
Revised Date: 10 February 2015
Accepted Date: 12 February 2015
Please cite this article as: Senchenkova S’yN, Popova AV, Shashkov AS, Shneider MM, Mei Z, Arbatsky
NP, Liu B, Miroshnikov KA, Volozhantsev NV, Knirel YA, Structure of a new pseudaminic acid-containing
capsular polysaccharide of Acinetobacter baumannii LUH5550 having the KL42 capsule biosynthesis
locus, Carbohydrate Research (2015), doi: 10.1016/j.carres.2015.02.006.
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ACCEPTED MANUSCRIPT
Structure of
a
new pseudaminic acid-containing capsular
polysaccharide of Acinetobacter baumannii LUH5550 having the KL42
capsule biosynthesis locus
Sofya N. Senchenkova, Anastasiya V. Popova, Alexander S. Shashkov, Mikhail
M. Shneider, Zhu Mei, Nikolay P. Arbatsky, Bin Liu, Konstantin A. Miroshnikov,
Nikolay V. Volozhantsev, Yuriy A. Knirel

ACCEPTED MANUSCRIPT
Note
Structure of a new pseudaminic acid-containing capsular polysaccharide of
Acinetobacter baumannii LUH5550 having the KL42 capsule biosynthesis locus
a
b
a
c
Sof’ya N. Senchenkova, Anastasiya V. Popova, Alexander S. Shashkov, Mikhail M. Shneider,
d
a
d
c
Zhu Mei, Nikolay P. Arbatsky, Bin Liu, Konstantin A. Miroshnikov, Nikolay V.
b
a,*
Volozhantsev, Yuriy A. Knirel
a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region,
b
Russia
c
M. M. Shemyakin & Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, Moscow, Russia
d
TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin,
China
Abstract
The capsular polysaccharide from Acinetobacter baumannii LUH5550 was studied by 1D and
1
13
2
D H and C NMR spectroscopy. The following structure of the branched trisaccharide
repeating unit was established:
→3)-β-D-Ribp-(1→3)-β-D-GalpNAc-(1→
4
↑
2
α-Psep5Ac7RHb
where Pse5Ac7RHb indicates 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutanoylamino]-
-glycero- -manno-non-2-ulosonic acid. The genes in the capsule biosynthesis locus designated
L
L
KL42 are consistent with the structure established.
*
Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328.
E-mail address: yknirel@gmail.com (Yuriy A. Knirel).
1

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Keywords: Acinetobacter baumannii; Capsular polysaccharide structure; Polysaccharide gene
locus; Pseudaminic acid
Acinetobacter baumannii is Gram-negative nonfermenting coccobacilli. Last decades, it
emerged as a new pathogen associated mainly with hospital and intensive care unit infections.
Therapy of A. baumannii infections is significantly complicated by the ability of the bacteria to
1
,2
accumulate efficiently various mechanisms of antibiotic resistance. A capsular polysaccharide
(CPS) shell covering the A. baumannii cell is highly important for bacteria survival and
3
infectivity. Recently, a great repertoire of pan-genome genes of A. baumannii has been
4
demonstrated. In accordance with these data, sequences of CPS biosynthesis gene loci of A.
5
-8
baumannii are highly variable, and >90 locus types (KL groups) have been recognized (J.
Kenyon, personal communication).
In this work, we established the CPS structure of A. baumannii LUH5550. The CPS gene
6
7
locus type specific to this strain has been designated KL42 or PSgc22.
A polysaccharide was isolated from A. baumannii LUH5550 by phenol-water extraction.
Sugar analysis by GLC of the alditol acetates derived after acid hydrolysis of the CPS revealed
ribose and galactosamine in the ratio 1.06:1.
Further studies by NMR spectroscopy indicated that the CPS also includes a derivative of
5
,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (pseudaminic acid, Pse)
in the side-chain. Therefore, the CPS was hydrolyzed under mild acidic conditions, and a
derivative of Pse was isolated by GPC on TSK HW-40. It carried one N-acetyl group (NAc) and
1
13
one N-(3-hydroxybutanoyl) group (NHb) and existed in two forms (1 and 2). The H and C
3
NMR chemical shifts and JH,H coupling constants of the major from 1 (Table 1) were identical
9
or close to the published data of the α-pyranose of Pse5Ac7Hb . Correlations between the
carbonyl carbons of the N-acyl substituents and protons at N-linked sugar carbons C-5 and C-7
1
13
in the H, C HMBC spectrum demonstrated NAc at N-5 and NHb at N-7 of Pse. Hence, 1 has
the structure shown in Chart 1.
1
3
1
13
No signal for the anomeric carbon was found in the C NMR and H, C HMBC spectra
of the minor form 2 owing to its low intensity, and the structure of this form could not be
unambiguously established. However, significantly lower-field positions of the signals for
C-3−C-5 and a higher-field position of the signal for C-6 as compared to the pyranose form
(Table 1) suggested that 2 was a pyrrolidinose having the structure shown in Chart 1.
The
D
configuration of Rib was determined by GLC of the acetylated (S)-2-octyl
1
0
13
glycosides, and that of GalN by known regularities in glycosylation effects on C NMR
1
1
chemical shifts. The absolute configurations of Pse was inferred from genetic data of the CPS
2

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(
see below) and was not confirmed chemically. GLC analysis of the trifluoroacetylated (S)-2-
1
2
octyl ester showed the R configuration of the Hb group. Therefore, the CPS contains
D-Rib,
D-GalNAc, and Pse5Ac7RHb.
1
13
The H NMR and C NMR spectra of the CPS (Figs. 1 and 2) showed signals for three
1
1
1
13
monosaccharides, which were assigned using 2D H, H COSY, TOCSY, ROESY, H, C
HSQC, and HMBC experiments (Table 1) and spin systems for one residue each of Rib, GalN,
and Pse, were identified, all being in the pyranose form. Relatively large J coupling constants
1
,2
of 7-8 Hz indicated the β configuration of Rib and GalN. The α configuration of Pse was
inferred by a relatively small difference (0.48 ppm) between the H-3ax and H-3eq chemical
1
3
shifts compared with published data.
A 2D ROESY experiment showed Rib H-1/GalN H-3 and GalN H-1/Rib H-3 correlations
at 4.47/3.91 and 4.74/3.71, respectively. Accordingly, the HMBC experiment revealed Rib C-
1
/GalN H-3 and GalN C-1/Rib H-3 correlations at δ 106.0/3.91 and 104.2/3.71, respectively.
These data defined the →3)-β-Ribp-(1→3)-β-GalpNAc-(1→ main chain structure of the CPS. A
low-field position at δ 80.6, 82.7, and 73.5 of the signals for the linkage carbons, namely C-3 of
GalN and C-3 and C-4 of Rib, as compared with their positions of the corresponding non-
substituted monosaccharides at δ 72.4, 69.7, and 68.2, respectively, was in agreement with the
ROESY and HMBC data and indicated that Pse5Ac7RHb was attached at position 4 of Rib.
Based on the data obtained, it was concluded that the CPS of A. baumannii LUH5550 has
the structure shown in Chart 2. Earlier, structure of another A. baumannii CPS that contains a Pse
1
4
6
derivative, namely Pse5Ac7Ac, has been elucidated in strains ACICU and A74 belonging to
the KL2 group (Chart 2).
6
7
The CPS gene locus for A. baumannii LUH5550 designated KL42 or PSgc22 has been
7
sequenced (GenBank accession number KC526903) and compared with KL2 (GenBank
accession number KJ459911) and other A. baumannii loci that include genes for synthesis of Pse
6
derivatives. The genes between gna and galU involved specifically with the synthesis of the
6
5
KL42 group CPS have been annotated using an extension of the proposed scheme (Fig. 3).
In KL42, there is a set of psa genes for synthesis of Pse5Ac7RHb, four of which share
high-level identity (>91% at the amino acid level) with the corresponding genes in KL2.
However, two gene pairs, psaD/psaG and psaE/psaH, are only 33-35% identical. psaE and psaH
encode putative acetyltransferases that transfer acetyl or 3-hydroxybutanoyl group in the
5
6
synthesis pathways of Pse5Ac7Ac in KL2 and Pse5Ac7RHb in KL42, respectively. A low
homology of nucleotidases PsaD and PsaG, which function downstream in the pathways,
evidently reflects a difference between their substrates produced by PsaE and PsaH, respectively.
3

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Genes galU, ugd, gpi, and pgm for synthesis of
D
-Glc and
D
-GlcNAc as well as gne1
5
gene for 4-epimerase that converts UDP- -GlcNAc into UDP-
D
D
-GalNAc are located
downstream of KL42-specific genes. Genes for synthesis of nucleotide precursors of
more genes for -GlcNAc synthesis map outside the CPS gene locus.
D-Rib and
D
The itrA2 gene encodes an initiating glycosylphosphatetransferase that links the first
5
sugar of the repeating unit to the lipid carrier. The substrate of ItrA2 was putatively identified as
6
UDP-
D
-GalNAc, and the occurrence of itrA2 homologs in KL42 and KL2 is in agreement with
the presence of β-D-GalNAc in the main chain of the CPSs of both groups.
KL42 includes kpsS2 gene, encoding a predicted Pse5Ac7RHb-specific
6
glycosyltransferase. KpsS2 is 51% identical to KpsS1, which is present in KL2 and assigned
6
putatively to a Pse5Ac7Ac-specific glycosyltransferase. These enzymes are distant homologs of
KpsS, which is known to function as a retaining 3-deoxy-β-D-manno-oct-2-ulosonic acid (Kdo)
1
5
transferase. It does not belong to any glycosyltransferase family and differs significantly from
glycosyltransferases that use an inverting mechanism. As judged by the presence of the α-Pse
derivatives in the KL2 and KL42 CPSs, both KpsS1 and KpsS2 also use a retaining mechanism
1
6
to transfer the nonulosonic acid from the known nucleotide precursor CMP-α-Pse5Ac7Ac or a
putative precursor CMP-α-Pse5Ac7RHb to Gal or Rib, respectively.
The remaining glycosyltransferase gene in KL42 is evidently responsible for construction
of the β-
D
-Ribp-(1→3)-
D
-GalpNAc linkage. It shares 86% identity with gtr5 in KL2, which
6
catalyzes formation of another linkage, β-
D
-Galp-(1→3)-
D
-GalpNAc. Based on rather high-
6
level identity, the gene in KL42 has been annotated as gtr5 too but as it transfers Rib rather than
Gal, it is suggested to revise its annotation to gtr64. The wzx and wzy processing genes also
occur in KL42. Therefore, the capsule gene locus of A. baumannii LUH5550 is fully appropriate
to the CPS structure established.
1
1
. Experimental
.1. Bacterial strain, cultivation, and isolation of CPS
7
A. baumannii LUH5550 (laboratory stock number G3782) was originally from the W. H.
Traub collection at the Institut für Medizinische Mikrobiologie und Hygiene, Universität des
Saarlandes (Saarland, Germany). 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
7
CPS was isolated by phenol-water extraction of bacterial cells, the extract was dialyzed
without layer separation and freed from insoluble contaminations by centrifugation. The
4

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resultant solution was treated with cold (4 °C) aq 50 % CCl CO H; after centrifugation the
3
2
supernatant was dialyzed against distilled water and freeze-dried to give CPS.
.2. Chemical analyses
A CPS sample (1 mg) was hydrolyzed with 2 M CF CO H (120 °C, 2 h).
1
3
2
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
-1
temperature program of 160 °C (1 min) to 290 °C at 7 °C min . The absolute configuration of Rib
1
0
was determined by GLC of the acetylated (S)-2-octyl glycosides as described. (R)-
3
-Hydroxybutanoic acid was identified by GLC of the trifluoroacetylated (S)-2-octyl ester
1
2
essentially as described.
1
.3. NMR spectroscopy
Samples were deuterium-exchanged by freeze-drying from 99.9 % D O and then
2
examined as solution in 99.95 % D O. NMR spectra were recorded on a Bruker Avance II 600
2
MHz spectrometer (Germany) at 20 °C. Sodium 3-trimethylsilylpropanoate-2,2,3,3-d (δ 0) and
4
H
acetone (δ 31.45) were used as internal references for calibration. 2D NMR spectra were
C
obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire
and process the NMR data. A mixing time of 150 ms was used in TOCSY and ROESY
experiments. A 60-ms delay was used for evolution of long-range couplings to optimize the 2D
1
13
H, C HMBC experiment for coupling constant JH,C 8 Hz.
Acknowledgements
Authors thank J. Kenyon for critical reading of the manuscript. This work was supported
by the Russian Foundation for Basic Research (project No. 14-04-00657), International Science
Technology Cooperation Program of China (2012DFG31680 and 2013DFR30640), National
&
Natural Science Foundation of China (NSFC) Program (31371259 and 31270003), and Research
Project of Chinese Ministry of Education (No. 113015A).
5

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1
0.1093/glycob/cwv007.
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0. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87-89.
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1. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26,
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27-535.
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4. Senchenkova, S. N.; Shashkov, A. S.; Shneider, M. M.; Arbatsky, N. P.; Popova, A. V.;
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Legends to Figures
1
Figure 1. H NMR spectrum of the CPS of A. baumannii LUH5550. Numbers refer to protons in
sugar and acyl residues denoted as follows: G, GalN; P, Pse; R, Rib; Ac, acetyl; Hb,
3
-hydroxybutanoyl.
1
3
Figure 2. C NMR spectrum of the CPS of A. baumannii LUH5550. Signals for CO groups are
not shown. Numbers refer to carbons in sugar and acyl residues denoted as follows: G, GalN; P,
Pse; R, Rib; Ac, acetyl; Hb, 3-hydroxybutanoyl
.
Figure 3. Organization of the CPS biosynthesis gene locus of A. baumannii LUH5550 (KL42)
compared to KL2 (adopted from ref. 6). Gray shading between the gene loci indicates genes
having the same function and high-level identity.
Chart 1. Structures of α-pyranose (1) and pyrrolidinose (2) forms of Pse5Ac7NHb.
1
2
6
Chart 2. Structures of the CPSs of A. baumannii LUH5550 (this work), ACICU, and A74
containing Pse derivatives.
7

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1
13
3
Table 1. H and C NMR chemical shifts (δ, ppm) and J coupling constants (Hz)
H.H
N-Acyl groups
NRHb
Monosaccharides
Residue
NAc
C-2 C-1
H-2
C-1
C-2 C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-1
C-2
C-3 C-4
H-1
H-2 H-3 (3ax, 3eq) H-4
H-5 (5a, 5b) H-6 (6a, 6b) H-7
H-8
H-9
H-2
H-3 H-4
(
J3ax,3eq, J3ax,4
)
(J3eq,4
)
(J , )
4 5
(J )
5,6
(J )
6,7
(J ) (J )
7,8 8.9
CPS
1
04.1 52.8 80.6
.74 4.04 3.91
69.4
4.16
73.5
3.78
66.5
4.16
75.9
62.4
175.8 23.6
→
3)-β-GalpNAc-(1→
4
3.69
3.78
2.02
→3,4)-β-Ribp-(1→
105.9 71.3 82.7
.47 3.61 3.71
α-Psep5NAc7NRHb-(2→ 176.3 101.7 36.4
62.7
4
3.49, 3.57
50.0
72.3
54.9
68.8 17.6 175.9 23.3 174.9 46.4
66.2 23.4
1
.62, 2.10
4.23
3.88
4.17
4.17 1.15 1.99 2.34, 2.38 4.17 1.23
Pse5NAc7NRHb
α-pyranose
177.6 97.7 36.0
66.6
4.17
(5.0)
69.7
4.08
(3.6)
50.1
4.23
(~3.5)
55.0
3.82
(~9)
71.3
4.03
(<2)
69.7
4.12
(<2)
54.2
68.3 16.8 175.9 23.1 175.0 46.4
4.13 1.10 2.01 ~2.38
66.2 23.4
1
.77, 1.93
4.19
4.20 1.25
(
13.3, ~12)
(10.7) (3.5) (6.5)
pyrrolidinose
43.0
55.7
69.1 17.5 175.0 23.2 175.0 46.4
4.17 1.17 2.00 ~2.38
66.2 23.4
2
.25, 2.45
3.98
4.20 1.25
(
15.3, 9.6)
(10.4) (4.2) (6.4)
8

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a
Pse5NAc7NHb
α-pyranose
175.0 97.7 36.0
.81, 1.94
12.7, 12.7)
66.6
4.18
(5.0)
50.1
4.25
(4.2)
71.3
4.06
(1.7)
54.2
4.19
68.4 16.8 175.9 23.2 177.4 46.4
66.2 23.4
1
4.14 1.13 2.03 2.39, 2.41 4.23 1.26
(
(10.5) (3.3) (6.5)
a
Data of ref. 13.
9

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ꢀ
ꢀ
ꢀ
ꢀ
Capsular polysaccharide was isolated from Acinetobacter baumannii LUH5550
The polysaccharide contains 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid
Structure of the polysaccharide was established by NMR and chemical approaches
Capsule biosynthesis locus was analyzed and found to match the structure established