Carbohydrate Research 409 (2015) 30e35
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structure of the K6 capsular polysaccharide from Acinetobacter baumannii isolate RBH4 Johanna J. Kenyon a, Alberto M. Marzaioli b, Ruth M. Hall a, Cristina De Castro b, * a b
School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia Department of Chemical Sciences, Complesso Universitario Monte Sant'Angelo, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2014 Received in revised form 23 March 2015 Accepted 27 March 2015 Available online 6 April 2015
The structure of the capsular polysaccharide (CPS) from an Acinetobacter baumannii global clone 2 (GC2) clinical isolate RBH4 that carries the KL6 gene cluster was elucidated by means of chemical and spectroscopical methods. The repeating unit of K6 CPS is linear and contains N-acetyl-D-galactosamine (DGalpNAc), two D-galactose (D-Galp) residues and 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac). The synthesis of these sugars could be attributed to genes in the KL6 capsule biosynthesis gene cluster, and the formation of the linkages between the sugars were assigned to glycosyltransferases or the Wzy polymerase encoded in KL6. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Capsular polysaccharide K locus KL6 gene cluster NMR spectroscopy Pseudaminic acid
1. Introduction Acinetobacter baumannii is an important opportunistic nosocomial Gram-negative pathogen that poses a significant threat to global health, as the majority of clinical isolates are resistant to almost all clinically used antibiotics.1 A. baumannii produces a capsular polysaccharide (CPS) in addition to the lipooligosaccharide (LOS) that decorates the outer membrane.2e5 The CPS is a known virulence determinant for A. baumannii, shown to be essential for survival in human serum and a rat soft tissue infection model, as well as for optimal growth in human ascites fluid.3 CPS surrounds the cell as high molecular weight polymers, and it is composed of a repeated oligosaccharide unit that contains different sugar residues, often aminosugars, connected together by
Abbreviations: COSY, correlation spectroscopy; CPS, capsular polysaccharide; HMBC, heteronuclear multiple bond coherence; HSQC, heteronuclear single quantum correlation; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LOS, lipooligosaccharide; NOESY, nuclear overhauser spectroscopy; PCP, phenolchloroformepetroleum ether; Pse5Ac7Ac, 5,7-di-N-acetylpseudaminic acid or 5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid; TOCSY, total correlation spectroscopy. * Corresponding author. Department of Chemical Sciences, Complesso Universitario Monte Sant'Angelo, Via Cintia, 4-80126 Napoli, Italy. Tel.: þ39 081674124. E-mail address: [email protected] (C. De Castro). http://dx.doi.org/10.1016/j.carres.2015.03.016 0008-6215/© 2015 Elsevier Ltd. All rights reserved.
specific linkages. This polysaccharide layer provides a protective coating that shields cells from the external environment, enhancing resistance to phagocytosis, disinfection and desiccation.6 Knowledge of CPS structure is fundamental to understanding its biosynthetic pathway, including the gene-to-function assignment. We previously reported that multiply antibiotic resistant A. baumannii isolates belonging to global clone 2 (GC2) can carry different genes in the K locus that directs the synthesis of the CPS.4 Two of the gene clusters, KL2 and KL6, included genes predicted to be involved in the synthesis of 5,7-di-Nacetylpseudaminic acid (Pse5Ac7Ac), and the K2 structure was subsequently shown to include Pse5Ac7Ac.7,8 KL2 and KL6 were also found to share further genes including itrA2 and gtr5 (Fig. 1). ItrA2 is an initiating transferase that links D-GalpNAc as the first sugar of the unit to the lipid carrier, and Gtr5 is a glycosyltransferase that catalyses the b-D-Galp-(1/3)-D-GalpNAc linkage (see Ref. 7 for details). However, KL2 and KL6 each encode two further glycosyltransferases that are not related, as well as unrelated Wzy polymerases. In this work, the structure of the CPS produced by A. baumannii RBH4, a GC2 isolate recovered in 2002 from the Royal Brisbane Hospital, Australia, which carries the KL6 capsule biosynthesis gene cluster [GenBank accession number KF130871] was determined and compared to K2.
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
31
Fig. 1. Alignment of KL2 and KL6 capsule biosynthesis gene clusters. KL6 and KL2, described previously,4 are drawn to scale from GenBank accession numbers KF130871 (this study) and KJ459911, respectively. Arrows represent genes and denote direction of transcription, with gene names shown above. Dashed boxes surround genes for the synthesis of nucleotide-linked sugar precursors, with sugar type shown above. Grey shading indicates DNA sequence that is more than 95% identical.
2. Results 2.1. Purification and chemical analysis CPS from A. baumannii RBH4 was isolated from the water layer obtained by hot phenol/water extraction, and further purified by enzymatic treatment as reported previously.7 In order to eliminate LOS contamination, the solution was ultracentrifuged. Polysaccharide was recovered from the supernatant, and used for structural analysis. Chemical analysis9 disclosed the occurrence of D-Galp and D-GalpNAc, linked at position 6 and position 3, respectively. An additional component was detected in the acetylated methyl glycosides mixture, with the fragmentation pattern comparable to that of a 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2ulosonic acid,7 which was later identified as Pse5Ac7Ac (5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid). Kdo, the marker of lipooligosaccharide, was not detected indicating absence or levels below the detection limit. 2.2. NMR analysis of pure CPS Proton spectrum (Fig. 2) of pure CPS contained three signals in the anomeric region: H-1 at 5.04 ppm (3JH1,H2 3.4 Hz) was attributed to an a-configured residue, and those at 4.47 ppm and 4.42 ppm (3JH1,H2 7.8 Hz in both cases), were attributed to two bconfigured residues. The spectrum also contained three N-acetyl signals at ca. 2 ppm, as well as two geminal deoxy protons at 1.63 and 2.55 ppm and one methyl group at 1.21 ppm, consistent with Pse5Ac7Ac occurrence. Complete assignment of proton and carbon chemical shifts (Table 1) was achieved by analysing the homo- and heteronuclear 2D NMR spectra. The three anomeric signals were labelled with a letter (AeC) in the order of their decreasing chemical shifts, and D
5.0
4.5
4.0
3.5
was assigned to Pse5Ac7Ac. For A, H-1 had only two correlations in the TOCSY spectrum (Fig. 3a), because of the partial overlap among H-2 and H-4 and because of the small H-4/H-5 coupling typical of the galacto ring stereochemistry, H-5 was identified because of the intra-residue H-5/H-3 and H-5/H-4 cross peaks present in the NOESY spectrum. C-2 resonated at 49.9 ppm indicating a nitrogenbearing carbon, for which the H-2 chemical shift (4.27 ppm) confirmed acetylation of this amino group. Therefore, A was found to be an N-acetylated a-galactosamine and the low field shift of its C-3 signal (78.9 ppm) with respect to the standard value (72.3 ppm)10 confirmed glycosylation at this position. With regard to residue B, H-1 (4.47 ppm) showed three cross peaks in the TOCSY spectrum (Fig. 3a) identified as H-2, H-3, H-4 by using information from COSY spectrum. Total assignment was possible by applying the same strategy so that this residue was identified as b-galactose on the basis of anomeric chemical shift and of its 3JH1,H2 coupling constant value (7.8 Hz), and because this proton showed the expected NOEs with H-3 and H-5; C-6 carbon chemical shift (71.0 ppm) proved glycosylation at this position. Residue C had the same spectroscopical pattern as B, and was also identified as a b-galactose (3JH1,H2¼7.7 Hz), glycosylated at C-6 (65.2 ppm): the modest downfield shift of this carbon was diagnostic of the occurrence of ketose sugar. Assignment of D started from the diastereotopic methylene protons at high field (2.55 and 1.63 ppm, in equatorial and axial position, respectively), which correlated with H-4 (3.86 ppm) in the COSY spectrum and with a signal at 4.29 ppm in the TOCSY spectrum, which was assigned to H-5. The resonances of the other protons were determined beginning with the methyl signal at 1.21 ppm. The HMBC spectrum correlated this methyl group to a carbon at 70.2 ppm assigned to C-8, and to a carbon at 54.9 assigned to C-7; the corresponding protons were found at 4.10 and 4.01 ppm, respectively, and COSY spectrum linked H-7 to H-6, which was at
3.0
2.5
2.0
1.5
Fig. 2. 1H NMR spectrum (600 MHz, D2O, 37 C) of pure CPS. Signals of impurities are crossed.
ppm
32
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
Table 1 Proton (600 MHz) and carbon (150 MHz) chemical shifts of CPS obtained by A. baumannii RBH4, measured at 37 C. Spectra were calibrated with respect to internal acetone (1H: 2.225 ppm, 13C: 31.45 ppm). Structure of the repeating unit of the CPS is in Fig. 5
A 3-a-GalpNAc B 6-b-Galp C 6-b-Galp
1
D 4-b-Psep5Ac7Ac
1
H C H 13 C 1 H 13 C 13 1
1
2
5.04 96.7 4.47 105.1 4.42 105.8
4.27 3.85 4.24 4.18 49.9 78.9 69.9 72.3 3.52 3.64 3.96 3.84 72.0 73.9 70.1 75.1 3.53 3.64 3.95 3.79 72.0 73.9 70.1 74.8
3ax; 3eq
3
4
4
5
5
6
7
0
6
6
3.762 62.9 3.81 71.0 3.60 65.2
d d 4.11 d 3.93 d
8
9
H 1.63; 2.56 3.86 4.29 4.06 4.01 4.10 13 C 34.4 73.1 48.8 74.8 54.9 70.2
1.21 18.3
4.06 ppm. Determination of the carbon chemical shifts of D indicated that both its amino groups were N-acetylated, as confirmed by the long-range correlation of H-5 and H-7 with a carbonyl (175.3 and 174.9, respectively). Comparison of H-3 proton chemical shifts with those published identified the anomeric configuration of the residue as b.11 The C-4 chemical shift at 73.1 ppm suggested that this position was glycosylated. HMBC spectrum analysis permitted also the identification of C-2 and C-1 at 102.8 ppm and 174.2 ppm, respectively. The sequence of the repeating unit was elucidated by the analysis of long-range correlations. The HMBC spectrum (Fig. 4) showed a correlation between H-1 of A and C-4 of D, H-1 of B and C3 of A, and H-1 of C and C-6 of B. These results were confirmed by
C4,1
ppm
C3,1
B4,1
4.6
C2,1 B2,1
B3,1
4.8 5.0
A2,1
B6’C1
ppm
4.6
A3,1
A4,1
*C4,1 B6C1 C5,1 *B4,1
A4B1
A3B1 B5,1
(a
C3,1 B3,1
4.8 5.0
A2,1
D4A1 4.0
4.2
3.8
(b 3.6
ppm
ppm
5.0
D3A1
D3’A1
(c
5.2 2.6
2.4
2.2
2.0
1.8
ppm
Fig. 3. (600 MHz, D2O, 37 C) Expansion of TOCSY (a) and NOESY (b and c) spectra of CPS from A. baumannii RBH4. Attribution of most of the cross peaks is indicated nearby the corresponding density. Letter labels reflect those reported in Table 1. The crosspeaks starred in panel b are probably intra-residue NOE spin diffusion effects, the possibility of inter-residue NOE effects arisen from polysaccharide conformation is not proved.
analysis of the NOESY spectrum (Fig. 3b and c), which displayed key correlations including: H-1 of A with H-4, H-3ax and H-3eq of D, H-1 of B with H-3 of A, H-1 of C with H-6 and H-60 of B. This data, combined with the linkage analysis and with the carbon chemical shift analysis, allowed elaboration of the structure of the oligosaccharide repeat unit of the K6 CPS shown in Fig. 5. 2.3. Assignment of function to glycosyltransferases encoded by KL6 The D-GalpNAc residue is drawn as the first sugar of the unit (Fig. 5), as KL6 includes itrA2, which encodes an initiating transferase specific for D-GalpNAc.7,12 Thus the Wzy polymerase would catalyse the a-D-GalpNAc-(1/4)-Pse5Ac7Ac linkage between oligosaccharide units (Fig. 6). The K6 structure contains three other glycosidic linkages, and KL6 contains three glycosyltransferase genes. One linkage, b-DGalp-(1/3)-D-GalpNAc, was previously predicted to be present in K6 based on the presence of a gtr5 glycosyltransferase gene in KL6 and KL2 (see Fig. 1).7 Gtr5a from KL2 and Gtr5b from KL6 differ somewhat in the first 71 amino acids (49/71 aa identical) but differ at only 2 positions in the remainder of the 23577 aa (278 aa) proteins (Fig. 7). However, it is clear that these differences do not alter the substrate and linkage specificities. The remaining b-Pse5Ac7Ac-(2/6)-D-Galp and b-D-Galp(1/6)-D-Galp linkages can be assigned tentatively to Gtr16 and Gtr17, respectively (Fig. 6), as follows. Gtr16 shares between 30 and 40% aa sequence identity with glycosyltransferases that use other nonulosonic acids as substrates. Gtr17 is 46% identical to Gtr4 encoded in KL2, that was previously assigned to the b-D-Glcp(1/6)-D-Galp linkage in K2,7 suggesting that Gtr17 carries out the b-D-Galp-(1/6)-D-Galp linkage in K6. 3. Discussion The structure of the oligosaccharide repeating-unit of the K6 CPS recovered from the A. baumannii GC2 clinical isolate, RBH4, was determined by integrating chemical and spectroscopical approaches. It is a linear tetrasaccharide containing D-GalpNAc, two D-Galp residues, and the acidic nine-carbon sugar, 5,7-di-N-acetyl pseudaminic acid (Fig. 5). Query of the Bacterial Carbohydrate Structure Database (BCSD, http://csdb.glycoscience.ru/bacterial/) with pseudaminic acid discloses that it occurs both in Gram-positive and Gram-negative bacteria. In the Gram-positive Kribbella and Actinoplanes genera, it constitutes the teichulosonic acid polymer of the cell wall. Regarding Gram-negative bacteria, the first report of pseudaminic acid dates back to 1985 when it was found in the lipopolysaccharide (LPS) of two different bacteria, Pseudomonas aeruginosa and Shigella boydii.13 Since then it has been reported in LPS or other carbohydrate-decorated molecules, such as the pili or flagellin, of other genera: Proteus, Campylobacter, Escherichia, Legionella, Helicobacter, Cellulophaga, Sinorhizobium, Pischiricckettsia, Pseudoalteromonas, and Vibrio. Only recently it has been found in two A. baumannii clinical isolates, A747 and ACICU,8 which share the same K2 CPS repeating unit. Indeed, taking only Gram-negative bacteria into account, it seems that pseudaminic acid is not a marker of a specific species, but it is associated with bacteria several of which are of concern for human health. The presence of Pse5Ac7Ac and a b-D-Galp-(1/3)-D-GalpNAc linkage confirmed our prediction that K6 and K2 are related, based on genes shared between the KL2 and KL6 capsule gene clusters.7 However in K6, Pse5Ac7Ac is part of the main-chain of the repeat-unit, whereas in K2, Pse5Ac7Ac together with D-Glcp form a side-branch (Fig. 6). The differences in structural arrangement can be attributed to different wzy and glycosyltransferase genes located in the central part of KL2 and KL6. The two Wzy proteins catalyse
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
D5
ppm
A4,2
A2
D8,7
55
D7
A5,6
C1B6
A4
A1D4
75
A1,3
A4,3
B1A3
C5,6 B6
B4 + C4
D8 A5
A6
C6’
65 A1,5
33
D4
B6’ D6
B5
D7,6
C5
C6 B2 + C2 B3 + C3
B2,3 C2,3
A3
85
95 A1 C2,1
105
B1
5.0
4.8
4.6
C1
4.4
B2,1
4.2
4.0
3.8
3.6
ppm
Fig. 4. (600 MHz, D2O, 37 C) Expansion of HSQC spectrum (black) overlapped to HMBC spectrum (grey) of pure CPS from A. baumannii RBH4. Attribution of most of the cross peaks is indicated nearby the corresponding density. The structure of the repeating unit of the CPS is reported in Fig. 5 together with the labels used. The signals crossed are impurities.
different linkages, and must have different substrate specificities. The formation of the remaining two internal linkages in the K6 unit were tentatively assigned to Gtr16 (b-Pse5Ac7Ac-(2/6)-D-Galp) and Gtr17 (b-D-Galp-(1/6)-D-Galp), which are not present in K2. Recently, we reported a further five capsule gene clusters that include genes for the synthesis of pseudaminic acid (psa genes), suggesting that pseudaminic acid is likely to be present in several different CPS structures in A. baumannii.7 In addition, the psa gene module is also found in the PSgc12 gene cluster of A. baumannii isolate LUH3713 and the PSgc26 gene cluster of LUH5553.14 The PSgc12 cluster, which we have designated KL81, differs from KL2 only in that it contains a pgt1 gene, encoding a phosphoglycerol
AcHN
CH3
8 7
NHAc
Pse5Ac7Ac -(2 6)
AcHN O
transferase, located between gne1 and pgm. The PSgc26 cluster, which we have designated KL90, is related to KL46, but differs in a single gtr, the itrA, and the wzy gene in the central portion, as well as gne1 in the right arm. All of these gene clusters except KL90 include itrA2, indicating that the CPS repeat units also begin with D-GalpNAc. Four of these gene clusters also have a form of gtr5. KL81 carries gtr5a and 3 further KL (KL31, KL33, and KL42) each carry a further variant of gtr5 that predicts a Gtr5 in which the N terminus differs (Fig. 7). Because of the differences, the sugars in the repeat unit cannot be predicted but it is likely that the linkage is b-(1/3). In conclusion, the K6 repeat unit structure is consistent with the content of the KL6 gene cluster, and confirms predictions that it shares three sugars and one linkage with K2. The disclosure of the structure of the K6 CPS repeat unit from A. baumannii RBH4 is valuable in understanding the polysaccharide determinants involved in the virulence of this bacterium and the interconnection of this information with those from other bacteria may shed light on common strategies used by all of them to circumvent host defenses. 4. Experimental section
O
D
3 O OH
4 HO
3
C
COOH
4.1. Bacterial isolate
Galp -(1 6)
The extensively antibiotic resistant A. baumannii isolate RBH4 was recovered from a patient at the Royal Brisbane Hospital in Brisbane, Australia in 2002. RBH4 was found to belong to GC2 using methods described previously.15
O
2 OH
O OH
4 HO
B
O
3 2 OH Galp -(1 3)
OH OH
4
A
O
O
3
2 NHAc
GalpNAc -(1 4)
O
Fig. 5. The structure of the repeating unit of the K6 CPS isolated from A. baumannii RBH4. For each residue, the chemical structure is drawn together with its abbreviated name and the label used in Table 1. To avoid crowding, most but not all of the carbon positions of each residue are numbered.
4.2. CPS purification and chemical analysis A. baumannii RBH4 cells (8.6 g) were extracted according to phenol-chloroformepetroleum ether (PCP) and hot phenol/water (1/1 v/v) extractions.16,17 LOS was recovered by the PCP method, while CPS (15% gCPS/gcells yield) was recovered in the water phase of the hot phenol/water extraction. CPS was purified from nucleic acids and proteins by enzymatic hydrolysis (8.0% gCPS/gcells) prior to
34
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
Fig. 6. The K6 and K2 repeat units. CPS names are shown on the left. Structures are drawn with abbreviated sugars names listed in abbreviations. Grey shading indicates features that are present in both structures. Wzy polymerases, glycosyltransferases, and the initiating transferase that were assigned to each linkage are shown in bold. Wzy proteins are distinguished by subscripts to indicate their origin.
ultracentrifugation (30,000 rpm, 4 C, 16 h) to remove LOS traces still present in the sample. The CPS-containing supernatant (6.4% gpure CPS/gcells yield) was freeze-dried and used for the chemical and spectroscopic analyses.
Monosaccharide composition analysis (acetylated methylglycoside), substitution pattern (partially methylated alditol acetylated) and absolute configuration (acetylated 2-octyl glycosides) were performed as reported.9 GCeMS analyses were performed
Fig. 7. Comparison of the different Gtr5 sequences encoded by different K gene clusters. Gtr5 type and the K gene cluster are indicated to the left of each sequence. Sequences are from GenPept accession numbers AGK44808 (Gtr5a, KL2), AGM37791 (Gtr5b, KL6), EKK11525 (Gtr5c, KL31), EKU40046 (Gtr5d, KL33), AHB32439 (Gtr5e, KL42). Alignment consensus is shown below, with * indicating amino acids that are conserved, : indicating amino acids with strongly similar properties, . indicating amino acids with weakly similar properties, and gaps representing no relationship. Grey shading shows amino acid differences.
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
with an Agilent instrument (GC instrument Agilent 6850 coupled to MS Agilent 5973), equipped with a SPB-5 capillary column (Supelco, 30 m0.25 i.d., flow rate, 0.8 mL min1) and He as carrier gas. Electron impact mass spectra were recorded with an ionization energy of 70 eV and an ionizing current of 0.2 m A. The temperature program used for the analyses was the following: 150 C for 5 min, 150/280 C at 3 C/min, 300 C for 5 min.
35
Acknowledgements C.D.C and A.M. acknowledge P.O.R. Campania FSE 2007e2013, Project CREMe for financial support. J.J.K and R.M.H were supported by grant APP1026189 from the National Health and Medical Research Council of Australia (NHMRC). We thank Matthew T. Wynn for assistance with bacterial growth, and Steven J. Nigro for determination of the clonal group.
4.3. NMR spectroscopy References NMR analyses were performed on a Bruker 600 MHz equipped with a cryogenic probe and spectra were recovered at 37 C using acetone as internal standard (1H 2.225 ppm, 13C 31.45 ppm). 2D spectra (DQFeCOSY, TOCSY, NOESY, gHSQC and gHMBC) were recorded using Bruker software (TopSpin 3.1). Homonuclear experiments were recorded using 512 FIDs of 2048 complex with 32 scans per FID, for TOCSY and NOESY spectra a mixing time of 100 and 200 ms was used, respectively. HSQC and HMBC spectra were acquired with 512 FIDs of 2048 complex point and 40 scans per FID for HSQC and 60 scans per FID for HMBC. Spectra were processed and analysed using a Bruker TopSpin 3 program. 4.4. Bioinformatics Sequence of the RBH4 capsule biosynthesis gene cluster was obtained from GenBank accession number KF138071. Glycosyltransferase functions were predicted by comparing the carbohydrate structures of isolates with similar glycosyltransferase sequences identified using BLASTp similarity searches.18 Sequences alignments were constructed using CLUSTALW2 (http://www.ebi. ac.uk/Tools/msa/clustalw2/).
1. Antunes LCS, Visca P, Towner KJ. Pathog Dis 2014;71:292e301. 2. Fregolino E, Gargiulo V, Lanzetta R, Parrilli M, Holst O, De Castro C. Carbohydr Res 2011;346:973e7. 3. Russo TA, Luke NR, Beanan JM, Olson R, Sauberan SL, MacDonald U, et al. Infect Immun 2010;78:3993e4000. 4. Kenyon JJ, Hall RM. PLoS One 2013;8:e62160. 5. Kenyon JJ, Holt KE, Pickard D, Dougan G, Hall RM. Res Micro 2014;165:472e5. 6. Cress BF, Englaender JA, He W, Kasper D, Linhardt RJ, Koffas MAG. FEMS Microbiol Rev 2014;38:660e97. 7. Kenyon JJ, Marzaioli A, Hall RM, De Castro C. Glycobiol 2014;24:554e63. 8. Senchenkova S, Shashkov A, Shneider M, Arbatsky N, Popova A, Miroshnikov KA, et al. Carbohydr Res 2014;391:89e92. 9. De Castro C, Parrilli M, Holst O, Molinaro A. Methods Enzymol 2010;480: 89e115. 10. Bock K, Pedersen C. Adv Carbohydr Chem Biochem 1983;48:27e66. 11. Tsvetkov YE, Shashkov AS, Knirel YA, Z€ ahringer U. Carbohydr Res 2001;335: 221e43. 12. Lees-Miller RG, Iwashkiw JA, Scott NE, Seper A, Vinogradov E, Schild S, et al. Mol Microbiol 2013;89:816e30. 13. Knirel YA, Vinogradov EV, Shashkov AS, Kochetkov NK, L'vov VL, Dmitriev BA. Carbohydr Res 1985;141:C1e3. 14. Hu D, Liu B, Dijkshoorn L, Wang L, Reeves PR. PLoS One 2013;8:e70329. 15. Post V, White PA, Hall RM. J Antimicrob Chemother 2010;65:1162e70. 16. Galanos C, Lüderitz O, Westphal O. Eur J Biochem 1969;9:245e9. 17. Westphal O, Jann K. Meth Carbohydr Chem 1965;5:83e91. 18. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J Mol Biol 1990;215: 403e10.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structure of the K6 capsular polysaccharide from Acinetobacter baumannii isolate RBH4 Johanna J. Kenyon a, Alberto M. Marzaioli b, Ruth M. Hall a, Cristina De Castro b, * a b
School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia Department of Chemical Sciences, Complesso Universitario Monte Sant'Angelo, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2014 Received in revised form 23 March 2015 Accepted 27 March 2015 Available online 6 April 2015
The structure of the capsular polysaccharide (CPS) from an Acinetobacter baumannii global clone 2 (GC2) clinical isolate RBH4 that carries the KL6 gene cluster was elucidated by means of chemical and spectroscopical methods. The repeating unit of K6 CPS is linear and contains N-acetyl-D-galactosamine (DGalpNAc), two D-galactose (D-Galp) residues and 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac). The synthesis of these sugars could be attributed to genes in the KL6 capsule biosynthesis gene cluster, and the formation of the linkages between the sugars were assigned to glycosyltransferases or the Wzy polymerase encoded in KL6. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Capsular polysaccharide K locus KL6 gene cluster NMR spectroscopy Pseudaminic acid
1. Introduction Acinetobacter baumannii is an important opportunistic nosocomial Gram-negative pathogen that poses a significant threat to global health, as the majority of clinical isolates are resistant to almost all clinically used antibiotics.1 A. baumannii produces a capsular polysaccharide (CPS) in addition to the lipooligosaccharide (LOS) that decorates the outer membrane.2e5 The CPS is a known virulence determinant for A. baumannii, shown to be essential for survival in human serum and a rat soft tissue infection model, as well as for optimal growth in human ascites fluid.3 CPS surrounds the cell as high molecular weight polymers, and it is composed of a repeated oligosaccharide unit that contains different sugar residues, often aminosugars, connected together by
Abbreviations: COSY, correlation spectroscopy; CPS, capsular polysaccharide; HMBC, heteronuclear multiple bond coherence; HSQC, heteronuclear single quantum correlation; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LOS, lipooligosaccharide; NOESY, nuclear overhauser spectroscopy; PCP, phenolchloroformepetroleum ether; Pse5Ac7Ac, 5,7-di-N-acetylpseudaminic acid or 5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid; TOCSY, total correlation spectroscopy. * Corresponding author. Department of Chemical Sciences, Complesso Universitario Monte Sant'Angelo, Via Cintia, 4-80126 Napoli, Italy. Tel.: þ39 081674124. E-mail address: [email protected] (C. De Castro). http://dx.doi.org/10.1016/j.carres.2015.03.016 0008-6215/© 2015 Elsevier Ltd. All rights reserved.
specific linkages. This polysaccharide layer provides a protective coating that shields cells from the external environment, enhancing resistance to phagocytosis, disinfection and desiccation.6 Knowledge of CPS structure is fundamental to understanding its biosynthetic pathway, including the gene-to-function assignment. We previously reported that multiply antibiotic resistant A. baumannii isolates belonging to global clone 2 (GC2) can carry different genes in the K locus that directs the synthesis of the CPS.4 Two of the gene clusters, KL2 and KL6, included genes predicted to be involved in the synthesis of 5,7-di-Nacetylpseudaminic acid (Pse5Ac7Ac), and the K2 structure was subsequently shown to include Pse5Ac7Ac.7,8 KL2 and KL6 were also found to share further genes including itrA2 and gtr5 (Fig. 1). ItrA2 is an initiating transferase that links D-GalpNAc as the first sugar of the unit to the lipid carrier, and Gtr5 is a glycosyltransferase that catalyses the b-D-Galp-(1/3)-D-GalpNAc linkage (see Ref. 7 for details). However, KL2 and KL6 each encode two further glycosyltransferases that are not related, as well as unrelated Wzy polymerases. In this work, the structure of the CPS produced by A. baumannii RBH4, a GC2 isolate recovered in 2002 from the Royal Brisbane Hospital, Australia, which carries the KL6 capsule biosynthesis gene cluster [GenBank accession number KF130871] was determined and compared to K2.
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
31
Fig. 1. Alignment of KL2 and KL6 capsule biosynthesis gene clusters. KL6 and KL2, described previously,4 are drawn to scale from GenBank accession numbers KF130871 (this study) and KJ459911, respectively. Arrows represent genes and denote direction of transcription, with gene names shown above. Dashed boxes surround genes for the synthesis of nucleotide-linked sugar precursors, with sugar type shown above. Grey shading indicates DNA sequence that is more than 95% identical.
2. Results 2.1. Purification and chemical analysis CPS from A. baumannii RBH4 was isolated from the water layer obtained by hot phenol/water extraction, and further purified by enzymatic treatment as reported previously.7 In order to eliminate LOS contamination, the solution was ultracentrifuged. Polysaccharide was recovered from the supernatant, and used for structural analysis. Chemical analysis9 disclosed the occurrence of D-Galp and D-GalpNAc, linked at position 6 and position 3, respectively. An additional component was detected in the acetylated methyl glycosides mixture, with the fragmentation pattern comparable to that of a 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2ulosonic acid,7 which was later identified as Pse5Ac7Ac (5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid). Kdo, the marker of lipooligosaccharide, was not detected indicating absence or levels below the detection limit. 2.2. NMR analysis of pure CPS Proton spectrum (Fig. 2) of pure CPS contained three signals in the anomeric region: H-1 at 5.04 ppm (3JH1,H2 3.4 Hz) was attributed to an a-configured residue, and those at 4.47 ppm and 4.42 ppm (3JH1,H2 7.8 Hz in both cases), were attributed to two bconfigured residues. The spectrum also contained three N-acetyl signals at ca. 2 ppm, as well as two geminal deoxy protons at 1.63 and 2.55 ppm and one methyl group at 1.21 ppm, consistent with Pse5Ac7Ac occurrence. Complete assignment of proton and carbon chemical shifts (Table 1) was achieved by analysing the homo- and heteronuclear 2D NMR spectra. The three anomeric signals were labelled with a letter (AeC) in the order of their decreasing chemical shifts, and D
5.0
4.5
4.0
3.5
was assigned to Pse5Ac7Ac. For A, H-1 had only two correlations in the TOCSY spectrum (Fig. 3a), because of the partial overlap among H-2 and H-4 and because of the small H-4/H-5 coupling typical of the galacto ring stereochemistry, H-5 was identified because of the intra-residue H-5/H-3 and H-5/H-4 cross peaks present in the NOESY spectrum. C-2 resonated at 49.9 ppm indicating a nitrogenbearing carbon, for which the H-2 chemical shift (4.27 ppm) confirmed acetylation of this amino group. Therefore, A was found to be an N-acetylated a-galactosamine and the low field shift of its C-3 signal (78.9 ppm) with respect to the standard value (72.3 ppm)10 confirmed glycosylation at this position. With regard to residue B, H-1 (4.47 ppm) showed three cross peaks in the TOCSY spectrum (Fig. 3a) identified as H-2, H-3, H-4 by using information from COSY spectrum. Total assignment was possible by applying the same strategy so that this residue was identified as b-galactose on the basis of anomeric chemical shift and of its 3JH1,H2 coupling constant value (7.8 Hz), and because this proton showed the expected NOEs with H-3 and H-5; C-6 carbon chemical shift (71.0 ppm) proved glycosylation at this position. Residue C had the same spectroscopical pattern as B, and was also identified as a b-galactose (3JH1,H2¼7.7 Hz), glycosylated at C-6 (65.2 ppm): the modest downfield shift of this carbon was diagnostic of the occurrence of ketose sugar. Assignment of D started from the diastereotopic methylene protons at high field (2.55 and 1.63 ppm, in equatorial and axial position, respectively), which correlated with H-4 (3.86 ppm) in the COSY spectrum and with a signal at 4.29 ppm in the TOCSY spectrum, which was assigned to H-5. The resonances of the other protons were determined beginning with the methyl signal at 1.21 ppm. The HMBC spectrum correlated this methyl group to a carbon at 70.2 ppm assigned to C-8, and to a carbon at 54.9 assigned to C-7; the corresponding protons were found at 4.10 and 4.01 ppm, respectively, and COSY spectrum linked H-7 to H-6, which was at
3.0
2.5
2.0
1.5
Fig. 2. 1H NMR spectrum (600 MHz, D2O, 37 C) of pure CPS. Signals of impurities are crossed.
ppm
32
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
Table 1 Proton (600 MHz) and carbon (150 MHz) chemical shifts of CPS obtained by A. baumannii RBH4, measured at 37 C. Spectra were calibrated with respect to internal acetone (1H: 2.225 ppm, 13C: 31.45 ppm). Structure of the repeating unit of the CPS is in Fig. 5
A 3-a-GalpNAc B 6-b-Galp C 6-b-Galp
1
D 4-b-Psep5Ac7Ac
1
H C H 13 C 1 H 13 C 13 1
1
2
5.04 96.7 4.47 105.1 4.42 105.8
4.27 3.85 4.24 4.18 49.9 78.9 69.9 72.3 3.52 3.64 3.96 3.84 72.0 73.9 70.1 75.1 3.53 3.64 3.95 3.79 72.0 73.9 70.1 74.8
3ax; 3eq
3
4
4
5
5
6
7
0
6
6
3.762 62.9 3.81 71.0 3.60 65.2
d d 4.11 d 3.93 d
8
9
H 1.63; 2.56 3.86 4.29 4.06 4.01 4.10 13 C 34.4 73.1 48.8 74.8 54.9 70.2
1.21 18.3
4.06 ppm. Determination of the carbon chemical shifts of D indicated that both its amino groups were N-acetylated, as confirmed by the long-range correlation of H-5 and H-7 with a carbonyl (175.3 and 174.9, respectively). Comparison of H-3 proton chemical shifts with those published identified the anomeric configuration of the residue as b.11 The C-4 chemical shift at 73.1 ppm suggested that this position was glycosylated. HMBC spectrum analysis permitted also the identification of C-2 and C-1 at 102.8 ppm and 174.2 ppm, respectively. The sequence of the repeating unit was elucidated by the analysis of long-range correlations. The HMBC spectrum (Fig. 4) showed a correlation between H-1 of A and C-4 of D, H-1 of B and C3 of A, and H-1 of C and C-6 of B. These results were confirmed by
C4,1
ppm
C3,1
B4,1
4.6
C2,1 B2,1
B3,1
4.8 5.0
A2,1
B6’C1
ppm
4.6
A3,1
A4,1
*C4,1 B6C1 C5,1 *B4,1
A4B1
A3B1 B5,1
(a
C3,1 B3,1
4.8 5.0
A2,1
D4A1 4.0
4.2
3.8
(b 3.6
ppm
ppm
5.0
D3A1
D3’A1
(c
5.2 2.6
2.4
2.2
2.0
1.8
ppm
Fig. 3. (600 MHz, D2O, 37 C) Expansion of TOCSY (a) and NOESY (b and c) spectra of CPS from A. baumannii RBH4. Attribution of most of the cross peaks is indicated nearby the corresponding density. Letter labels reflect those reported in Table 1. The crosspeaks starred in panel b are probably intra-residue NOE spin diffusion effects, the possibility of inter-residue NOE effects arisen from polysaccharide conformation is not proved.
analysis of the NOESY spectrum (Fig. 3b and c), which displayed key correlations including: H-1 of A with H-4, H-3ax and H-3eq of D, H-1 of B with H-3 of A, H-1 of C with H-6 and H-60 of B. This data, combined with the linkage analysis and with the carbon chemical shift analysis, allowed elaboration of the structure of the oligosaccharide repeat unit of the K6 CPS shown in Fig. 5. 2.3. Assignment of function to glycosyltransferases encoded by KL6 The D-GalpNAc residue is drawn as the first sugar of the unit (Fig. 5), as KL6 includes itrA2, which encodes an initiating transferase specific for D-GalpNAc.7,12 Thus the Wzy polymerase would catalyse the a-D-GalpNAc-(1/4)-Pse5Ac7Ac linkage between oligosaccharide units (Fig. 6). The K6 structure contains three other glycosidic linkages, and KL6 contains three glycosyltransferase genes. One linkage, b-DGalp-(1/3)-D-GalpNAc, was previously predicted to be present in K6 based on the presence of a gtr5 glycosyltransferase gene in KL6 and KL2 (see Fig. 1).7 Gtr5a from KL2 and Gtr5b from KL6 differ somewhat in the first 71 amino acids (49/71 aa identical) but differ at only 2 positions in the remainder of the 23577 aa (278 aa) proteins (Fig. 7). However, it is clear that these differences do not alter the substrate and linkage specificities. The remaining b-Pse5Ac7Ac-(2/6)-D-Galp and b-D-Galp(1/6)-D-Galp linkages can be assigned tentatively to Gtr16 and Gtr17, respectively (Fig. 6), as follows. Gtr16 shares between 30 and 40% aa sequence identity with glycosyltransferases that use other nonulosonic acids as substrates. Gtr17 is 46% identical to Gtr4 encoded in KL2, that was previously assigned to the b-D-Glcp(1/6)-D-Galp linkage in K2,7 suggesting that Gtr17 carries out the b-D-Galp-(1/6)-D-Galp linkage in K6. 3. Discussion The structure of the oligosaccharide repeating-unit of the K6 CPS recovered from the A. baumannii GC2 clinical isolate, RBH4, was determined by integrating chemical and spectroscopical approaches. It is a linear tetrasaccharide containing D-GalpNAc, two D-Galp residues, and the acidic nine-carbon sugar, 5,7-di-N-acetyl pseudaminic acid (Fig. 5). Query of the Bacterial Carbohydrate Structure Database (BCSD, http://csdb.glycoscience.ru/bacterial/) with pseudaminic acid discloses that it occurs both in Gram-positive and Gram-negative bacteria. In the Gram-positive Kribbella and Actinoplanes genera, it constitutes the teichulosonic acid polymer of the cell wall. Regarding Gram-negative bacteria, the first report of pseudaminic acid dates back to 1985 when it was found in the lipopolysaccharide (LPS) of two different bacteria, Pseudomonas aeruginosa and Shigella boydii.13 Since then it has been reported in LPS or other carbohydrate-decorated molecules, such as the pili or flagellin, of other genera: Proteus, Campylobacter, Escherichia, Legionella, Helicobacter, Cellulophaga, Sinorhizobium, Pischiricckettsia, Pseudoalteromonas, and Vibrio. Only recently it has been found in two A. baumannii clinical isolates, A747 and ACICU,8 which share the same K2 CPS repeating unit. Indeed, taking only Gram-negative bacteria into account, it seems that pseudaminic acid is not a marker of a specific species, but it is associated with bacteria several of which are of concern for human health. The presence of Pse5Ac7Ac and a b-D-Galp-(1/3)-D-GalpNAc linkage confirmed our prediction that K6 and K2 are related, based on genes shared between the KL2 and KL6 capsule gene clusters.7 However in K6, Pse5Ac7Ac is part of the main-chain of the repeat-unit, whereas in K2, Pse5Ac7Ac together with D-Glcp form a side-branch (Fig. 6). The differences in structural arrangement can be attributed to different wzy and glycosyltransferase genes located in the central part of KL2 and KL6. The two Wzy proteins catalyse
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
D5
ppm
A4,2
A2
D8,7
55
D7
A5,6
C1B6
A4
A1D4
75
A1,3
A4,3
B1A3
C5,6 B6
B4 + C4
D8 A5
A6
C6’
65 A1,5
33
D4
B6’ D6
B5
D7,6
C5
C6 B2 + C2 B3 + C3
B2,3 C2,3
A3
85
95 A1 C2,1
105
B1
5.0
4.8
4.6
C1
4.4
B2,1
4.2
4.0
3.8
3.6
ppm
Fig. 4. (600 MHz, D2O, 37 C) Expansion of HSQC spectrum (black) overlapped to HMBC spectrum (grey) of pure CPS from A. baumannii RBH4. Attribution of most of the cross peaks is indicated nearby the corresponding density. The structure of the repeating unit of the CPS is reported in Fig. 5 together with the labels used. The signals crossed are impurities.
different linkages, and must have different substrate specificities. The formation of the remaining two internal linkages in the K6 unit were tentatively assigned to Gtr16 (b-Pse5Ac7Ac-(2/6)-D-Galp) and Gtr17 (b-D-Galp-(1/6)-D-Galp), which are not present in K2. Recently, we reported a further five capsule gene clusters that include genes for the synthesis of pseudaminic acid (psa genes), suggesting that pseudaminic acid is likely to be present in several different CPS structures in A. baumannii.7 In addition, the psa gene module is also found in the PSgc12 gene cluster of A. baumannii isolate LUH3713 and the PSgc26 gene cluster of LUH5553.14 The PSgc12 cluster, which we have designated KL81, differs from KL2 only in that it contains a pgt1 gene, encoding a phosphoglycerol
AcHN
CH3
8 7
NHAc
Pse5Ac7Ac -(2 6)
AcHN O
transferase, located between gne1 and pgm. The PSgc26 cluster, which we have designated KL90, is related to KL46, but differs in a single gtr, the itrA, and the wzy gene in the central portion, as well as gne1 in the right arm. All of these gene clusters except KL90 include itrA2, indicating that the CPS repeat units also begin with D-GalpNAc. Four of these gene clusters also have a form of gtr5. KL81 carries gtr5a and 3 further KL (KL31, KL33, and KL42) each carry a further variant of gtr5 that predicts a Gtr5 in which the N terminus differs (Fig. 7). Because of the differences, the sugars in the repeat unit cannot be predicted but it is likely that the linkage is b-(1/3). In conclusion, the K6 repeat unit structure is consistent with the content of the KL6 gene cluster, and confirms predictions that it shares three sugars and one linkage with K2. The disclosure of the structure of the K6 CPS repeat unit from A. baumannii RBH4 is valuable in understanding the polysaccharide determinants involved in the virulence of this bacterium and the interconnection of this information with those from other bacteria may shed light on common strategies used by all of them to circumvent host defenses. 4. Experimental section
O
D
3 O OH
4 HO
3
C
COOH
4.1. Bacterial isolate
Galp -(1 6)
The extensively antibiotic resistant A. baumannii isolate RBH4 was recovered from a patient at the Royal Brisbane Hospital in Brisbane, Australia in 2002. RBH4 was found to belong to GC2 using methods described previously.15
O
2 OH
O OH
4 HO
B
O
3 2 OH Galp -(1 3)
OH OH
4
A
O
O
3
2 NHAc
GalpNAc -(1 4)
O
Fig. 5. The structure of the repeating unit of the K6 CPS isolated from A. baumannii RBH4. For each residue, the chemical structure is drawn together with its abbreviated name and the label used in Table 1. To avoid crowding, most but not all of the carbon positions of each residue are numbered.
4.2. CPS purification and chemical analysis A. baumannii RBH4 cells (8.6 g) were extracted according to phenol-chloroformepetroleum ether (PCP) and hot phenol/water (1/1 v/v) extractions.16,17 LOS was recovered by the PCP method, while CPS (15% gCPS/gcells yield) was recovered in the water phase of the hot phenol/water extraction. CPS was purified from nucleic acids and proteins by enzymatic hydrolysis (8.0% gCPS/gcells) prior to
34
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
Fig. 6. The K6 and K2 repeat units. CPS names are shown on the left. Structures are drawn with abbreviated sugars names listed in abbreviations. Grey shading indicates features that are present in both structures. Wzy polymerases, glycosyltransferases, and the initiating transferase that were assigned to each linkage are shown in bold. Wzy proteins are distinguished by subscripts to indicate their origin.
ultracentrifugation (30,000 rpm, 4 C, 16 h) to remove LOS traces still present in the sample. The CPS-containing supernatant (6.4% gpure CPS/gcells yield) was freeze-dried and used for the chemical and spectroscopic analyses.
Monosaccharide composition analysis (acetylated methylglycoside), substitution pattern (partially methylated alditol acetylated) and absolute configuration (acetylated 2-octyl glycosides) were performed as reported.9 GCeMS analyses were performed
Fig. 7. Comparison of the different Gtr5 sequences encoded by different K gene clusters. Gtr5 type and the K gene cluster are indicated to the left of each sequence. Sequences are from GenPept accession numbers AGK44808 (Gtr5a, KL2), AGM37791 (Gtr5b, KL6), EKK11525 (Gtr5c, KL31), EKU40046 (Gtr5d, KL33), AHB32439 (Gtr5e, KL42). Alignment consensus is shown below, with * indicating amino acids that are conserved, : indicating amino acids with strongly similar properties, . indicating amino acids with weakly similar properties, and gaps representing no relationship. Grey shading shows amino acid differences.
J.J. Kenyon et al. / Carbohydrate Research 409 (2015) 30e35
with an Agilent instrument (GC instrument Agilent 6850 coupled to MS Agilent 5973), equipped with a SPB-5 capillary column (Supelco, 30 m0.25 i.d., flow rate, 0.8 mL min1) and He as carrier gas. Electron impact mass spectra were recorded with an ionization energy of 70 eV and an ionizing current of 0.2 m A. The temperature program used for the analyses was the following: 150 C for 5 min, 150/280 C at 3 C/min, 300 C for 5 min.
35
Acknowledgements C.D.C and A.M. acknowledge P.O.R. Campania FSE 2007e2013, Project CREMe for financial support. J.J.K and R.M.H were supported by grant APP1026189 from the National Health and Medical Research Council of Australia (NHMRC). We thank Matthew T. Wynn for assistance with bacterial growth, and Steven J. Nigro for determination of the clonal group.
4.3. NMR spectroscopy References NMR analyses were performed on a Bruker 600 MHz equipped with a cryogenic probe and spectra were recovered at 37 C using acetone as internal standard (1H 2.225 ppm, 13C 31.45 ppm). 2D spectra (DQFeCOSY, TOCSY, NOESY, gHSQC and gHMBC) were recorded using Bruker software (TopSpin 3.1). Homonuclear experiments were recorded using 512 FIDs of 2048 complex with 32 scans per FID, for TOCSY and NOESY spectra a mixing time of 100 and 200 ms was used, respectively. HSQC and HMBC spectra were acquired with 512 FIDs of 2048 complex point and 40 scans per FID for HSQC and 60 scans per FID for HMBC. Spectra were processed and analysed using a Bruker TopSpin 3 program. 4.4. Bioinformatics Sequence of the RBH4 capsule biosynthesis gene cluster was obtained from GenBank accession number KF138071. Glycosyltransferase functions were predicted by comparing the carbohydrate structures of isolates with similar glycosyltransferase sequences identified using BLASTp similarity searches.18 Sequences alignments were constructed using CLUSTALW2 (http://www.ebi. ac.uk/Tools/msa/clustalw2/).
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