Anastasiya V. Popova

Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid

Capsular polysaccharide was isolated by the phenol-water extraction of Acinetobacter baumannii ACICU cells and studied by sugar analysis, partial acid hydrolysis, and 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-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.

Structure elucidation of the capsular polysaccharide of Acinetobacter baumannii AB5075 having the KL25 capsule biosynthesis locus

Capsular polysaccharide was isolated by the phenol-water extraction of Acinetobacter baumannii AB5075 and studied by 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established: → 3)-β-D-ManpNAcA-(1 → 4)-β-D-ManpNAcA-(1 → 3)-α-D-QuipNAc4NR-(1 → where R indicates (S)-3-hydroxybutanoyl or acetyl in the ratio ∼ 2.5:1. The genes in the polysaccharide biosynthesis locus designated KL25 are appropriate to the established CPS structure.

Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

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

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

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

The capsular polysaccharide from Acinetobacter baumannii LUH5550 was studied by 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the branched trisaccharide repeating unit was established: [structure: see text] 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.

K19 capsular polysaccharide of Acinetobacter baumannii is produced via a Wzy polymerase encoded in a small genomic island rather than the KL19 capsule gene cluster.

Polymerization of the oligosaccharides (K units) of complex capsular polysaccharides (CPSs) requires a Wzy polymerase, which is usually encoded in the gene cluster that directs K unit synthesis. Here, a gene cluster at the Acinetobacter K locus (KL) that lacks a wzy gene, KL19, was found in Acinetobacter baumannii ST111 isolates 28 and RBH2 recovered from hospitals in the Russian Federation and Australia, respectively. However, these isolates produced long-chain capsule, and a wzy gene was found in a 6.1 kb genomic island (GI) located adjacent to the cpn60 gene. The GI also includes an acetyltransferase gene, atr25, which is interrupted by an insertion sequence (IS) in RBH2. The capsule structure from both strains was →3)-α-d-GalpNAc-(1→4)-α-d-GalpNAcA-(1→3)-β-d-QuipNAc4NAc-(1→, determined using NMR spectroscopy. Biosynthesis of the K unit was inferred to be initiated with QuiNAc4NAc, and hence the Wzy forms the β-(1→3) linkage between QuipNAc4NAc and GalpNAc. The GalpNAc residue is 6-O-acetylated in isolate 28 only, showing that atr25 is responsible for this acetylation. The same GI with or without an IS in atr25 was found in draft genomes of other KL19 isolates, as well as ones carrying a closely related CPS gene cluster, KL39, which differs from KL19 only in a gene for an acyltransferase in the QuiNAc4NR synthesis pathway. Isolates carrying a KL1 variant with the wzy and atr genes each interrupted by an ISAba125 also have this GI. To our knowledge, this study is the first report of genes involved in capsule biosynthesis normally found at the KL located elsewhere in A. baumannii genomes.

The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51

The whole genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near cpn60 gene. This GI is in precisely the same location as a GI carrying wzy and atr genes recently found in several A. baumannii isolates, but does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with D-Fuc3NAc as a side branch, and the K units are linked via a β-D-GlcpNAc-(1→3)-β-D-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.The whole-genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-d-galactose (d-Fuc3NAc), and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near the cpn60 gene. This GI is in precisely the same location as another GI carrying wzy and atr genes recently found in several A. baumannii isolates, but it does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with d-Fuc3NAc as a side branch, and the K units are linked via a β-d-GlcpNAc-(1→3)-β-d-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

Structures of three different neutral polysaccharides of Acinetobacter baumannii, NIPH190, NIPH201, and NIPH615, assigned to K30, K45, and K48 capsule types, respectively, based on capsule biosynthesis gene clusters.

Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β-d-glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D (1)H and (13)C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit.

Revised structure of the capsular polysaccharide of Acinetobacter baumannii LUH5533 (serogroup O1) containing di-N-acetyllegionaminic acid

A glycopolymer classified as an O-specific polysaccharide had earlier been isolated from the multi-drug resistant nosocomial pathogen Acinetobacter baumannii LUH5533 (serogroup O1) and its chemical structure had been established. In the present work, we found that the glycopolymer was a capsular polysaccharide (CPS), its structure was revised, and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (di-N-acetyllegionaminic acid) was identified as its component, which had been overlooked earlier. The revised structure is consistent with predicted functions of genes in the CPS biosynthesis cluster classified into the KL7 or PSgc1 group, which is present in the LUH 5533 genome.

Acinetobacter baumannii K11 and K83 capsular polysaccharides have the same 6-deoxy-l-talose-containing pentasaccharide K units but different linkages between the K units

Acinetobacter baumannii produces a variety of capsular polysaccharides (CPS) via genes located at the chromosomal K locus and some KL gene clusters include genes for the synthesis of specific sugars. The structures of K11 and K83 CPS produced by isolates LUH5545 and LUH5538, which carry related KL11a and KL83 gene clusters, respectively, were established by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy. Both CPS contain l-rhamnose (l-Rha) and 6-deoxy-l-talose (l-6dTal), and both KL gene clusters include genes for dTDP-l-Rhap synthesis and a tle (talose epimerase) gene encoding an epimerase that converts dTDP-l-Rhap to dTDP-l-6dTalp. The K11 and K83 repeat units are the same pentasaccharide, consisting of d-glucose, l-Rha, l-6dTal, and N-acetyl-d-glucosamine, except that l-6dTal is 2-O-acetylated in K83. However, the K units are linked differently, with l-Rha in the main chain in K11, but as a side-branch in K83. KL11 and KL83 encode unrelated Wzy polymerases that link the K units together and different acetyltransferases, though only Atr8 from KL83 is active. The substrate specificity of each Wzy polymerase was assigned, and the functions of all glycosyltransferases were predicted. The CPS structures produced by three closely related K loci, KL29, KL105 and KL106, were also predicted.