Structure of the Acinetobacter baumannii MAR20-4513 (K104) capsular polysaccharide (CPS) was established using Smith degradation, 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC and HMBC experiments, and modeling. The K104 CPS is composed of a branched pentasaccharide K unit containing two d-Glcp residues, and one residue each of d-GalpNAc, d-GlcpNAc and d-Galp. The following structure of the pentasaccharide repeating unit of the CPS was established:Functions of genes in the K locus of A. baumannii K104 were assigned by a comparison with sequences in the available databases and found to be in agreement with the CPS structure.

Acinetobacter baumannii is a widely distributed nosocomial pathogen that causes various acute and chronic infections, particularly in immunocompromised patients. In this study, the activities of the K9-specific virulent phage AM24 and phage-encoded depolymerase DepAPK09 were assessed using in vivo mouse sepsis and burn skin infection models. In the mouse sepsis model, in the case of prevention or early treatment, a single K9-specific phage or recombinant depolymerase injection was able to protect 100% of the mice after parenteral infection with a lethal dose of A. baumannii of the K9-type, with complete eradication of the pathogen. In the case of delayed treatment, mouse survival decreased to 70% when injected with the phage and to 40% when treated with the recombinant enzyme. In the mouse burn skin infection model, the number of A. baumannii cells on the surface of the wound and in the deep layers of the skin decreased by several-fold after treatment with both the K9-specific phage and the recombinant depolymerase. The phage and recombinant depolymerase were highly stable and retained activity under a wide range of temperatures and pH values. The results obtained contribute to expanding our understanding of the in vivo therapeutic potential of specific phages and phage-derived depolymerases interacting with A. baumannii of different capsular types.

Capsular polysaccharide (CPS) produced by two Acinetobacter baumannii isolates carrying the KL58 locus were recently determined and found to differ due to the presence of additional CPS biosynthesis genes found in prophage. Here, we have examined the CPS produced by a third KL58-carrying A. baumannii isolate, BAL114, which was recovered in 2009 in Vietnam. CPS extracted from BAL114 was studied by sugar analysis, selective solvolysis with trifluoroacetic acid in the presence of 2-methyl-1-propanol, and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. Consistent with the genetic content of KL58, the polysaccharide was found to include tetrasaccharide K58 units consisting of a 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid, d-Gal, d-Glc, and d-GalNAc. However, the linkage between the K58 units was different to the β-d-GalNAc-(1→3)-d-Glc linkage seen previously for other K58 forms. There were two CPS types in the ratio ∼1 : 1 with K58 units linked by either β-(1→3) or β-(1→4) linkages between the d-GalNAc and d-Gal residues.

The whole genome sequence from Acinetobacter baumannii MAR18-2212, an isolate recovered in 2018 from a human respiratory tract specimen in Russia, was obtained and found to carry KL95 at the capsular polysaccharide (CPS) biosynthesis K locus. KL95 includes a module of four genes (rmlB-rmlA-qdtE-qdtB) predicting synthesis of 3-acetamido-3,6-dideoxy-d-glucose (d-Quip3NAc). The structure of the K95 CPS isolated from MAR18-2212 was established using 1H and 13C NMR spectroscopy, including two-dimensional 1Н,1Н COSY, 1Н,1Н TOCSY, 1Н,1Н ROESY, 1Н,13C HSQC, and 1Н,13C HMBC experiments, and was confirmed by Smith degradation. K95 is composed of branched hexasaccharide K-units containing three d-GalpNAc residues, two d-Galp residues and one residue of d-Quip3NAc. Assignment of the enzymes encoded by KL95 established the role of rmlB-rmlA-qdtE-qdtB in the synthesis of d-Quip3NAc. Five encoded glycosyltransferases and the Wzy polymerase were also assigned to the glycosidic linkages in K95.

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 β-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Acinetobacter baumannii is a leading cause of multidrug-resistant bacterial infections worldwide, and the capsular polysaccharide (CPS) is a major virulence determinant. A previous study of A. baumannii from intubated and asymptomatic patients admitted to the intensive care unit (ICU) at the Hospital for Tropical Diseases in Ho Chi Minh City in Vietnam revealed multiple lineages with diverse antibiotic resistance profiles and CPS biosynthesis loci. Here, we show that 48_n, an asymptomatic nasal carriage isolate belonging to ST142, is extensively antibiotic resistant and carries acquired resistance determinants accounting for the resistance profile. 48_n carries the novel KL71 CPS biosynthesis locus in the chromosome. The structure of the CPS produced by 48_n was established using 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional 1Н,1Н COSY, 1Н,1Н TOCSY, 1Н,1Н ROESY, 1Н,13C HSQC, and 1Н,13C HMBC experiments, and confirmed by Smith degradation. Consistent with the genetic content of KL71, the K71 CPS was found to be made up of octasaccharide K units containing six l-rhamnose residues and one residue each of N-acetyl-d-glucosamine and d-glucuronic acid. K71 CPS was branched and closely related to the K74 CPS produced by BAL_309, an antibiotic susceptible ST142 isolate recovered from an intubated patient in the same ICU 7 years later. K71 and K74 differ only in the linkage between K units, and this is due to the replacement of a single gene at the K locus that codes for the Wzy polymerase. IMPORTANCE:The majority of Acinetobacter baumannii genomes sequenced and analyzed to develop an understanding of extensively drug-resistant (XDR) isolates belong to the globally disseminated CC2 clonal complex. While XDR isolates belonging to rarer lineages are often unexplored, detailed analyses could provide novel insights into the spread of resistance, as well as cell surface features such as the CPS that determine the specificity of non-antibiotic therapeutics required to treat XDR infections that resist antimicrobial chemotherapy. Here, we describe the properties of an XDR asymptomatic nasal carriage isolate recovered in Vietnam that belongs to ST142, a rarely encountered sequence type. We report the resistance profile and correlate this with detected resistance determinants. We also solve the structure of the CPS and reveal its relationship with CPS produced by other A. baumannii isolates.

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 β-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.

Two novel virulent phages of the genus Obolenskvirus infecting Acinetobacter baumannii, a significant nosocomial pathogen, have been isolated and studied. Phages Brutus and Scipio were able to infect A. baumannii strains belonging to the K116 and K82 capsular types, respectively. The biological properties and genomic organization of the phages were characterized. Comparative genomic, phylogenetic, and pangenomic analyses were performed to investigate the relationship of Brutus and Scipio to other bacterial viruses and to trace the possible origin and evolutionary history of these phages and other representatives of the genus Obolenskvirus. The investigation of enzymatic activity of the tailspike depolymerase encoded in the genome of phage Scipio, the first reported virus infecting A. baumannii of the K82 capsular type, was performed. The study of new representatives of the genus Obolenskvirus and mechanisms of action of depolymerases encoded in their genomes expands knowledge about the diversity of viruses within this taxonomic group and strategies of Obolenskvirus–host bacteria interaction.

We report the genome sequence of bacteriophage KpS110, which infects Klebsiella pneumoniae, a multidrug-resistant encapsulated bacterium that causes severe community-acquired and hospital-acquired infections. The phage genome is 156,801 bp, with 201 open reading frames. KpS110 is most closely related to phages of the family Ackermannviridae at the genome and proteome levels.

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.

Curtobacterium is a genus of Gram-positive bacteria within the order Actinomycetales. Some Curtobacterium species (C. flaccumfaciens, C. plantarum) are harmful pathogens of agricultural crops such as soybean, dry beans, peas, sugar beet and beetroot, which occur throughout the world. Bacteriophages (bacterial viruses) are considered to be potential curative agents to control the spread of harmful bacteria. Temperate bacteriophages integrate their genomes into bacterial chromosomes (prophages), sometimes substantially influencing bacterial lifestyle and pathogenicity. About 200 publicly available genomes of Curtobacterium species, including environmental metagenomic sequences, were inspected for the presence of sequences of possible prophage origin using bioinformatic methods. The comparison of the search results with several ubiquitous bacterial groups showed the relatively low level of the presence of prophage traces in Curtobacterium genomes. Genomic and phylogenetic analyses were undertaken for the evaluation of the evolutionary and taxonomic positioning of predicted prophages. The analyses indicated the relatedness of Curtobacterium prophage-derived sequences with temperate actinophages of siphoviral morphology. In most cases, the predicted prophages can represent novel phage taxa not described previously. One of the predicted temperate phages was induced from the Curtobacterium genome. Bioinformatic analysis of the modelled proteins encoded in prophage-derived regions led to the discovery of some 100 putative glycopolymer-degrading enzymes that contained enzymatic domains with predicted cell-wall- and cell-envelope-degrading activity; these included glycosidases and peptidases. These proteins can be considered for the experimental design of new antibacterials against Curtobacterium phytopathogens.

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.

ABSTRACT The extensively antibiotic-resistant Acinetobacter baumannii GC1(ST1IP) isolate SGH0807 from Singapore carries the KL70 gene cluster. The structure of the K70 capsular polysaccharide (CPS) produced by SGH0807 was determined using sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy. The K70 CPS consists of branched tetrasaccharide K-units and is closely related to the previously reported K9 CPS. The KL70 and KL9 loci differ in a short segment that encodes the initiating transferase for d-FucpNAc in K70 and d-GlcpNAc in K9. The two structures differ only in the identity of the “first” sugar of the K-unit, d-FucpNAc in K70 and d-GlcpNAc in K9. This difference alters the identity of one of the sugars involved in the linkage between K-units formed by the Wzy polymerase. However, KL70 and KL9 encode an identical Wzy polymerase, designated as WzyKL9, indicating that the differences between d-FucpNAc and d-GlcpNAc do not affect its function. Whether the difference in the first sugars was recognized by the depolymerases encoded by three K9-specific bacteriophages, AM24, BS46, and APK09, that hydrolyze the bond in K9 CPS formed by WzyKL9 was also examined. Purified depolymerases incubated with K70 CPS purified from SGH0807 formed oligosaccharide fragments that were monomers and dimers of the CPS cleaved at the linkage between K-units. As depolymerases encoded by phage determine host specificity by hydrolyzing specific CPS types, these phages could infect and lyse the SGH0807 K70 isolate. A. baumannii carrying KL70 were found in Singapore hospitals between 2006 and 2009. 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.

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 β-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.

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 → 6)-α-Gal disaccharide branching from a →3)-α-D-GlcpNAc-(1 → 3)-β-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.

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.

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)-β-d-GlcpNAc-(1→ main chain with a β-d-Glcp-(1→2)-β-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.

Acinetobacter baumannii is one of the most significant pathogens causing nosocomial infections. In this study, we present the characterization of novel virulent bacteriophage Cato, which specifically infects A. baumannii strains with a K102 capsular polysaccharide structure. The phage genome was annotated and analyzed. The phage shares similar genome organisation with the viruses belonging to the genus Obolenskvirus of the family Myoviridae. Linear double-stranded DNA genome of phage Cato is 45,188 bp-long, and contains 82 open reading frames. Putative functions were assigned for products of 27 predicted genes. The N-terminal domains of the tailspikes show a high level of homology, whereas the CPS-recognizing/degrading parts of the proteins are more versatile. HMM-HMM motif comparison and structural modelling of tailspike trimer (Fig. 4) confirm this complex structure. The tail fiber protein encoded in the Cato genome, can also participate in the attachment of the phage to A. baumannii bacterial surface. The gene encoding phage tailspike depolymerase was cloned, expressed, and purified. The recombinant depolymerase was found to be highly specific and formed opaque halos only on the bacterial lawn of the host bacterial strain.