5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic Acids in the Capsular Polysaccharides of Acinetobacter baumannii.

The polysaccharide capsule surrounding bacterial cell plays an important role in pathogenesis of infections caused by the opportunistic pathogen Acinetobacter baumannii by providing protection from external factors. The structures of the capsular polysaccharide (CPS) produced by A. baumannii isolates and the corresponding CPS biosynthesis gene clusters are highly diverse, although many of them are related. Many types of A. baumannii CPSs contain isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid (DTNA). Three of these isomers, namely acinetaminic acid (l-glycero-l-altro isomer), 8-epiacinetaminic acid (d-glycero-l-altro isomer), and 8-epipseudaminic acid (d-glycero-l-manno isomer), have not been found so far in naturally occurring carbohydrates from other species. In A. baumannii CPSs, DTNAs carry N-acyl substituents at positions 5 and 7; in some CPSs, both N-acetyl and N-(3-hydroxybutanoyl) groups are present. Remarkably, pseudaminic acid carries the (R)-isomer and legionaminic acid carries the (S)-isomer of the 3-hydroxybutanoyl group. The review addresses the structure and genetics of biosynthesis of A. baumannii CPSs containing di-N-acyl derivatives of DTNA.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Acinetobacter baumannii K20 and K21 capsular polysaccharide structures establish roles for UDP-glucose dehydrogenase Ugd2, pyruvyl transferase Ptr2 and two glycosyltransferases.

Infections caused by Acinetobacter baumannii isolates from the major global clones, GC1 and GC2, are difficult to treat with antibiotics, and phage therapy, which requires extensive knowledge of the variation in the surface polysaccharides, is an option under consideration. The gene clusters directing the synthesis of capsular polysaccharide (CPS) in A. baumannii GC1 isolate A388 and GC2 isolate G21 differ by a single glycosyltransferase (gtr) gene. They include genes encoding a novel UDP-glucose dehydrogenase (Ugd2) and a putative pyruvyl transferase (Ptr2). The composition and structures of the linear K20 and K21 tetrasaccharide repeats (K units) of the CPSs isolated from A338 and G21, respectively, were established by sugar analyses and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The K20 and K21 CPSs are the first known to include GlcpA produced by Ugd2 and d-galactose with an (R)-configured 4,6-pyruvic acid acetal added by Prt2. The first sugar in the tetrasaccharide K units is 2-acetamido-4-amino-2,4,6-trideoxy-d-glucose (d-QuipNAc4N) that carries a 4-N-[(S)-3-hydroxybutanoyl] group in some K units and a 4-N-acetyl group in the others. Accordingly, K unit polymerases WzyK20 and WzyK21 form a ß-d-QuipNAc4NR-(1→2)-d-Galp bond. The K20 and K21 units differ only in the configuration of the glycosidic linkages of d-GlcpNAc allowing the unique inverting glycosyltransferases Gtr43 and the retaining glycosyltransferase Gtr45 to be assigned to the formation of the ß-d-GlcpNAc-(1→4)-d-GlcpA and α-d-GlcpNAc-(1→4)-d-GlcpA linkages, respectively.

Genetics of biosynthesis and structure of the K53 capsular polysaccharide of Acinetobacter baumannii D23 made up of a disaccharide K unit.

The KL53 capsular polysaccharide (CPS) gene cluster of Acinetobacter baumannii D23 was sequenced, and includes a single gtr gene encoding the glycosyltransferase Gtr2, and the itrA1 gene for ItrA1 that is known to initiate CPS biosynthesis with d-QuiNAc4NAc. The K53 CPS was isolated and studied by one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy before and after O-deacetylation. The disaccharide K unit of the CPS was established as →3)-α-d-GalpNAcA4Ac-(1→3)-ß-d-QuipNAc4NAc-(1→, where GalNAcA and QuiNAc4NAc indicate 2-acetamido-2-deoxygalacturonic acid and 2,4-diacetamido-2,4,6-trideoxyglucose, respectively. This established the linkage formed by Gtr2. The degree of 4-O-acetylation of d-GalNAcA by Atr18, encoded at the KL53 locus, is ~55 %.

Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells-peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor-lipopolysaccharide.

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 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 ß-d-GlcpNAc-(1→3)-ß-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.

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.

Role of capsular modified heptose in the virulence of Campylobacter jejuni.

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

Immunochemical characterization of the O antigens of two Proteus strains, O8-related antigen of Proteus mirabilis 12 B-r and O2-related antigen of Proteus genomospecies 5/6 12 B-k, infecting a hospitalized patient in Poland.

A hospitalized 73-year-old woman was infected with a Proteus mirabilis strain, 12 B-r, isolated from the place of injection of a blood catheter. Another strain, 12 B-k, recognized as Proteus genomospecies 5 or 6, was isolated from the patient's faeces, which was an example of a nosocomial infection rather than an auto-infection. Serological investigation using ELISA and Western blotting showed that strain 12 B-k from faeces belonged to the Proteus O2 serogroup. Strain 12 B-r from the wound displayed cross-reactions with several Proteus O serogroups due to common epitopes on the core or O-specific parts of the lipopolysaccharide. Studies of the isolated 12 B-r O-specific polysaccharide by NMR spectroscopy revealed its close structural similarity to that of Proteus O8. The only difference in 12 B-r was the presence of an additional GlcNAc-linked phosphoethanolamine residue, which creates a putative epitope responsible for the cross-reactivity with Pt. mirabilis O16. The new O-antigen form could appear as a result of adaptation of the bacterium to a changing environment. On the basis of the data obtained, we suggest division of the O8 serogroup into two subgroups: O8a for strains of various Proteus species that have been previously classified into the O8 serogroup, and O8a,b for Pt. mirabilis 12 B-r, where 'a' is a common epitope and 'b' is a phosphoethanolamine-associated epitope. These findings further confirm serological and structural heterogeneity of O antigens of Proteus strains isolated lately from patients in Poland.

O antigen of FranconibacterpulverisG3872 (O1) is a 4-deoxy-d-arabino-hexose-containing polysaccharide synthesized by the ABC-transporter-dependent pathway.

Franconibacter (Enterobacter, Cronobacter) pulveris bacteria share several typical characteristics with, and hence pose a challenge for the detection of, Cronobacter sakazakii, an emerging opportunistic pathogen, which can cause severe infections in neonates. A structurally variable O-specific polysaccharide (OPS) called O antigen provides the major basis for the typing of Gram-negative bacteria. We investigated the structure and genetics of the O antigen of F. pulveris G3872 (designated O1). An OPS was isolated by mild alkaline degradation of the LPS, whereas the same polysaccharide and its oligosaccharide fragments were obtained by mild acid degradation. Studies by sugar analysis and NMR spectroscopy showed that the OPS contained d-ribose, l-rhamnose (l-Rha) and a rarely occurring monosaccharide 4-deoxy-d-arabino-hexose, and the OPS structure was established. The O-antigen gene cluster of F. pulveris G3872 between JUMPStart and gnd genes includes putative genes for glycosyltransferases, ATP-binding cassette (ABC)-transporter genes wzm and wzt, and genes for the synthesis of l-Rha, but no genes for the synthesis of 4-deoxy-d-arabino-hexose. A mutation test with the wzm gene confirmed that the OPS is synthesized and exported by the ABC-transporter-dependent pathway. A trifunctional transferase was suggested to catalyse formation of two glycosidic linkages and add a methyl group to the non-reducing end of the OPS to terminate the chain elongation. A carbohydrate-binding module that presumably recognizes the terminal methyl-modified monosaccharide was found at the C-terminus of Wzt. Primers specific for F. pulveris G3872 were designed based on the wzm gene, which has potential to be used for identification and detection of the O1 serogroup.

Elucidation of a masked repeating structure of the O-specific polysaccharide of the halotolerant soil bacteria Azospirillum halopraeferens Au4.

An O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide isolated by the phenol-water extraction from the halotolerant soil bacteria Azospirillum halopraeferens type strain Au4. The polysaccharide was studied by sugar and methylation analyses, selective cleavages by Smith degradation and solvolysis with trifluoroacetic acid, one- and two-dimensional (1)H and (13)C NMR spectroscopy. The following masked repeating structure of the O-specific polysaccharide was established: →3)-α-L-Rhap2Me-(1→3)-[ß-D-Glcp-(1→4)]-α-D-Fucp-(1→2)-ß-D-Xylp-(1→, where non-stoichiometric substituents, an O-methyl group (~45%) and a side-chain glucose residue (~65%), are shown in italics.

Revised structure of the polysaccharide from Acinetobacter baumannii LUH5551 assigned as the K63 type capsular polysaccharide.

K63 capsular polysaccharide produced by Acinetobacter baumannii isolate LUH5551 (previously designated isolate O24) was re-examined using sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. Though previously reported as O24 consisting of linear tetrasaccharide units that include a 7-acetamido-5-acylamino form of 8-epilegionaminic acid [8eLeg5R7Ac, acylated at C5 with (S)-3-hydroxybutanoyl or acetyl (1:1)], the elucidated structure of the K63 type capsule was found to include a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid, Leg5Ac7R, where R is either (S)-3-hydroxybutanoyl or an acetyl group (∼1:1 ratio). This finding is consistent with the presence of the lgaABCHIFG gene module for Leg5Ac7R biosynthesis in the KL63 gene cluster at the capsular polysaccharide (CPS) biosynthesis K locus in the LUH5551 genome. The glycosyltransferases (Gtrs) and Wzy polymerase encoded by KL63 were assigned to linkages in the linear K63 tetrasaccharide unit and linkage of the K63 units.

Structure of the alanopine-containing O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Proteus vulgaris HSC 438 classified into a new Proteus serogroup, O76.

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Proteus vulgaris HSC 438, and the following structure was established by chemical methods and one- and two-dimensional (1)H and (13)C NMR spectroscopy: →3)-ß-d-Quip4NAlo-(1→3)-α-d-Galp6Ac-(1→6)-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-ß-d-GlcpNAc-(1→, where d-Qui4N stands for 4-amino-4,6-dideoxy-d-glucose and Alo for N-((S)-1-carboxyethyl)-l-alanine (alanopine); only about half of the Gal residues are O-acetylated. This structure is unique among the Proteus O-polysaccharides, and therefore it is proposed to classify P. vulgaris HSC 438 into a new Proteus serogroup, O76. A serological cross-reactivity of HSC 438 O-antiserum and lipopolysaccharides of some other Proteus serogroups was observed and accounted for by shared epitopes on the O-polysaccharides or lipopolysaccharide core regions, including that associated with d-Qui4NAlo.

Complete chemical structure of the K135 capsular polysaccharide produced by Acinetobacter baumannii RES-546 that contains 5,7-di-N-acetyl-8-epipseudaminic acid.

A structurally diverse capsular polysaccharide (CPS) in the outer cell envelope plays an important role in the virulence of the important bacterial pathogen, Acinetobacter baumannii. More than 75 different CPS structures have been determined for the species to date, and many CPSs include isomers of a higher sugar, namely 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid. Recently, a novel isomer having the d-glycero-l-manno configuration (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac) has been identified in the CPS from A. baumannii clinical isolate RES-546 [Carbohydr. Res. 513 (2022) 108,531]. Here, the complete chemical structure of this CPS, designated K135, was elucidated. The CPS was found to have a branched tetrasaccharide K unit and to include the higher sugar as part of a 8ePse5Ac7Ac-(2 â†’ 6)-α-Gal disaccharide branching from a →3)-α-D-GlcpNAc-(1 â†’ 3)-ß-D-GlcpNAc-(1→ main chain. Assignment of glycosyltransferases encoded by the CPS biosynthesis gene cluster in the RES-546 genome enabled the first sugar of the K unit, and hence the topology of the K135 CPS, to be determined.

Loss of a Branch Sugar in the Acinetobacter baumannii K3-Type Capsular Polysaccharide Due To Frameshifts in the gtr6 Glycosyltransferase Gene Leads To Susceptibility To Phage APK37.1.

The type of capsular polysaccharide (CPS) on the cell surface of Acinetobacter baumannii can determine the specificity of lytic bacteriophage under consideration for therapeutic use. Here, we report the isolation of a phage on an extensively antibiotic resistant ST2 A. baumannii isolate AB5001 that carries the KL3 CPS biosynthesis gene cluster predicting a K3-type CPS. As the phage did not infect isolates carrying KL3 or KL22 and known to produce K3 CPS, the structure of the CPS isolated from A. baumannii AB5001 was determined. AB5001 produced a variant CPS form, K3-v1, that lacks the ß-d-GlсpNAc side chain attached to the d-Galp residue in the K3 structure. Inspection of the KL3 sequence in the genomes of AB5001 and other phage-susceptible isolates with a KL3 locus revealed single-base deletions in gtr6, causing loss of the Gtr6 glycosyltransferase that adds the missing d-GlсpNAc side chain to the K3 CPS. Hence, the presence of this sugar profoundly restricts the ability of the phage to digest the CPS. The 41-kb linear double-stranded DNA (dsDNA) phage genome was identical to the genome of a phage isolated on a K37-producing isolate and thus was named APK37.1. APK37.1 also infected isolates carrying KL116. Consistent with this, K3-v1 resembles the K37 and K116 structures. APK37.1 is a Friunavirus belonging to the Autographiviridae family. The phage-encoded tail spike depolymerase DpoAPK37.1 was not closely related to Dpo encoded by other sequenced Friunaviruses, including APK37 and APK116. IMPORTANCE Lytic bacteriophage have potential for the treatment of otherwise untreatable extensively antibiotic-resistant bacteria. For Acinetobacter baumannii, most phage exhibit specificity for the type of capsular polysaccharide (CPS) produced on the cell surface. However, resistance can arise via mutations in CPS genes that abolish this phage receptor. Here, we show that single-base deletions in a CPS gene result in alteration of the final structure rather than deletion of the capsule layer and hence affect the ability of a newly reported podophage to infect strains producing the K3 CPS.

Structure of the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) containing a 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose moiety.

A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) and identified by 1D and 2D (1)H and (13)C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

Involvement of a Phage-Encoded Wzy Protein in the Polymerization of K127 Units To Form the Capsular Polysaccharide of Acinetobacter baumannii Isolate 36-1454.

A comprehensive understanding of capsular polysaccharide (CPS) diversity is critical to implementation of phage therapy to treat panresistant Acinetobacter baumannii infections. Predictions from genome sequences can assist identification of the CPS type but can be complicated if genes outside the K locus (CPS biosynthesis gene cluster) are involved. Here, the CPS produced by A. baumannii clinical isolate 36-1454 carrying a novel K locus, KL127, was determined and compared to other CPSs. KL127 differs from KL128 in only two of the glycosyltransferase (gtr) genes. The K127 unit in 36-1454 CPS was the pentasaccharide ß-d-Glcp-(1→6)-d-ß-GalpNAc-(1→6)-α-d-Galp-(1→6)-ß-d-Glсp-(1→3)-ß-d-GalpNAc in which d-Glcp at position 4 replaces d-Galp in K128, and the glycosyltransferases encoded by the different gtr genes form the surrounding linkages. However, although the KL127 and KL128 gene clusters encode nearly identical Wzy polymerases, the linkages between K units that form the CPS chains are different, i.e., ß-d-GalpNAc-(1→3)-d-Galp in 36-1454 (K127) and ß-d-GalpNAc-(1→4)-d-Galp in KZ-1093 (K128). The linkage between K127 units in 36-1454 is the same as the K-unit linkage in five known CPS structures, and a gene encoding a Wzy protein related to the Wzy of the corresponding K loci was found encoded in a prophage genome in the 36-1454 chromosome. Closely related Wzy proteins were encoded in unrelated phage in available KL127-carrying genomes. However, a clinical isolate, KZ-1257, carrying KL127 but not the prophage was found, and K127 units in the KZ-1257 CPS were ß-d-GalpNAc-(1→4)-d-Galp linked, confirming that WzyKL127 forms this linkage and thus that the phage-encoded WzyPh1 forms the ß-d-GalpNAc-(1→3)-d-Galp linkage in 36-1454. IMPORTANCE Bacteriophage therapy is an attractive innovative treatment for infections caused by extensively drug resistant Acinetobacter baumannii, for which there are few effective antibiotic treatments remaining. Capsular polysaccharide (CPS) is a primary receptor for many lytic bacteriophages, and thus knowledge of the chemical structures of CPS produced by the species will underpin the identification of suitable phages for therapeutic cocktails. However, recent research has shown that some isolates carry additional genes outside of the CPS biosynthesis K locus, which can modify the CPS structure. These changes can subsequently alter phage receptor sites and may be a method utilized for natural phage resistance. Hence, it is critical to understand the genetics that drive CPS synthesis and the extent to which genes outside of the K locus can affect the CPS structure.

NoteIdentification of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-ulosonic acid (di-N-acetyl-8-epipseudaminic acid) in the capsular polysaccharide of Acinetobacter baumannii Res546.

A structurally diverse capsular polysaccharide that surrounds the bacterial cell plays an important role in virulence of Acinetobacter baumannii, a cause of nosocomial infections worldwide. Various isomers of 5,7-diacylamido-3,5,7,9-tetradeoxynon-2-ulosonic acid have been identified as components of bacterial polysaccharides. In this work, we report on the identification of a new isomer having the d-glycero-l-manno configuration (8-epipseudaminic acid) in the capsular polysaccharide of A. baumannii Res546. The higher sugar was isolated by Smith degradation of the polysaccharide followed by mild acid hydrolysis and identified by a comparison with all isomers using NMR spectroscopy and optical rotation.

The K89 capsular polysaccharide produced by Acinetobacter baumannii LUH5552 consists of a pentameric repeat-unit that includes a 3-acetamido-3,6-dideoxy-d-galactose residue.

Acinetobacter baumannii isolate LUH5552 carries the KL89 capsule biosynthesis gene cluster. Capsular polysaccharide (CPS) isolated from LUH5552 was analyzed by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K89 CPS structure has not been seen before in A. baumannii CPS structures resolved to date and includes a 3-acetamido-3,6-dideoxy-d-galactose (d-Fucp3NAc) residue which is rare amongst A. baumannii CPS. The K89 CPS has a →3)-α-d-GalpNAc-(1→3)-ß-d-GlcpNAc-(1→ main chain with a ß-d-Glcp-(1→2)-ß-d-Fucp3NAc-(1→6)-d-Glcp side branch that is α-(1→4) linked to d-GalpNAc. The roles of the Wzy polymerase and the four glycosyltransferases encoded by the KL89 gene cluster in the biosynthesis of the K89 CPS were assigned. Two glycosyltransferases, Gtr121 and Gtr122, link the d-Fucp3NAc to its neighboring sugars.