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1.
Carbohydr Res ; 545: 109273, 2024 Sep 17.
Article in English | MEDLINE | ID: mdl-39326204

ABSTRACT

Capsular polysaccharide (CPS) is a heteroglycan that coats the cell surface of most isolates of the important Gram-negative bacterial pathogen, Acinetobacter baumannii. Strain MAR 15-4076, a clinical isolate recovered in Russia in 2015, was found to carry the KL129 sequence at the CPS biosynthesis K locus. The CPS was isolated from the strain and studied by sugar analysis, Smith degradation, one- and two-dimensional 1H and 13C NMR spectroscopy. It was composed of branched pentasaccharide units that include a →3)-α-l-Rhap-(1 â†’ 3)-α-l-Rhap-(1 â†’ 3)-ß-d-GlcpNAc-(1→ mainchain and α-d-ManpNAc-(1 â†’ 3)-l-Rhap side branch. Though the pentasaccharide units are identical to those that make up the K84 CPS produced by A. baumannii LUH5540, the units are linked differently via the substitution of an alternate l-Rhap residue, resulting in a difference in the overall topology of the CPS. This was due to the replacement of the Wzy polymerase gene encoded at the K locus.

2.
mSystems ; : e0094124, 2024 Sep 10.
Article in English | MEDLINE | ID: mdl-39254035

ABSTRACT

The carbapenem-resistant Acinetobacter baumannii isolate BAL062 is a clinical reference isolate used in several recent experimental studies. It is from a ventilator-associated pneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B sub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B outbreak isolates were extensively antibiotic-resistant and carry GC2-associated genomic resistance islands, AbGRI1, AbGRI2, and AbGRI3, BAL062 has lost AbGRI3 and three aminoglycoside resistance genes, armA, aacA4, and aphA1, leading to amikacin, tobramycin and kanamycin susceptibility. The location of Tn2008VAR found in the chromosome of this sub-lineage was also corrected. Like many of the outbreak isolates, BAL062 carries the KL58 gene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is provided. As information about K type is important for the development of novel CPS-targeting therapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It is most closely related to K2 and K93, sharing similar configurations and linkages between K units, and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-ulosonic acid (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetylpseudaminic acid). Inspection of publicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in geographically diverse isolates belonging to several sequence types that were recovered over two decades from clinical, animal, and environmental sources.IMPORTANCEMany published experimental studies aimed at developing a clearer understanding of the pathogenicity of carbapenem-resistant Acinetobacter baumannii strains currently causing treatment failure due to extensive antibiotic resistance are undertaken using historic, laboratory-adapted isolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. The clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone causing extensively resistant infections and has been used in various recent studies. The correlation of resistance profiles and resistance gene data is key to identifying genes available for gene knockout and complementation analyses, and we have mapped the antibiotic resistance genes to find candidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently under investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat CRAb infections often exhibit specificity for specific structural epitopes of the capsular polysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of the CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming and annotation are important for locus identification and interpretation of experimental studies, we also have correlated automatic annotations to the standard gene names.

3.
J Antimicrob Chemother ; 79(5): 1014-1018, 2024 05 02.
Article in English | MEDLINE | ID: mdl-38530861

ABSTRACT

BACKGROUND: The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. OBJECTIVES: To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. METHODS: The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. RESULTS: SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. CONCLUSIONS: This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.


Subject(s)
Acinetobacter baumannii , Aminoglycosides , Anti-Bacterial Agents , Chromosomes, Bacterial , Drug Resistance, Bacterial , Acinetobacter baumannii/genetics , Acinetobacter baumannii/drug effects , Aminoglycosides/pharmacology , Italy , Anti-Bacterial Agents/pharmacology , Chromosomes, Bacterial/genetics , Drug Resistance, Bacterial/genetics , Humans , Genomic Islands/genetics , DNA Transposable Elements/genetics , Genes, Bacterial/genetics , Sequence Analysis, DNA , Microbial Sensitivity Tests , Acinetobacter Infections/microbiology , Polymerase Chain Reaction , Genome, Bacterial , DNA, Bacterial/genetics
4.
Carbohydr Res ; 538: 109097, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38555658

ABSTRACT

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure: This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the ß-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/genetics , Acinetobacter baumannii/chemistry , Bacterial Capsules/chemistry , Polysaccharides/analysis , Magnetic Resonance Spectroscopy , Multigene Family , Sugars , Polysaccharides, Bacterial/chemistry
5.
Int J Biol Macromol ; 262(Pt 1): 130045, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38336317

ABSTRACT

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. K239 consists of branched heptasaccharide repeats (K-units) comprised of five residues of l-rhamnose (l-Rhap), and one residue each of d-glucuronic acid (d-GlcpA) and N-acetyl-d-glucosamine (d-GlcpNAc). The structure of K239 is closely related to that of the A. baumannii K86 CPS type, though the two differ in the 2,3-substitution patterns on the l-Rhap residue that is involved in the linkage between K-units in the CPS polymer. This structural difference was attributed to the presence of a gtr221 glycosyltransferase gene and a wzyKL239 polymerase gene in KL239 that replaces the gtr80 and wzyKL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel WzyKL239 polymerase encoded by KL239 that forms the ß-d-GlcpNAc-(1→2)-l-Rhap linkage between K239 units. A. baumannii MAR19-4435 was found to be non-susceptible to infection by the APK86 bacteriophage, which encodes a depolymerase that specifically cleaves the linkage between K-units in the K86 CPS, indicating that the difference in 2,3-substitution of l-Rhap influences the susceptibility of this isolate to bacteriophage activity.


Subject(s)
Acinetobacter baumannii , Polysaccharides, Bacterial , Polysaccharides, Bacterial/chemistry , Acinetobacter baumannii/genetics , Acinetobacter baumannii/chemistry , Bacterial Capsules/chemistry , Nucleotidyltransferases/genetics , Multigene Family
6.
Carbohydr Res ; 535: 109020, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38150754

ABSTRACT

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.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/chemistry , Bacterial Capsules/chemistry , Polysaccharides/analysis , Sialic Acids/chemistry , Multigene Family , Polysaccharides, Bacterial/chemistry
7.
Microbiol Spectr ; 11(6): e0302523, 2023 Dec 12.
Article in English | MEDLINE | ID: mdl-37975684

ABSTRACT

IMPORTANCE: Bacteriophage show promise for the treatment of Acinetobacter baumannii infections that resist all therapeutically suitable antibiotics. Many tail-spike depolymerases encoded by phage that are able to degrade A. baumannii capsular polysaccharide (CPS) exhibit specificity for the linkage present between K-units that make up CPS polymers. This linkage is formed by a specific Wzy polymerase, and the ability to predict this linkage using sequence-based methods that identify the Wzy at the K locus could assist with the selection of phage for therapy. However, little is known about the specificity of Wzy polymerase enzymes. Here, we describe a Wzy polymerase that can accommodate two different but similar sugars as one of the residues it links and phage depolymerases that can cleave both types of bond that Wzy forms.


Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Bacteriophages , Humans , Acinetobacter baumannii/genetics , Bacterial Capsules/metabolism , Multigene Family , Polysaccharides, Bacterial/analysis
8.
Microb Genom ; 9(6)2023 06.
Article in English | MEDLINE | ID: mdl-37310786

ABSTRACT

The outer core locus (OCL) that includes genes for the synthesis of the variable outer core region of the lipooligosaccharide (LOS) is one of the key epidemiological markers used for tracing the spread of Acinetobacter baumannii, a bacterial pathogen of global concern. In this study, we screened 12 476 publicly available A. baumannii genome assemblies for novel OCL sequences, detecting six new OCL types that were designated OCL17-OCL22. These were compiled with previously characterized OCL sequences to create an updated version of the A. baumannii OCL reference database, providing a total of 22 OCL reference sequences for use with the bioinformatics tool Kaptive. Use of this database against the 12 476 downloaded assemblies found OCL1 to be the most common locus, present in 73.6 % of sequenced genomes assigned by Kaptive with a match confidence score of good or above. OCL1 was most common amongst isolates belonging to sequence types (STs) ST1, ST2, ST3 and ST78, all of which are over-represented clonal lineages. The highest level of diversity in OCL types was found in ST2, with eight different OCLs identified. The updated OCL reference database is available for download from GitHub (https://github.com/klebgenomics/Kaptive; under version v. 2.0.5), and has been integrated for use on Kaptive-Web (https://kaptive-web.erc.monash.edu/) and PathogenWatch (https://pathogen.watch/), enhancing current methods for A. baumannii strain identification, classification and surveillance.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/genetics , Interleukin-1 Receptor-Like 1 Protein , Databases, Nucleic Acid , Lipopolysaccharides/genetics
9.
FEMS Microbes ; 4: xtad009, 2023.
Article in English | MEDLINE | ID: mdl-37333444

ABSTRACT

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

10.
Microbiol Spectr ; 11(1): e0363122, 2023 02 14.
Article in English | MEDLINE | ID: mdl-36651782

ABSTRACT

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.


Subject(s)
Acinetobacter baumannii , Bacteriophages , Acinetobacter baumannii/metabolism , Sugars/metabolism , Polysaccharides, Bacterial/genetics , Myoviridae , Bacteriophages/genetics , Bacteriophages/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Bacterial Capsules/metabolism
11.
Carbohydr Res ; 523: 108726, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36446189

ABSTRACT

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.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/chemistry , Bacterial Capsules/chemistry , Polysaccharides/analysis , Glycosyltransferases/genetics , Multigene Family , Sugars , Polysaccharides, Bacterial/chemistry
12.
Microb Genom ; 8(10)2022 10.
Article in English | MEDLINE | ID: mdl-36214673

ABSTRACT

Several novel non-antibiotic therapeutics for the critical priority bacterial pathogen, Acinetobacter baumannii, rely on specificity to the cell-surface capsular polysaccharide (CPS). Hence, prediction of CPS type deduced from genes in whole genome sequence data underpins the development and application of these therapies. In this study, we provide a comprehensive update to the A. baumannii K locus reference sequence database for CPS typing (available in Kaptive v. 2.0.1) to include 145 new KL, providing a total of 237 KL reference sequences. The database was also reconfigured for compatibility with the updated Kaptive v. 2.0.0 code that enables prediction of 'K type' from special logic parameters defined by detected combinations of KL and additional genes outside the K locus. Validation of the database against 8994 publicly available A. baumannii genome assemblies from NCBI databases identified the specific KL in 73.45 % of genomes with perfect, very high or high confidence. Poor sequence quality or the presence of insertion sequences were the main reasons for lower confidence levels. Overall, 17 KL were overrepresented in available genomes, with KL2 the most common followed by the related KL3 and KL22. Substantial variation in gene content of the central portion of the K locus, that usually includes genes specific to the CPS type, included 34 distinct groups of genes for synthesis of various complex sugars and >400 genes for forming linkages between sugars or adding non-sugar substituents. A repertoire of 681 gene types were found across the 237 KL, with 88.4 % found in <5 % of KL.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/genetics , Bacterial Capsules/genetics , DNA Transposable Elements , Multigene Family , Polysaccharides, Bacterial/genetics
13.
Int J Biol Macromol ; 217: 515-521, 2022 Sep 30.
Article in English | MEDLINE | ID: mdl-35843396

ABSTRACT

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.


Subject(s)
Acinetobacter baumannii , Acetylgalactosamine/analogs & derivatives , Acinetobacter baumannii/chemistry , Acinetobacter baumannii/genetics , Bacterial Capsules/chemistry , Bacterial Capsules/genetics , Fucose/analogs & derivatives , Galactose/analysis , Glycosyltransferases/genetics , Polysaccharides, Bacterial/chemistry
14.
Int J Biol Macromol ; 218: 310-316, 2022 Oct 01.
Article in English | MEDLINE | ID: mdl-35872309

ABSTRACT

Two acylated forms of the higher sugar, 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid called pseudaminic acid, Pse5Ac7Ac and Pse5Ac7RHb where R indicates (R)-3-hydroxybutanoyl, have been found to occur in many capsular polysaccharide (CPS) types produced by isolates of an important human pathogen, Acinetobacter baumannii. The presence of either a psaABCEDF or psaABCGHF gene module at the K locus (KL) for CPS biosynthesis determines the type of the variant produced. Here, an A. baumannii clinical isolate 52-249, recovered in 2015 in Moscow, Russia, was found to include a novel psaABCIJF gene module in the KL218 sequence at the K locus. The CPS from 52-249 was extracted and studied by sugar analysis and partial acid hydrolysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. A branched tetrasaccharide repeating unit was identified, which included a →3)-α-d-Galp-(1→6)-α-d-GlcpNAc-(1→3)-ß-d-GalpNAc-(1→ main chain and Pse5Ac7Ac attached as a side branch, indicating that the psaABCIJF gene module is associated with synthesis of this variant. The K218 CPS was found to be structurally related to the K46 CPS of A. baumannii, and a comparison of the two structures enabled the assignment of glycosyltransferases. A KpsS3 protein for the α-(2→6) linkage of the Pse5Ac7Ac residue to D-Galp in K218 was identified.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/chemistry , Bacterial Capsules/chemistry , Dietary Carbohydrates/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Polysaccharides, Bacterial/chemistry , Sialic Acids , Sugars/metabolism
15.
Int J Biol Macromol ; 218: 447-455, 2022 Oct 01.
Article in English | MEDLINE | ID: mdl-35872312

ABSTRACT

The K98 capsular polysaccharide (CPS) from the Acinetobacter baumannii clinical isolate, REV-1184, was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The CPS was found to consist of linear tetrasaccharide repeats (K-units) that include one residue each of d-GlcpNAc, d-GalpNAc, 2-acetamido-2-deoxy-d-galacturonic acid (d-GalpNAcA), and 2-acetamido-2,6-dideoxy-d-glucose (N-acetylquinovosamine, d-QuipNAc), with the GalpNAc residue decorated with a (R)-configurated 4,6-pyruvic acid acetal group. The CPS has a similar composition to that of A. baumannii K4 but the topology of the tetrasaccharide K-unit is different (linear in K98 versus branched in K4). This was due to a difference in sequence for the Wzy polymerases encoded by the CPS biosynthesis gene clusters KL98 and KL4, with the WzyK98 polymerase forming a ß-d-QuipNAc-(1→3)-d-GalpNAc linkage between the K98 units.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/chemistry , Acinetobacter baumannii/genetics , Multigene Family , Polysaccharides, Bacterial/chemistry , Pyruvates
16.
Microbiol Spectr ; 10(3): e0150321, 2022 06 29.
Article in English | MEDLINE | ID: mdl-35475638

ABSTRACT

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.


Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Bacteriophages , Acinetobacter baumannii/chemistry , Acinetobacter baumannii/genetics , Acinetobacter baumannii/metabolism , Bacterial Capsules/metabolism , Humans , Polymerization , Polysaccharides, Bacterial/metabolism
17.
Antimicrob Agents Chemother ; 66(1): e0180721, 2022 01 18.
Article in English | MEDLINE | ID: mdl-34662195

ABSTRACT

To enhance the utility of the genetically diverse panel of Acinetobacter baumannii isolates reported recently by Galac and coworkers (M. R. Galac, E. Snesrud, F. Lebreton, J. Stam, et al., Antimicrob Agents Chemother 64:e00840-20, 2020, https://doi.org/10.1128/AAC.00840-20) and to identify the novel KL and OCL, all of the gene clusters that direct the biosynthesis of capsular polysaccharide and of the outer core of lipooligosaccharide, respectively, were reexamined. The nine KL and one OCL previously recorded as novel were identified, and nine further novel KL and two OCL were found.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/genetics , Multigene Family
18.
Microb Genom ; 7(12)2021 12.
Article in English | MEDLINE | ID: mdl-34874246

ABSTRACT

Acinetobacter baumannii is an opportunistic pathogen that is difficult to treat due to its resistance to extreme conditions, including desiccation and antibiotics. Most strains causing outbreaks around the world belong to two main global lineages, namely global clones 1 and 2 (GC1 and GC2). Here, we used a combination of Illumina short read and MinION (Oxford Nanopore) long-read sequence data with a hybrid assembly approach to complete the genome sequence of two antibiotic-sensitive GC1 strains, Ex003 and Ax270, recovered in Lebanon from water and a rectal swab of a cat, respectively. Phylogenetic analysis of Ax270 and Ex003 with 186 publicly available GC1 genomes revealed two major clades, including five main lineages (L1-L5), and four single-isolate lineages outside of the two clades. Ax270 and Ex003, along with AB307-0294 and MRSN7213 (both predicted antibiotic-susceptible isolates) represent these individual lineages. Antibiotic resistance islands and transposons interrupting the comM gene remain important features in L1-L5, with L1 associated with the AbaR-type resistance islands, L2 with AbaR4, L3 strains containing either AbaR4 or its variants as well as Tn6022::ISAba42, and L4 and L5 associated with Tn6022 or its variants. Analysis of the capsule (KL) and outer core (OCL) polysaccharide loci further revealed a complex evolutionary history probably involving many recombination events. As more genomes become available, more GC1 lineages continue to emerge. However, genome sequence data from more diverse geographical regions are needed to draw a more accurate population structure of this globally distributed clone.


Subject(s)
Acinetobacter baumannii/classification , Anti-Bacterial Agents/pharmacology , Whole Genome Sequencing/methods , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Evolution, Molecular , Genome Size , Genome, Bacterial , High-Throughput Nucleotide Sequencing , Lebanon , Microbial Sensitivity Tests , Phylogeny
19.
Int J Biol Macromol ; 193(Pt B): 1294-1300, 2021 Dec 15.
Article in English | MEDLINE | ID: mdl-34757131

ABSTRACT

Whole genome sequence from Acinetobacter baumannii isolate Ab-46-1632 reveals a novel KL144 capsular polysaccharide (CPS) biosynthesis gene cluster, which carries genes for d-glucuronic acid (D-GlcA) and l-rhamnose (l-Rha) synthesis. The CPS was extracted from Ab-46-1632 and studied by 1H and 13C NMR spectroscopy, including a two-dimensional 1H,13C HMBC experiment and Smith degradation. The CPS was found to have a hexasaccharide repeat unit composed of four l-Rhap residues and one residue each of d-GlcpA and N-acetyl-d-glucosamine (D-GlcpNAc) consistent with sugar synthesis genes present in KL144. The K144 CPS structure was established and found to be related to those of A. baumannii K55, K74, K85, and K86. A comparison of the corresponding gene clusters to KL144 revealed a number of shared glycosyltransferase genes correlating to shared glycosidic linkages in the structures. One from the enzymes, encoded by only KL144 and KL86, is proposed to be a novel multifunctional rhamnosyltransfaerase likely responsible for synthesis of a shared α-l-Rhap-(1 â†’ 2)-α-L-Rhap-(1 â†’ 3)-L-Rhap trisaccharide fragment in the K144 and K86 structures.


Subject(s)
Acinetobacter baumannii/genetics , Bacterial Proteins/genetics , Polysaccharides, Bacterial/genetics , Genetic Linkage/genetics , Glycosyltransferases/genetics , Magnetic Resonance Spectroscopy/methods , Multigene Family/genetics , Whole Genome Sequencing/methods
20.
Sci Rep ; 11(1): 21741, 2021 11 05.
Article in English | MEDLINE | ID: mdl-34741090

ABSTRACT

Identification of novel therapeutic targets is required for developing alternate strategies to treat infections caused by the extensively drug-resistant bacterial pathogen, Acinetobacter baumannii. As capsular polysaccharide (CPS) is a prime virulence determinant required for evasion of host immune defenses, understanding the pathways for synthesis and assembly of this discrete cell-surface barrier is important. In this study, we assess cell-bound and cell-free CPS material from A. baumannii AB5075 wildtype and transposon library mutants and demonstrate that the Wzi outer membrane protein is required for the proper assembly of the CPS layer on the cell surface. Loss of Wzi resulted in an estimated 4.4-fold reduction in cell-associated CPS with a reciprocal increase in CPS material shed in the extracellular surrounds. Transmission electron microscopy revealed a disrupted CPS layer with sparse patches of CPS on the external face of the outer membrane when Wzi function was lost. However, this genotype did not have a significant effect on biofilm formation. Genetic analysis demonstrated that the wzi gene is ubiquitous in the species, though the nucleotide sequences were surprisingly diverse. Though divergence was not concomitant with variation at the CPS biosynthesis K locus, an association between wzi type and the first sugar of the CPS representing the base of the structure most likely to interact with Wzi was observed.


Subject(s)
Acinetobacter baumannii/metabolism , Bacterial Capsules/metabolism , Bacterial Outer Membrane Proteins/metabolism , Polysaccharides, Bacterial/metabolism , Acinetobacter baumannii/genetics , Acinetobacter baumannii/ultrastructure , Bacterial Outer Membrane Proteins/genetics , Biofilms , Genetic Complementation Test , Genetic Variation
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