Structural insights and therapeutic targets in Acinetobacter baumannii capsule biosynthesis

Abstract

Gram‐negative bacteria produce nonulosonic acids, a family of nine‐carbon sugars that mimic host sialic acids and may contribute to bacterial virulence. The nonulosonic acid C8‐epimeric 5,7‐di‐N‐acetyllegionaminic acid (8‐epi‐Leg5,7Ac2) is a component of the K‐locus 49 (KL49) capsule of hypervirulent Acinetobacter baumannii, and its epimerization is proposed to be mediated by ElaA, ElaB, and ElaC. We identified a KL49 strain that induces lethal sepsis in mice and found deletion of elaA, elaB, or elaC abolished lethality and markedly reduced capsule production. Crystal structures of ElaB, ElaA, and ElaC revealed conserved N‐acetyl recognition underlying CMP‐Leg5,7Ac2 synthesis and regioselective C8 epimerization. Together, these genetic, structural, and in vivo findings identify ElaABC as potential antivirulence targets in emerging hypervirulent KL49 A. baumannii strains.

Hypervirulent KL49 A. baumannii's capsular polysaccharide contains the nonulosonic acid 8‐epi‐Leg5,7Ac2, synthesized by epimerization via ElaA, ElaB, and ElaC. Crystal structures of ElaA, ElaB, and ElaC reveal their role in CMP‐Leg5,7Ac2 synthesis and regioselective C8 epimerization. Deletion of elaA, elaB, or elaC leads to reduced capsule formation, highlighting potential antivirulence targets in hypervirulent strains.

Abbreviations

CFU, colony‐forming unit

CMP, Cytidine 5′‐monophosphate

CPS, capsular polysaccharide

CRAB, carbapenem‐resistant Acinetobacter baumannii

CTP, Cytidine 5′‐triphosphate

HGT, horizontal gene transfer

IPTG, Isopropyl β‐D‐1‐thiogalactopyranoside

NAD ⁺, nicotinamide adenine dinucleotide

NADH, reduced nicotinamide adenine dinucleotide

NADP ⁺, nicotinamide adenine dinucleotide phosphate

NulO, nonulosonic acid

PEG, polyethylene glycol

RMSD, root‐mean‐square deviation

Antimicrobial resistance is a critical global health threat that increasingly limits treatment options for bacterial infections. The ESKAPE pathogens comprise six highly virulent, multidrug‐resistant bacteria that account for a large proportion of hospital‐acquired infections worldwide. This group includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. Owing to their diverse resistance mechanisms, these pathogens represent major clinical threats and frequently lead to infections that are difficult to treat [1]. A central virulence determinant among these organisms is the polysaccharide capsule, which functions as a physical and immunological shield. For example, Klebsiella pneumoniae capsules layer physically prevents phagocytosis and inhibits complement activation, promoting bloodstream and pulmonary persistence [2], while Pseudomonas aeruginosa produces exopolysaccharides such as alginate that enable robust biofilm formation on medical devices, increasing resistance to both antibiotics and environmental stress [3]. In A. baumannii, the capsule has been identified as an important virulence factor that contributes to resistance against complement and provides protection in human ascites fluid or serum [4].

Recently, persistent infection by hypervirulent A. baumannii strains, such as the lethal A. baumannii LAC‐4, isolated from Los Angeles hospitals, has been reported [5, 6, 7]. LAC‐4 causes fatal outcomes in mouse models, with multiple lesions in the spleen and lungs [8]. Genomic analysis of A. baumannii LAC‐4 suggests that the K‐locus KL49, which is involved in capsular polysaccharide (CPS) biosynthesis and transport, is a key determinant of hypervirulence (Fig. 1A) [9]. Compared to other pathogenic A. baumannii genomes, LAC‐4 uniquely possesses two potential virulence factors, ABLAC_37000 and ABLAC_37010, renamed as ElaC and ElaB, respectively. In subsequent studies, knock out of one of the KL49 genes responsible for putative glycosyltransferase activity (gtr100, Fig. 1A) was noted to significantly reduce virulence, although the exact mechanism remains unclear [10]. The repeating unit structure of A. baumannii LAC‐4 polysaccharide has been verified using NMR analysis, revealing a linear linkage of trisaccharides composed of α‐L‐fucosamine, α‐D‐glucosamine, and 8‐epi‐Leg5,7Ac2 in CPS [11, 12]. The molecular mechanisms underlying this linkage and its contribution to pathogenicity remain to be fully elucidated.

Fig. 1.

Structure of the KL49 locus in CRAB 1707 and reactions mediated by ElaA, ElaB, and ElaC. (A) Genetic organization of the KL49 locus in CRAB 1707. Arrows indicate the transcription direction. Genes involved in Leg5,7Ac2 biosynthesis and 8‐epi‐Leg5,7Ac2 epimerization are labeled in blue and red, respectively; the qnr3 (wbpV) pseudogene, split into two ORFs, is shown in white. The position of the termination codon is indicated by a black arrow. A schematic representation of ElaA, ElaB, and ElaC illustrates the biosynthetic pathway of 8‐epi‐Leg5,7Ac2. (B) Comparison of the OCLs from CRAB 1707 and LAC‐4. (C) clinker analysis of CRAB 1707 KL49 and V. vulnificus YJ016 nab gene cluster.

Bacterial analogs of sialic acid, collectively termed nonulosonic acids (NulOs), are nine‐carbon monosaccharides observed on the surfaces of various human pathogens [13]. NulOs are typically present at the terminal position of repeating units in polysaccharide. These polysaccharides exist as lipooligosaccharides or CPS, both of which are key virulence factors in A. baumannii infection [14, 15]. A stereochemical variant of Leg5,7Ac2, 8‐epi‐Leg5,7Ac2, is present in A. baumannii strain LAC‐4 [16]. This stereochemical variation is proposed to be mediated by three enzymes, ElaA, ElaB, and ElaC at the KL49 locus (Fig. 1A) [16]. Based on amino acid sequence homology, ElaC and ElaA are predicted to function as NAD(P)H‐dependent enzymes, while ElaB is predicted to be a CTP‐transferase. ElaA and ElaC are predicted to be responsible for the reduction and oxidation at the carbon‐8 position of Leg5,7Ac2, thus playing a key role in the stereochemical modification that influences bacterial virulence [16]. ElaB participates in the activation of Leg5,7Ac2 by CTP, thereby facilitating the formation of CMP‐Leg5,7Ac2 (Fig. 1A).

In this study, we aimed to elucidate the substrate specificity underlying the the virulence‐associated sugar 8‐epi‐Leg5,7Ac2 in KL49 A. baumannii. We identified a hypervirulent, carbapenem‐resistant A. baumannii (CRAB) KL49 strain, CRAB1707, from a national pathogen resource bank in Korea. Pronounced virulence in vivo CRAB1707 was confirmed in a mouse septic shock model, prompting us to investigate the molecular basis and origin of its hypervirulence [17, 18] Mutational analysis demonstrated that elaABC genes are essential for capsule biosynthesis. In a mouse infection model, 1 × 10⁷ CFU of CRAB1707 mutants lacking elaA, elaB, or elaC, an inoculum that causes severe morbidity and mortality in the wild‐type strain, showed no significant pathogenicity. We determined the crystal structures of ElaA, ElaB, and ElaC to understand the molecular mechanisms underlying biosynthesis of 8‐epi‐Leg5,7Ac2 [16, 19]. To the best of our knowledge, this is the first attempt to explore the structure–function relationship of enzymes responsible for CPS NulO modification in A. baumannii. A comprehensive investigation of the polysaccharide‐modifying enzymes reported herein is crucial for understanding their evolution and functional significance in 8‐epi‐Leg5,7Ac2 biosynthesis and provides a foundation for developing targeted inhibitors. This study provides new insight into the virulence mechanisms of hypervirulent KL49 A. baumannii and paves the way for developing targeted treatments to combat multidrug resistance and severe pathogenicity.

Materials and methods

Genome sequencing and analysis

Whole‐genome sequencing was performed by Macrogen (Seoul, Republic of Korea) using both the HiSeq X Ten (Illumina, San Diego, CA, USA) and PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA) platforms. The genome sequence data of Acinetobacter baumannii strain CRAB1707 generated in this study have been deposited at DDBJ/ENA/GenBank under the accession JBWQFK000000000. Homology comparisons between CRAB 1707 and LAC‐4 or V. vulnificus YJ016 were performed by Clinker program, which was facilitated through the CAGECAT server [20, 21]. Phylogenetic analysis was performed using the NCBI BLAST algorithm against the refseq_select database. The resulting distance trees were visualized and annotated using iTOL [22]. Additionally, the GC content at the third codon position (GC3) was calculated using the CAIcal server [23].

Generation of deletion mutants of A. baumannii CRAB 1707

The elaA, elaB, and elaC genes of A. baumannii CRAB 1707 were deleted using an overlap extension PCR method, as previously described [24]. A mutated DNA fragment without each gene was amplified and combined with a gene that confers hygromycin resistance PCR. The combined DNA fragments were ligated into FspI‐digested suicide vector pHKD 01 to generate pOH‐elaA/pOH‐elaB/pOH‐elaC. The Escherichia coli S17‐1 λ pir strain was used as a conjugal donor to A. baumannii CRAB 1707. Following conjugation, sucrose counter‐selection was performed to resolve the integrated suicide vector and generate precise in‐frame deletion mutants.

Capsule isolation

Bacterial cells were negatively stained with nigrosin for capsule comparison between CRAB 1707 and the deletion mutants, then observed under a bright‐field microscope using a 100× oil immersion objective (NA 1.25). To minimize diffraction‐related optical effects caused by the slit‐like halo structure, all images were captured by focusing on the plane where the halo appeared thinnest for each strain. CRAB 1707 and the deletion mutants were grown overnight in 7 mL Luria Bertani (LB) medium. Cells were harvested, resuspended in 2 mL lysis buffer, and processed as previously described [25]. Precipitated capsules were analyzed using gradient sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE, 8–14%).

Cloning, protein expression and purification

elaA, elaB, and elaC genes were amplified from the genomic DNA of CRAB 1707 and cloned into the pET28a vector (Novagen, Madison, WI, USA) using the restriction sites NdeI and XhoI. Each protein contained an N‐terminal His₆ tag followed by a thrombin cleavage site. Proteins were expressed in E. coli BL21 (DE3) strain by inducing protein expression with 0.2 mm IPTG at 25 °C for 10 h. For purification, the cells were resuspended in 20 mm Tris pH 8 and 100 mm NaCl and homogenized by sonication. Cell lysates were purified using HisTrap HP affinity chromatography (GE Healthcare, Chicago, IL, USA). Thrombin was used to cleave the His‐tag after affinity chromatography, and the proteins were further purified by anion chromatography.

Ethics statement

All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Konkuk University, Republic of Korea (Approval No. KU25226‐1).

Survival test

Forty female ICR mice (6 weeks, n = 10 per group) were randomly assigned to four groups. Mice were housed under specific pathogen‐free conditions in individually ventilated cages with five animals per cage controlled environmental conditions. Sterilized rodent chow and distilled water were provided ad libitum throughout the study. Animals were monitored for clinical signs of infection or distress throughout the experimental period. All procedures were conducted in accordance with institutional animal care guidelines to minimize animal suffering. Mice were infected intraperitoneally with CRAB NMS 1707 or the respective mutant strains at a dose of 1 × 10⁷ CFU per mouse in a total volume of 200 μL. Mice were monitored every hour for the first 36 h, and subsequently every 6 h up to 96 h. At the end of the survival study, surviving mice were euthanized by CO₂ inhalation in accordance with institutional animal care and use guidelines.

Crystallization and crystal structure determination

ElaB‐CTP complex was crystallized using 0.2 m magnesium formate and 20% w/v polyethyleneglycol (PEG) 3350 in the presence of 5 mm CTP disodium salt and 10 mm magnesium chloride. ElaC in complex with 5 mm NADH was cocrystallized with 0.2 m sodium acetate and 20% w/v PEG 3350. ElaA in complex with 5 mm NADP⁺ was co‐crystallized with 18% (v/v) 2‐propanol, 0.1 m sodium citrate tribasic at pH 5.5, and 20% (w/v) PEG 4000. All protein concentrations were set to 15 mg/mL.

Crystals were soaked in 25% ethylene glycol containing crystallization buffer prior to the data collection at BL 7A (Pohang Accelerator Laboratory, Pohang, Republic of Korea). All the diffraction images were processed using XDS program package [26]. The structures of ElaA, ElaB, and ElaC were solved by molecular replacement using the coordinates of PDB entries 3EZY, 6IFD, and 6UH2 as search models, respectively, with Phaser [27]. All the structures were modeled using program Coot and refined by program Phenix [28, 29]. Data collection and refinement statistics are summarized in Table S1. Molecular models shown in Figs 3, 4, 5 were rendered using pymol [30]. Multiple sequence alignment was visualized by ESPript [31].

Fig. 3.

Structural basis of CMP‐Leg5,7Ac2 binding by ElaB. (A) Dimeric structure of ElaB in complex with CTP. (B) CTP and ethylene glycol (EG) bound in the active site of ElaB. (C) CTP and Leg5,7Ac2 (LGA) modeled in the active site of ElaB. (D) Structure of PseF (gray ribbon) in complex with CMP‐Pse (PDB ID: 6IFD). Residues involved in Leg5,7Ac2 binding are rendered as sticks and labeled. Molecular graphics were rendered using pymol.

Fig. 4.

Structure of ElaA. (A) The dimeric structure of ElaA, depicted in a ribbon model. (B) Active site structure of ElaA in complex with NADP⁺. The modeled adenosine‐2′,5′‐diphosphate (light orange) is overlaid with the electron density map mesh. The nicotinamide mononucleotide moiety (dark gray) was modeled by structural superposition with the structure of an oxidoreductase from Listeria innocua WlbA, in complex with NAD⁺ (PDB entry 3E18). Residues relevant to catalytic activity are rendered as sticks and labeled. (C) The active site structure of WlbA which shows the binding site of the sugar nucleotide, UDP‐GlnNAc. (D) Presumed binding mode of CMP‐Leg5,7Ac2 in ElaA shown with a transparent electrostatic potential surface. Residues involved with CMP‐Leg5,7Ac2 are rendered and labeled. Catalytic residues of H181 and K91 are indicated by arrows. Molecular graphics were rendered using pymol.

Fig. 5.

Structural features of ElaC in epimerization at the C8 position of Leg5,7Ac2. (A) Tetrameric assembly of ElaC. NAD⁺ is shown as spheres. (B) Close‐up view of the ElaC active site. The acetate ion, ethylene glycol (EG), and NADH are rendered as stick models and overlaid with the electron density map mesh. (C) Structural superposition between ElaC (light pink) and human mtKAR (PDB ID 4CQL, chain L, gray). The elongated V150–P165 loop is enclosed by a dashed line, with individual residues indicated by arrows. (D) Modeled CMP‐8‐keto‐Leg5,7Ac2 (gray sticks) in the ElaC active site, displayed with a transparent electrostatic potential surface. The ElaC residues involved in catalysis and recognition of the Leg5,7Ac2 carboxylate group are shown as sticks. CMP‐8‐keto‐Leg5,7Ac2 is depicted in gray sticks. Molecular graphics were rendered using pymol.

Structural modeling of substrates bound to ElaABC

Docking studies were performed using Maestro (version 14.4.138). Ligands were prepared by LigPrep in which pH 7.4 +/−2. Induced Fit Docking was employed to simulate ligand binding without constraints. The resulting docking poses were analyzed and filtered based on the distance between the 8‐hydroxyl group of the substrate and the nicotinamide ring of NAD(P)H, ensuring the selection of biologically relevant conformations for further structural and mechanistic insights.

Results

Hypervirulence of the KL‐49 strain

A CRAB strain carrying the KL49 capsule type was screened from strains obtained from the Pathogen Resource Bank of the Korea Disease Control and Prevention Agency (KDCA), by polymerase chain reaction (PCR) to genotype gtr100, gtr20 and elaA genes, leading to the identification of the NMS 1707 strain as a KL49 strain (CRAB 1707). To determine the genotype of this strain, we performed whole‐genome sequencing of CRAB 1707. The assembled genome consisted of two contigs corresponding to the chromosome (3 956 900 bp) and a plasmid (88 559 bp). The isolate belonged to ST2 (Pasteur scheme), corresponding to ST74 (Oxford scheme), consistent with the global clone 2 lineage [32]. Kaptive analysis confirmed CRAB 1707 to belong to KL49 with 100.0% coverage and 99.97% amino acid identity (Fig. 1A) [33, 34].

We found the key enzymes that mediate conversion of Leg5,7Ac2 to 8‐epi‐Leg5,7Ac2 in the locus, ElaB, ElaA, and ElaC, in the predicted order (Fig. 1A). All three proteins matched the Kaptive reference (A. baumannii BAL_173) with 100% amino acid identity except for ElaB, which has a single amino acid variation (R219 instead of S219). A premature stop codon in the middle of wbpV renders it a pseudogene in CRAB 1707, indicating that CRAB 1707 is KL49‐type similar to A. baumannii LAC‐4 [9]. This differs from ISAba1 insertion observed in the wbpL gene of the ST10‐type A. baumannii NL6 strain, which is also identified as KL49 (Fig. 1A) [9, 11]. The inactivation of wbpV in LAC‐4 and the ISAba1‐mediated disruption of wbpL in A. baumannii NL6 likely represent convergent evolutionary events [11]. These distinct genetic alterations appear to be strategic adaptations to maintain or modify the KL49 capsule phenotype, potentially enhancing strain fitness or immune evasion.

Comparison of outer core locus (OCL) showed CRAB1707 carries OCL1 (100% coverage, 98.88% identity) by Kaptive [33, 35], whereas LAC‐4 carries OCL2‐type outer core locus (Fig. 1B) [9], indicating that KL49 capsules can be associated with distinct OCL types. The first six ORFs are conserved between strains (Fig. 1B), suggesting the KL49 capsule is conserved while the OCL region appears more variable and may have undergone independent reassortment.

We searched for homologs of elaABC genes in other species and found multiple V. vulnificus species to have these genes. Genomic analysis of Vibrio vulnificus YJ016, which is known to cause severe sepsis in human hosts, revealed a specialized O‐antigen/capsule biosynthetic locus (VV0311–VV0326) integrated into the LPS core biosynthesis cluster (Fig. 1C) [36, 37]. This locus encompasses a complete set of genes for Leg5,7Ac2 biosynthesis, notably including the ElaABC cassette (CMP‐Leg5,7Ac2 8‐epimerase). The amino acid sequence identities of ElaA, ElaB, and ElaC to VV0315, Nab1, and PseA are 62%, 77%, and 78%, respectively. The remarkable synteny observed between the elaABC cassette of A. baumannii CRAB1707 and its homologs in V. vulnificus YJ016 suggests that this cluster operates as a discrete functional module acquired through horizontal gene transfer (HGT). This is further supported by the phylogenetic placement of the CRAB1707 ElaC within the Vibrio lineage (Fig. S1), a horizontal acquisition that potentially diversifies the bacterial surface glycan profile with the incorporation of 8‐epi‐Leg5,7Ac2 [38]. The GC3s content of the elaABC cluster in CRAB1707 (17.49%–22.00%) was significantly lower than that of its V. vulnificus homologs (37.00%–41.67%). This drastic reduction in GC3s suggests that upon horizontal transfer from a Vibrio‐like marine donor, these genes underwent rapid amelioration to accommodate the extreme AT‐biased mutational pressure of the Acinetobacter genome (Fig. S2). The divergent codon usage patterns, combined with the high amino acid identity, provide robust evidence for the ancient capture and subsequent adaptive evolution of this nonulosonic acid biosynthetic machinery.

Infection model of CRAB1707 elaA/elaB/elaC mutants

We generated deletion mutants lacking elaA, elaB, or elaC to assess whether nonulosonic acid 8‐epi‐Leg5,7Ac2 is essential for capsule biosynthesis. Optical microscopy of nigrosine‐stained cells showed that CRAB mutants lacking any of the elaA, elaB, or elaC genes displayed reduced capsule formation compared to wild‐type CRAB (Fig. 2A). Capsule isolation and electrophoretic analysis with alcian blue staining further confirmed the loss of capsules in these mutants (Fig. 2B).

Fig. 2.

Effects of elaA, elaB, or elaC mutations on capsule formation in CRAB 1707. (A) Bright field microscopy of negatively stained CRAB 1707 strain and its deletion mutants reveals capsules formed as bright halos around the bacteria. (B) CPS, indicated by the red arrow, isolated from CRAB and mutants, separated using SDS/PAGE and stained with alcian blue. (C) Survival rate of CRAB‐infected mice compared to elaA‐, elaB‐, or elaC‐deficient strains (n = 10 mice per group).

To assess the impact of elaA, elaB, or elaC mutations in vivo, mice were inoculated with CRAB1707 mutants at a dose of 1 × 10⁷ CFU, which is typically lethal in mouse infection model of CRAB1707 [17]. In this model, only the wild‐type CRAB1707 induced death, whereas none of the mutants caused mortality or exhibited severe infection symptoms (Fig. 2C).

The structure of ElaB

ElaB participates in the first step of Leg5,7Ac2 biosynthesis by coupling CTP with Leg5,7Ac2 to produce CMP‐Leg5,7Ac2. The crystal structure of ElaB was determined in the presence of substrate CTP, at the resolution of 1.8 Å. The asymmetric unit contains a dimer, and the lid domain (residues 139–144) is disordered and is not visible in the electron density map (Fig. 3A). In the ElaB‐CTP complex, each ElaB molecule is bound to a CTP molecule. Ethylene glycol (EDO) molecules were observed adjacent to the CTP‐binding site in the A chain of the homodimer (Fig. 3B). Additionally, two magnesium ions were directly coordinated to the pyrophosphate moiety of CTP, with one ion (Mg²⁺) further coordinating to D211 and D213 to anchor the CTP molecule (Fig. 3B).

To investigate how Leg5,7Ac2 binds to ElaB, we performed induced fit docking implemented in Maestro (version 14.4.138). Among the nine docking poses with docking scores ranging from −6.139 to −3.194, one pose (docking score = −4.552) aligned with our catalytic model, where the 2‐hydroxyl group faces CTP and the two N‐acetyl groups fit into hydrophobic pockets. In this model, the two N‐acetyl groups interacted with the hydrophobic surface formed by W81, W84, and L194 (Figs 3C and S3). Compared with PseF (Protein Data Bank [PDB] ID: 6IFD), a CMP‐Pse synthetase from Aeromonas caviae [39], the Pse moiety binds with its two N‐acetyl groups facing a hydrophobic pocket (Fig. 3D). The docking pose revealed that the carboxylate group of Leg5,7Ac2 coordinates with the catalytic Mg²⁺ ion, which is also complexed with the α‐phosphate of CTP. This interaction likely stabilizes the transition state by neutralizing the negative charge that develops during the nucleophilic attack of the C1‐hydroxyl group on the α‐phosphorus atom.

ElaA structure and CMP‐Leg5,7Ac2 binding

ElaA is proposed to act on CMP‐Leg5,7Ac2 to generate CMP‐8‐keto‐Leg5,7Ac2 [16]. Among the ElaABC proteins, ElaA is the largest, consisting of 323 residues. When searching for similar structures in the PDB, TM_0414 from Thermotoga maritima (PDB ID: 3EZY) was identified as the most similar, with only 23.6% sequence identity and 91% coverage. TM_0414 is annotated as a putative dehydrogenase with no defined biological function and belongs to the Gfo/Idh/MocA family fold with an inserted β‐sheet domain in its substrate binding region [40].

The crystal structure of ElaA was determined by molecular replacement, using the crystal structure of TM_0414 with an RMSD of 2.432 Å (210 aligned atoms). ElaA forms a homodimer in its asymmetric unit and its structure is composed of an N‐terminal Rossman fold domain responsible for NAD(P)⁺ and substrate binding, and a C‐terminal α/β domain involved in substrate binding and the dimeric interface (Fig. 4A) [40]. In our attempts to crystallize ElaA in the presence or absence of the cofactors NAD⁺ or NADP⁺, crystals were obtained only through cocrystallization with NADP⁺. In the electron density map, the nicotinamide‐ribose portion was disordered, and NADP⁺ was modeled as adenosine‐2′,5′‐diphosphate (Fig. 4B). NADP⁺ binding involved interactions between its adenine moiety and V165 and L171, whereas its two phosphate groups engaged R156 and the backbone amides of L9 and A34 (Fig. 4B). Residues 34–41 are disordered in the electron density map. Notably, the adenine ring of NADP⁺ bent approximately 90 degrees away from the phosphate‐binding site, a conformation similar to NADP⁺ binding observed in this family protein (Fig. 4A) [41].

To understand the structural basis of CMP‐NulO recognition, we searched for a related nucleotide sugar dehydrogenase within the same enzyme family. WlbA, an enzyme from Chromobacterium violaceum involved in the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐D‐mannuronic acid [42] (Fig. 4C), was identified as a suitable reference. In the structure of WlbA complexed with UDP‐GlcNAc, the UDP moiety binds to the inserted β‐sheet domain. Notably, the substrate‐binding region undergoes a conformational rearrangement to accommodate the UDP moiety. To investigate how CMP‐Leg5,7Ac2 binds to ElaA, we performed induced fit docking using Maestro (version 14.4.138). Prior to docking, we modeled the nicotinamide‐ribose portion of NADP⁺ based on the structure of an oxidoreductase from Listeria innocua (PDB ID: 3E18) (Fig. 4B). The docking resulted in a pose in which the 8‐hydroxyl group is positioned near the nicotinamide ring, with a docking score of −5.304 and a Glide Emodel score of −77.131. In this model, CMP‐Leg5,7Ac2 adopts an extended conformation, with the 5‐N‐acetyl group interacting with hydrophobic residues I148, W155, and L179, and the 7‐N‐acetyl group contacting L120, M281, and Y282 within the substrate‐binding site (Figs 4D and S4). The CMP moiety binds to the inserted β‐sheet domain, consistent with the binding mode observed in the WlbA–UDP‐GlcNAc complex.

The structure of ElaC

The structure of ElaC was determined in the presence of the reaction product, NAD⁺. The structure of ElaC was a tetramer, as observed in short‐chain dehydrogenase (SDR) family proteins (Fig. 5A). NAD⁺ was observed with an acetate ion from the crystallization buffer stacked on the nicotinamide ring, thereby mimicking the reactive group of the native substrate (Fig. 5B). Notably, an ethylene glycol molecule was also identified in the pocket adjacent to the nicotinamide ring, which appears to correspond to the binding motif of Leg5,7Ac2. The catalytic residue, Y168, is positioned in a charge‐relay configuration with K172 and the acetate ion (Fig. 5B). Y91, Y97, Y147, and Y157, conserved among ElaC homologs, are likely involved in substrate recognition (Fig. S5).

Unlike SDR family epimerases such as GDP‐mannose‐3,5‐epimerase, which catalyze both oxidation and reduction within the same active site [43], ElaC seems to perform only the final reduction step. It is structurally related to ketoacyl reductases (KARs) from type II fatty acid synthases [44, 45]. To gain insight into its functional divergence from KARs to a nucleotide‐NulO reductase, the structure of human mitochondrial KAR (mtKAR) was superposed (Fig. 5C). The overall structures are highly similar (RMSD = 1.320 Å over 178 aligned atoms), except for a longer loop spanning residues V150–P165 in ElaC, which may correspond to the CMP‐binding site (Fig. 5C).

Using induced fit docking in Maestro (ver. 14.4.138), CMP‐8‐keto‐Leg5,7Ac2 was modeled into the active site of ElaC. Among six poses with scores from −9.757 to −7.215, the pose with the 8‐acetyl group within 3 Å of the nicotinamide ring (score = −9.547) was selected. In this conformation, the 8‐acetyl group superimposes with the acetate ion observed in the crystal structure, and the 7‐N‐acetyl group occupies the ethylene glycol‐binding site formed by Y97, Y147, M164, and P165. The 5‐N‐acetyl group is positioned within a separate hydrophobic cavity lined by I156, Y157, F209, and Y213. Q205 contributes to interactions with the 4‐hydroxyl group (Fig. 5D). The cytosine moiety engages in stacking interactions with the Leg5,7Ac2 fragment, positioning the 8‐acetyl group optimally for hydride transfer from NADH to the 8‐epi hydroxyl group during catalysis. The cytosine moiety binds within a pocket formed by the elongated V150–P165 loop (Fig. 5D). In contrast to the substrate orientation in ElaA‐catalyzed 8‐hydroxyl oxidation, which is less orientation‐dependent, the 8‐keto reduction by ElaC likely requires a specific alignment so that the re face of the 8‐keto group is directed toward the nicotinamide ring, yielding 8‐epi‐Leg5,7Ac2. Our docking results suggest that, in addition to accommodating the two acetyl groups within hydrophobic pockets, the 8‐keto group interacts with S145 and Y147 in a manner consistent with the stereochemistry necessary for reduction (Fig. 5D).

Discussion

Deletion of elaA/elaB/elaC in CRAB 1707 caused a marked reduction in capsule production, revealing that the production of 8‐epi‐Leg5,7Ac2 by the ElaABC system is essential for capsule formation in hypervirulent CRAB strains. Although the precise role of C8 epimerization, whether it is essential for the glycosidic linkage between 8‐epi‐Leg5,7Ac2 and L‐FucNAc mediated by Gtr100, or for efficient polymerization of the capsule repeat unit by Wzy, remains to be fully elucidated, it significantly influences capsule generation. How attenuation of the KL49 capsule reduces virulence and prevents progression to sepsis warrants further investigation.

The CTP‐bound ElaB structure reveals hydrophobic pockets likely to accommodate the two characteristic 5,7‐N‐acetyl groups. Docking of substrate into the active site yielded a model consistent with related pseudaminic acid synthetases. We identified key structural features, including a hydrophobic pocket, that could be targeted for the development of inhibitors. A Gfo/Idh/MocA‐type enzyme ElaA provides a rigid scaffold for oxidation of the sugar hydroxyl group, whereas a downstream SDR fine‐tunes stereochemical outcomes by enforcing face‐selective hydride re‐delivery to the ketone intermediate. Structural models of CMP‐Leg5,7Ac2 in the ElaA active site and of CMP‐8‐keto‐Leg5,7Ac2 bound to ElaC indicate that Leg5,7Ac2 orientation is tightly regulated by regiospecific interactions of key functional groups. Notably, the two N‐acetyl groups of Leg5,7 AC2 appear to play a critical role in substrate binding, facilitating the proper orientation of CMP‐Leg5,7 AC2 for epimerization. In the case of ElaC, precise positioning of the 8‐hydroxyl group is essential for hydride transfer during epimerization. Furthermore, the serendipitous observation of acetate and ethylene glycol molecules in the crystal structure—occupying positions corresponding to the 8‐keto and 5‐NAc binding sites in our model—supports our proposed model for Leg5,7Ac2 binding. ElaA homologs, termed ElaA2, are found in KL62, KL79, KL97, KL110, KL183, and KL192 with 84% sequence identity [34], and ElaA structure will be valuable for understanding the function of ElaA2. High‐resolution structures of ElaA and ElaC offer a foundation for structure‐guided drug development, given the high selectivity of ElaA‐ and ElaC‐mediated epimerization for bacterial NulOs, thereby highlighting these enzymes as promising antimicrobial targets.

The identification of the KL49‐like NulO biosynthesis gene cluster within V. vulnificus YJ016, particularly the conserved ElaABC (CMP‐Leg5,7Ac2 8‐epimerase) operon, underscores a complex evolutionary adaptation to environmental pressures. The markedly lower GC3 content of this region compared to the V. vulnificus core genome strongly suggests HGT, likely from Vibrio pathogens to an Acinetobacter‐related lineage. Due to the absence of obvious insertion sequences (ISs) flanking the elaABC locus, we speculate that dissemination of the elaABC‐containing region likely occurred through homologous recombination rather than IS‐mediated mobilization. This genetic acquisition is not limited to uptake; the integration of ElaABC enzymes into the LPS and CPS biosynthetic pathways suggests an evolutionarily refined mechanism of surface glycan modification via Leg5,7Ac2 epimerization. ElaC homologs are also found in Aeromonas, Allivibrio, and Idiomarina (Fig. S1), suggesting that 8‐epi‐Leg5,7Ac2‐containing NulO structures may have originally functioned in symbiosis or environmental adaptation in aquatic microorganisms before being co‐opted for a distinct role in the Acinetobacter capsule.

Previous studies have shown that nab1 in V. vulnificus YJ016, a homolog of elaB in CRAB1707, resulted in accumulation of NulOs in the cytosol without incorporation into the LPS polymer, consistent with our observation of capsule deficiency in the elaB deletion mutant [36]. Furthermore, deletion of nab2, which corresponds to lgaD in CRAB1707 (Fig. 1C) and is essential for Leg5,7Ac2 biosynthesis, compromises bacterial survival during bloodstream infection and tissue dissemination in vivo [46]. The unexpected convergence of distantly related pathogens such as A. baumannii and V. vulnificus on producing 8‐epi‐Leg5,7Ac2 or related NulOs suggests that ElaABC‐mediated synthesis of specific 8‐epimers functions as a shared virulence strategy. This molecular mimicry allows these bacteria to evade host innate immune responses by masquerading as host‐like sialic acid structures [47, 48]. This finding not only highlights the genomic plasticity of V. vulnificus in diversifying its glycan repertoire but also positions the Ela‐mediated epimerization as a potential key virulence determinant in sepsis‐prone pathogens. Understanding this mechanism provides new insights into bacterial immune evasion strategies and highlights potential targets for therapeutic intervention against sepsis‐causing bacteria.

The loss of lethality in elaA, elaB, or elaC mutants at the same inoculum that is fatal for wild‐type CRAB1707 demonstrates that the 8‐epi‐Leg5,7Ac2 biosynthetic pathway is critical for KL49 hypervirulence and represents promising drug targets. Pharmacological inhibition of ElaA, ElaB, and ElaC, either individually or in combination, offers a direct, mechanism‐based strategy to disable the KL49 capsule, thereby reducing virulence and preventing progression to systemic infection and sepsis. Our structural and functional data validate ElaABC as feasible, pathogen‐specific antivirulence targets. They also highlight the essential role of stereochemical modifications in A. baumannii virulence, offering a valuable foundation for rational inhibitor design. Further development of small molecules that inhibit 8‐epi‐Leg5,7Ac2 formation could offer an innovative therapeutic approach against hypervirulent, KL49 A. baumannii. Such strategies have the potential to reduce mortality rates and address a significant, urgent global health threat.

Author contributions

YK conceived and supervised the study; WCL and YK designed experiments; WCL, JJ, CYL, HC, MHO, and JYL acquired the data; WCL, JJ, and CYL analyzed and interpreted the data; JL provided resources; WCL and YK wrote the manuscript. All authors reviewed and approved the final manuscript.

Supporting information

Table S1. X‐ray diffraction data collection and crystallographic refinement statistics.

Fig. S1. Phylogenetic analysis of ElaC from Acinetobacter baumannii CRAB1707.

Fig. S2. Comparison of GC3s content between elaABC clusters.

Fig. S3. Pairwise amino acid sequence alignment of ElaB and VV0316 coded by nab1 gene from Vibrio vulnificus YJ016.

Fig. S4. Multiple sequence alignment of ElaA, ElaA2 (from KL62), and VV0315 (from V. vulnificus YJ016).

Fig. S5. Pairwise sequence alignment of ElaC and VV0317 from V. vulnificus YJ016.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.