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In bacteria, the ankyrin-like protein AnkB helps overcome stress by regulating catalase activity when expressed under stressful conditions. As the structural properties of AnkB are largely unexplored, our understanding of various AnkB-mediated functions in bacteria remains limited. In the present study, we describe the structure of AnkB from Acinetobacter baumannii, hereafter referred to as "AbAnkB," which has a unique tertiary configuration compared with that of other ankyrin domain-containing proteins. Structural analysis revealed that AbAnkB has a relatively long loop between AKR3 and AKR4 and an oppositely positioned α 8 helix. Based on amino acid conservation and protein surface analyses, we identified a hydrophobic patch that might be critical for the function of AbAnkB. To the best of our knowledge, our study is the first to report the structure of a bacterial AnkB protein; our findings will markedly enhance our understanding of its functions in bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Penicillin-binding protein 2 (PBP2), a vital protein involved in bacterial cell-wall synthesis, serves a target for β-lactam antibiotics. Acinetobacter baumannii is a pathogen notorious for multidrug resistance; therefore, exploration of PBPs is pivotal in the development of new antimicrobial strategies. In this study, the tertiary structure of PBP2 from A. baumannii (abPBP2) was elucidated using X-ray crystallography. The structural analysis demonstrated notable movement in the head domain, potentially critical for its glycosyltransferase function, suggesting that abPBP2 assumes a fully closed conformation. Our findings offer valuable information for developing novel antimicrobial agents targeting abPBP2 that are applicable in combating multidrug-resistant infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An efficient synthetic strategy has been developed to achieve a pyruvic acid acetal containing tetrasaccharide repeating unit corresponding to the K82 capsular polysaccharide of Acinetobacter baumannii LUH5534 strain in very good yield. The synthetic scheme involves the use of suitably functionalized monosaccharide thioglycosides as glycosyl donors and a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic glycosylation activator to furnish satisfactory yield of the products with appropriate stereochemistry at the glycosidic linkages. Incorporation of the (R)-pyruvic acid acetal in the d-galactose moiety was achieved in very good yield by the treatment of the diol derivative with methyl 2,2-bis(p-methylphenylthio)propionate in the presence of a combination of NIS and triflic acid., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acinetobacter baumannii poses a serious threat to human health. Pathogenic bacterial lipopolysaccharides (LPSs) are potent immunogens for the development of antibacterial vaccines. To investigate the antigenic properties of A. baumannii LPS, five well-defined core oligosaccharide fragments from the LPS of A. baumannii SMAL and ATCC 19606 were synthesized. A divergent synthesis strategy based on orthogonally protected α-(2 → 5)-linked Kdo dimer 6 was developed. Selective exposure of different positions in this key precursor and then elongation of sugar chains via stereocontrolled formation of both 1,2- trans and 1,2- cis -2-aminoglycosidic linkages permitted the efficient synthesis of the targets. The synthetic route also highlights a 4- O and then 7- O glycosylation sequence for assembly of the novel 4,7-branched Kdo framework. Antigenicity assay using the glycan microarray technique disclosed that tetrasaccharide 3 featuring both 4,7-branch and α-(2 → 5)-Kdo-Kdo structural elements was a potential antigenic determinant.

Rhodanese-like domains (RLDs) represent a widespread protein family canonically involved in sulfur transfer reactions between diverse donor and acceptor molecules. RLDs mediate these transsulfuration reactions via a transient persulfide intermediate, created by modifying a conserved cysteine residue in their active sites. RLDs are involved in various aspects of sulfur metabolism, including sulfide oxidation in mitochondria, iron-sulfur cluster biogenesis, and thio-cofactor biosynthesis. However, due to the inherent complexity of sulfur metabolism caused by the intrinsically high nucleophilicity and redox sensitivity of thiol-containing compounds, the physiological functions of many RLDs remain to be explored. Here, we focus on a single domain Acinetobacter baumannii RLD (Ab-RLD) associated with a desulfurase encapsulin which is able to store substantial amounts of sulfur inside its protein shell. We determine the 1.6 Å x-ray crystal structure of Ab-RLD, highlighting a homodimeric structure with a number of unusual features. We show through kinetic analysis that Ab-RLD exhibits thiosulfate sulfurtransferase activity with both cyanide and glutathione acceptors. Using native mass spectrometry and in vitro assays, we provide evidence that Ab-RLD can stably carry a persulfide and thiosulfate modification and may employ a ternary catalytic mechanism. Our results will inform future studies aimed at investigating the functional link between Ab-RLD and the desulfurase encapsulin., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The Gram-negative bacterium Acinetobacter baumannii is one of the most resilient multidrug-resistant pathogens in hospitals. Among Gram-negative bacteria, it is particularly resistant to dehydration (anhydrobiosis), and this feature allows A. baumannii to persist in hospital environments for long periods, subjected to unfavorable conditions. We leverage the combination of μ-Raman spectroscopy and atomic force microscopy (AFM) to investigate the anhydrobiotic mechanisms in A. baumannii cells by monitoring the membrane (both inner and outer membranes) properties of four A. baumannii strains during a 16-week dehydration period and in response to temperature excursions. We noted that the membranes of A. baumannii remained intact during the dehydration period despite undergoing a liquid-crystal-to-gel-phase transition, accompanied by changes in the mechanical properties of the membrane. This was evident from the AFM images, which showed the morphology of the bacterial cells alongside modifications of their superficial mechanical properties, and from the alteration in the intensity ratio of μ-Raman features linked to the CH 3 and CH 2 symmetric stretching modes. Furthermore, employing a universal power law revealed a significant correlation between this ratio and bacterial fitness across all tested strains. Additionally, we subjected dry A. baumannii to a temperature-dependent experiment, the results of which supported the correlation between the Raman ratio and culturability, demonstrating that the phase transition becomes irreversible when A. baumannii cells undergo different temperature cycles. Besides the relevance to the present study, we argue that μ-Raman can be used as a powerful nondestructive tool to assess the health status of bacterial cells based on membrane properties with a relatively high throughput.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii , O -linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O -linked glycosylation.

Acinetobacter species encode for extracellularly secreted Biofilm-associated protein (Bap), a multi-domain protein with variable molecular weights reaching several hundred kilodaltons. Bap is crucial for the development of multi-dimensional structures of mature biofilms. In our investigation, we analyzed 7338 sequences of A. baumannii from the NCBI database and found that Bap or Bap-like protein (BLP) was present in 6422 (87.52%) isolates. Further classification revealed that 12.12% carried Type-1 Bap, 68.44% had Type-2, 6.91% had Type-3, 0.05% had Type-6 or SDF-Type, and 12.51% lacked Bap or BLP. The majority of isolates with Type-1, Type-2, and Type-3 Bap belonged to ST1, ST2, and ST25, respectively. Phylogenetic analysis suggested that Type-1 Bap is the most ancient, while Type-3 and SDF-Type have evolved recently. Studying the interaction of predicted Bap structures with human CEACAM-1 and PIgR showed that Bap with its BIg13 and BIg6 domains interact with the N-terminal domain of CEACAM-1, involving Arg 43 and Glu 40 , involved in CEACAM-1 dimerization. Also, we found that recently evolved Type-3 and SDF-Type Bap showed greater interaction with CEACAM-1 and PIgR. It can be asserted that the evolution of Bap has conferred enhanced virulence characteristics to A. baumannii with increased interaction with CEACAM-1 and PIgR. Using in silico approaches, this study explores the evolutionary, physicochemical, and structural features of A. baumannii Bap and unravels its crucial role in mediating interaction with human CEACAM-1 and PIgR through detailed structure modelling. These findings advance our understanding of A. baumannii Bap and highlight its role in pathogenesis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 1 H and 13 C 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 Wzy KL141 polymerase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1 H and 13 C 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 wzy KL239 polymerase gene in KL239 that replaces the gtr80 and wzy KL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel Wzy KL239 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 1 H and 13 C 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Herein, we report a concise synthetic approach for the first total synthesis of a pentasaccharide repeating unit of Acinetobacter baumannii K11 capsular polysaccharides containing a rare sugar 6-deoxy-l-talose. The pentasaccharide was synthesized in a convergent manner using a [3 + 2] block glycosylation strategy. During this synthetic strive, we used a 2,2,2-trichloroethoxycarbonyl (Troc)-protected monosaccharide unit to achieve a high yield during the glycosylation to synthesize a trisaccharide, and chemoselective deprotection of the Troc group from the trisaccharide was carried out under a mild, pH-neutral condition, keeping the O -glycosidic bond, azido, and acid/base sensitive group intact. A thiotolylglycoside disaccharide donor containing 6-deoxy-l-talose was synthesized for the first time by the armed-disarmed glycosylation method between two thiotolylglycosides.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been used to identify microorganisms and predict antibiotic resistance. The preprocessing method for the MS spectrum is key to extracting critical information from complicated MS spectral data. Different preprocessing methods yield different data, and the optimal approach is unclear. In this study, we adopted an ensemble of multiple preprocessing methods--FlexAnalysis, MALDIquant, and continuous wavelet transform-based methods--to detect peaks and build machine learning classifiers, including logistic regressions, naïve Bayes classifiers, random forests, and a support vector machine. The aim was to identify antibiotic resistance in Acinetobacter baumannii , Acinetobacter nosocomialis , Enterococcus faecium , and Group B Streptococci (GBS) based on MALDI-TOF MS spectra collected from two branches of a referral tertiary medical center. The ensemble method was compared with the individual methods. Random forest models built with the data preprocessed by the ensemble method outperformed individual preprocessing methods and achieved the highest accuracy, with values of 84.37% ( A. baumannii ), 90.96% ( A. nosocomialis ), 78.54% ( E. faecium ), and 70.12% (GBS) on independent testing datasets. Through feature selection, important peaks related to antibiotic resistance could be detected from integrated information. The prediction model can provide an opinion for clinicians. The discriminative peaks enabling better prediction performance can provide a reference for further investigation of the resistance mechanism.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 1 H and 13 C NMR spectroscopy. A branched tetrasaccharide repeating unit was identified, which included a →3)-α-d-Galp-(1→6)-α-d-GlcpNAc-(1→3)-β-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., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 1 H and 13 C 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 Wzy K98 polymerase forming a β-d-QuipNAc-(1→3)-d-GalpNAc linkage between the K98 units., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 1 H and 13 C 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 β-d-Glc p -(1→6)-d-β-Gal p NAc-(1→6)-α-d-Gal p -(1→6)-β-d-Glс p -(1→3)-β-d-Gal p NAc in which d-Glc p at position 4 replaces d-Gal p 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., β-d-Gal p NAc-(1→3)-d-Gal p in 36-1454 (K127) and β- d- Gal p NAc-(1→4)-d-Gal p 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 β-d-Gal p NAc-(1→4)-d-Gal p linked, confirming that Wzy KL127 forms this linkage and thus that the phage-encoded Wzy Ph1 forms the β-d-Gal p NAc-(1→3)-d-Gal p 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.

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.

A structurally diverse capsular polysaccharide that surrounds the bacterial cell plays an important role in virulence of Acinetobacter baumannii, a cause of nosocomial infections worldwide. Various isomers of 5,7-diacylamido-3,5,7,9-tetradeoxynon-2-ulosonic acid have been identified as components of bacterial polysaccharides. In this work, we report on the identification of a new isomer having the d-glycero-l-manno configuration (8-epipseudaminic acid) in the capsular polysaccharide of A. baumannii Res546. The higher sugar was isolated by Smith degradation of the polysaccharide followed by mild acid hydrolysis and identified by a comparison with all isomers using NMR spectroscopy and optical rotation., (Copyright © 2022. Published by Elsevier Ltd.)

Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two-component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon that indicated that the A. baumannii PmrA consensus box is 5'-HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography and we present a full-length model using molecular modelling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies. Graphical Abstract., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

Acinetobacter baumannii has become a leading cause of bacterial nosocomial infections, in part, due to its ability to resist desiccation, disinfection and antibiotics. Several factors contribute to the tenacity and virulence of this pathogen, including production of a broad range of surface glycoconjugates, secretory systems and efflux pumps. We became interested in examining the importance of trehalose in A. baumannii after comparing intact bacterial cells by high-resolution magic angle spinning nuclear magnetic resonance and by noting high levels of this disaccharide, obscuring all other resonances in the spectrum. Since this was observed under normal growth conditions, we speculated that trehalose must serve additional functions beyond osmolyte homeostasis. Using the virulent isolate A. baumannii AB5075 and mutants in the trehalose synthesis pathway, osmoregulatory trehalose synthesis proteins A and B (△otsA and △otsB), we found that the trehalose-deficient △otsA showed increased sensitivity to desiccation, colistin, serum complement and peripheral blood mononuclear cells, while trehalose-6-phosphate producing △otsB behaved similar to the wild-type. The △otsA mutant also demonstrated increased membrane permeability and loss of capsular polysaccharide. These findings demonstrate that trehalose deficiency leads to loss of virulence in A. baumannii AB5075., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The majority of bacteria and archaea contains Toxin-Antitoxin system (TA) that codes for the stable Toxin and unstable Antitoxin components forming a complex. The Antitoxin inhibits the catalytic activities of the Toxin. In general, the Antitoxin will be degraded by the proteases leading to the Toxin activation that subsequently targets essential cellular processes, including transcription, translation, replication, cell division, and cell wall biosynthesis. The Zeta Toxin-Epsilon Antitoxin system in ESKAPE pathogen stabilizes the resistance plasmid and promotes pathogenicity. The known TA system in Acinetobacter baumannii are known to be involved in the replication and translation, however, the mechanism of Zeta Toxin-Epsilon Antitoxin in cell wall biosynthesis remains unknown. In the present study, molecular docking and molecular dynamic (MD) simulations were employed to demonstrate whether Zeta Toxin can impair cell wall synthesis in A. baumannii. Further, the degradation mechanism of Antitoxin in the presence and absence of adenosine triphosphate (ATP) molecules are explained through MD simulation. The result reveals that the cleavage of Antitoxin could be possible with the presence of ATP by displaying its response from 20 ns, whereas the Zeta Toxin/Epsilon was unstable after 90 ns. The obtained results demonstrate that Zeta Toxin is "temporarily favorable" for ATP to undergo phosphorylation at UNAG kinase through the substrate tunneling process. The study further evidenced that phosphorylated UNAG prevents the binding of MurA, the enzyme that catalyzes the initial step of bacterial peptidoglycan biosynthesis. Therefore, the present study explores the binding mechanism of Zeta Toxin/Epsilon Antitoxin, which could be beneficial for preventing cell wall biosynthesis as well as for unveiling the alternative treatment options to antibiotics., (© 2021 Wiley Periodicals LLC.)

Background: Infection caused by Acinetobacter baumannii has emerged as a significant clinical problem with unacceptably high mortality rate due to the increase in antibiotic-resistant strains. Producing novel monoclonal antibody (MAb) against outer membrane protein A (OmpA) could be considered as a potential tool to improve treatment of A. baumannii infections., Objectives: In this study, we aimed to produce murine MAbs against OmpA peptide of A. baumannii., Materials and Methods: BALB/c mice were immunized with 18-mer amino acid peptide as a part of the OmpA protein. Four antibody-secreting hybridomas were obtained using hybridoma technology and then characterized according to isotypes, affinity constant, reactivity in ELISA, flow cytometry, indirect immunofluorescence (IFA) and opsonophagocytic killing assays., Results: All four produced MAbs (1A1-D10, 1G1-E7, 2C11-F10, and 4H2-H9) had IgG1 isotype with Kappa light chain. One of these MAbs, 1G1-E7 was purified and selected for further characterizations. 1G1-E7 showed a high reactivity with both immunogenic peptide and A. baumannii in ELISA. Our results indicated that 1G1-E7 MAb reacted with 95.3% of A. baumannii in flow cytometry as well as IFA. Moreover, the affinity of the 1G1-E7 MAb was measured 1.37 × 10 8  M -1 . The 1G1-E7 significantly improved opsonophagocytic killing of a clinical isolate of A. baumannii., Conclusion: Our findings showed that the OmpA can be identified by produced MAbs. The efficacy of novel anti-OmpA antibodies in A. baumannii targeting needs to be further investigated in challenging models, and then could be subjected for genetic engineering to produce therapeutic antibody against A. baumannii., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acinetobacter baumannii has become a major healthcare threat that causes nosocomial infections, especially in critically ill patients. The spread of carbapenem-resistant A. baumannii (CRAB) strains has long been a clinical concern. It is important to study the epidemiology and virulence characteristics of different CRAB isolates in order to tailor infection prevention and antibiotic prescribing. In this study, a total of 71 CRAB isolates were collected in the hospital, and clinical characteristics of infections were analyzed. The genomic characteristics and phylogenetic relationships were elucidated based on genome sequencing and analysis. The isolates were assigned to three sequence types (STs, Pasteur) and nine capsular polysaccharide (KL) types, among which ST2/KL22 was the most prevalent CRAB in the hospital. Even though all the ST2/KL22 isolates contained the same reported virulence genes, one specific clade of ST2/KL22 showed more pathogenic in mouse infection model. Complete genomic analysis revealed differences at the oprD locus between the low- and high-virulent isolates. More specifically, a premature stop codon in the low-virulence strains resulted in truncated OprD expression. By evaluating pathogenicity in C57BL/6 J mice, knock-out of oprD in high-virulent isolate resulted in virulence attenuation, and complementing the avirulent strain with full-length oprD from high-virulent isolate enhanced virulence of the former. The oprD gene may be associated with the enhanced virulence of the specific ST2/KL22 clone, which provides a potential molecular marker for screening the hypervirulent A. baumannii strains.

The KL26 gene cluster responsible for the synthesis of the K26 capsular polysaccharide (CPS) of Acinetobacter baumannii includes rmlBDAC genes for l-rhamnose (l-Rhap) synthesis, tle to generate 6-deoxy-l-talose (l-6dTalp) from l-Rhap, and a manC gene for D-mannose (D-Manp) that is rare in Acinetobacter CPS. K26 CPS material was isolated from A. baumannii isolate KZ-1098, and studied by sugar analysis, Smith degradation, and one and two-dimensional 1 H and 13 C NMR spectroscopy before and after O-deacetylation with aqueous ammonia. The following structure of the branched hexasaccharide repeating unit of the CPS was established: →2)-β-D-Manp-1→4-β-D-Glcp-1→3-α-L-6dTalp-1→3-β-D-GlcpNAc-(1→3↑14│Acα-L-Rhap-2←1-α-D-Glcp The structural depolymerase of phage vB_AbaP_APK26 cleaved selectively the β-GlcpNAc-(1 → 2)-α-Manp linkage in the K26 CPS formed by Wzy K26 to give monomer, dimer, and trimer of the CPS repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as 1 H and 13 C NMR spectroscopy. The wzy K26 gene responsible for this linkage and the manC gene were only found in six A. baumannii genomes carrying KL26 and one carrying the novel KL148 gene cluster, indicating the rare occurrence of β-GlcpNAc-(1 → 2)-α-Manp in A. baumannii CPS structures. However, K26 shares a β-d-Glcp-(1 → 3)-α-l-6dTalp-(1 → 3)-β-d-GlcpNAc trisaccharide fragment with a group of related A. baumannii CPSs that have varying patterns of acetylation of l-6dTalp., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acinetobacter baumannii infections are associated with a high mortality rate. Sulbactam, a beta-lactamase inhibitor, is commonly used to treat A. baumannii infections, but its underlying mechanisms are unclear. Two-component regulatory systems (TCSs) are important for bacterial adaptability and response ability. In this study, we focused on two TCSs, namely AdeSR and BaeSR, and identified a protein highly similar to the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains of the TCSs by using Swiss-Model. Sulbactam and β-lactamase inhibitors, which are structurally similar to sulbactam, were docked with the selected sequence 4JAS using the simulation tools SwissDock and ArgusLab. Analysis with both these analytical tools showed that sulbactam can react on the active sites of 4JAS at a relatively steady level (ΔG -7 to -10 kcal/mol). Sulbactam likely interacts with the active sites of BaeSR and AdeSR, and owing to its smaller size and ability to form ionic bonds with Mg 2+ , it may potentially compete with ATP/ADP in BaeSR and AdeSR and consequently interfere with A. baumannii multiplication. This is the first study to investigate the association between sulbactam and TCSs in A. baumannii using molecular docking and simulation analyses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The human pathogen Acinetobacter baumannii produces and utilizes acinetobactin for iron assimilation. Although two isomeric structures of acinetobactin, one featuring an oxazoline (Oxa) and the other with an isoxazolidinone (Isox) at the core, have been identified, their differential roles as virulence factors for successful infection have yet to be established. This study provides direct evidence that Oxa supplies iron more efficiently than Isox, primarily owing to its specific recognition by the cognate outer membrane receptor, BauA. The other components in the acinetobactin uptake machinery appear not to discriminate these isomers. Interestingly, Oxa was found to form a stable iron complex that is resistant to release of the chelated iron upon competition by Isox, despite their comparable apparent affinities to Fe(III). In addition, both Oxa and Isox were found to be competent iron chelators successfully scavenging iron from host metal sequestering proteins responsible for nutritional immunity. These observations collectively led us to propose a new model for acinetobactin-based iron assimilation at infection sites. Namely, Oxa is the principal siderophore mediating the core Fe(III) supply chain for A. baumannii, whereas Isox plays a minor role in the iron delivery and, alternatively, functions as an auxiliary iron collector that channels the iron pool toward Oxa. The unique siderophore utilization mechanism proposed here represents an intriguing strategy for pathogen adaptation under the various nutritional stresses encountered at infection sites. IMPORTANCE Acinetobacter baumannii has acquired antibiotic resistance at an alarming rate, and it is becoming a serious threat to society, particularly due to the paucity of effective treatment options. Acinetobactin is a siderophore of Acinetobacter baumannii, responsible for active iron supply, and it serves as a key virulence factor to counter host nutritional immunity during infection. While two acinetobactin isomers were identified, their distinctive roles for successful infection of Acinetobacter baumannii remained unsettled. This study clearly identified the isomer containing an oxazoline core as the principal siderophore based on comparative analysis of the specificity of the acinetobactin uptake machinery, the stability of the corresponding iron complexes, and the iron scavenging activity against the host iron sequestering proteins. Our findings are anticipated to stimulate efforts to discover a potent antivirulence agent against Acinetobacter baumannii that exploits the acinetobactin-based iron assimilation mechanism.

Aryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production., (© 2021. The Author(s).)

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

Bacterial fatty acids are critical components of the cellular membrane. A shift in environmental conditions or in the bacterium's lifestyle may result in the requirement for a distinct pool of fatty acids with unique biophysical properties. This can be achieved by the modification of existing fatty acids or via de novo synthesis. Furthermore, bacteria have evolved efficient means to acquire these energy-rich molecules from their environment. However, the balance between de novo fatty acid synthesis and exogenous acquisition during pathogenesis is poorly understood. Here, we studied the mouse fatty acid landscape prior to and after infection with Acinetobacter baumannii, a Gram-negative, opportunistic human pathogen. The lipid fluxes observed following infection revealed fatty acid- and niche-specific changes. Lipidomic profiling of A. baumannii isolated from the pleural cavity of mice identified novel A. baumannii membrane phospholipid species and an overall increased abundance of unsaturated fatty acid species. Importantly, we found that A. baumannii relies largely upon fatty acid acquisition in all but one of the studied niches, the blood, where the pathogen biosynthesizes its own fatty acids. This work is the first to reveal the significance of balancing the making and taking of fatty acids in a Gram-negative bacterium during infection, which provides new insights into the validity of targeting fatty acid synthesis as a treatment strategy. IMPORTANCE Acinetobacter baumannii is one of the world's most problematic superbugs and is associated with significant morbidity and mortality in the hospital environment. The critical need for new antimicrobial strategies is recognized, but our understanding of its behavior and adaptation to a changing environment during infection is limited. Here, we investigated the role of fatty acids at the host-pathogen interface using a mouse model of disease. We provide comprehensive insights into the bacterial membrane composition when the bacteria colonize the pleural cavity. Furthermore, we show that A. baumannii heavily relies upon making its own fatty acids when residing in the blood, whereas the bacterium favors fatty acid acquisition in most other host niches. Our new knowledge aids in understanding the importance of host fatty acids in infectious diseases. Furthermore, fatty acid synthesis is an attractive target for the development of new antimicrobial strategies, but our work emphasizes the critical need to understand microbial lipid homeostasis before this can be deemed suitable.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

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

The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Acinetobacter baumannii Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was ∼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.', Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Jiang et al.)

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 ( Ab -54149) to form a glycoconjugate for preparing anti- Ab -54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab -54149 than an antibody purified from anti- Ab -54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides ( e.g ., Helicobacter pylori and Enterobacter cloacae ), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti- Ab -54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae , indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Acinetobacter baumannii is a Gram-negative bacterium primarily associated with hospital-acquired, often multidrug-resistant (MDR) infections. The ribosome-targeting antibiotics amikacin and tigecycline are among the limited arsenal of drugs available for treatment of such infections. We present high-resolution structures of the 70S ribosome from A. baumannii in complex with these antibiotics, as determined by cryoelectron microscopy. Comparison with the ribosomes of other bacteria reveals several unique structural features at functionally important sites, including around the exit of the polypeptide tunnel and the periphery of the subunit interface. The structures also reveal the mode and site of interaction of these drugs with the ribosome. This work paves the way for the design of new inhibitors of translation to address infections caused by MDR A. baumannii., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N -linked glycopeptides of Campylobacter fetus subsp. fetus as well as O -linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species ( B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans ). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Ahmad Izaham and Scott.)

The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K + , in osmostress response. Growth of A. baumannii was K + -dependent and the K + -dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K + and K + accumulation increased with the salinity of the medium. K + uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K + concentration: mannitol and glutamate concentrations increased with increasing K + concentrations whereas trehalose was highest at low K + . After osmotic upshock, cells first accumulated K + followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K + and glutamate. These experiments demonstrate K + uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K + and different organic solutes., (© 2020 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Few antibiotics are effective against Acinetobacter baumannii , one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii , is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI ( Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR ( Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention., Competing Interests: Competing interest statement: C.V.R. is a cofounder of and consultant at OMass Therapeutics., (Copyright © 2020 the Author(s). Published by PNAS.)

Magnetic trapping has been employed in the development of analytical methods owing to its ease and simplicity in handling samples. Nevertheless, the generation of functional probes is usually time consuming. A new and simple affinity method that uses gadolinium ion (Gd 3+ ), a magnetic ion, as affinity probe for magnetic tapping of pathogenic bacteria was demonstrated in the present study. Escherichia coli O157:H7, Staphylococcus aureus, and Acinetobacter baumannii were selected as model bacteria. The model bacteria were magnetically isolated after incubation in Tris buffer (pH 8) containing Gd 3+ (0.1 M) under microwave heating (power: 180 W, 90 s × 3). The resultant Gd 3+ -bacterium conjugates possessed sufficient magnetism, resulting in magnetic aggregations by an external magnet (∼4,000 Gauss). For ease of magnetic isolation, the sample containing Gd 3+ -bacterium complexes was stirred by a small magnet. After 1 h, the magnet attached with precipitates, i.e., Gd 3+ -bacterium conjugates, was readily removed using a pair of tweezers. The bacteria in the resultant conjugates were characterized by matrix-assisted laser desorption/ionization mass spectrometry. The limits of detection of the current approach toward E. coli O157:H7, S. aureus, and A. baumannii in complex samples were ∼10 4 -10 5  cells mL -1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7-residue sequence motif to identify mis-annotation of multiple DHDPS genes in the high-priority Gram-negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis-annotations using a combination of enzyme kinetics and X-ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens. PDB ID: 6UE0., (© 2020 Federation of European Biochemical Societies.)

The need for detailed structural characterization of glycerophospholipids (GPLs) for many types of biologically motivated applications has led to the development of novel mass spectrometry-based methodologies that utilize alternative ion activation methods. Ultraviolet photodissociation (UVPD) has shown great utility for localizing sites of unsaturation within acyl chains and to date has predominantly been used for positive mode analysis of GPLs. In the present work, UVPD is used to localize sites of unsaturation in GPL anions. Similar to UVPD mass spectra of GPL cations, UVPD of deprotonated or formate-adducted GPLs yields diagnostic fragment ions spaced 24 Da apart. This method was integrated into a liquid chromatography workflow and used to evaluate profiles of sites of unsaturation of lipids in Escherichia coli ( E. coli ) and Acinetobacter baumannii (A. baumannii) . When assigning sites of unsaturation, E. coli was found to contain all unsaturation elements at the same position relative to the terminal methyl carbon of the acyl chain; the first carbon participating in a site of unsaturation was consistently seven carbons along the acyl chain when counting carbons from the terminal methyl carbon. GPLs from A. baumannii exhibited more variability in locations of unsaturation. For GPLs containing sites of unsaturation in both acyl chains, an MS 3 method was devised to assign sites to specific acyl chains.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism's OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

The stereoselective synthesis of fimsbactin A, a siderophore of the human pathogen Acinetobacter baumannii , was established. Based on this synthetic route, various fimsbactin stereoisomeric analogues were generated and tested for their iron delivery activity for A. baumannii . This investigation revealed that the fimsbactin uptake machinery in this bacterium was indeed highly stereoselective in substrate recognition.

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii., (© 2020 The Protein Society.)

An efficient α-selective glycosylation method was developed for the synthesis of 2-deoxy-2-amino-d-glucosides based on synergetic α-directing effects of the TolSCl/AgOTf promotion system and the functional groups at the corresponding azido donor 6- O -position to exert steric β-shielding effect or remote participation in the glycosylation reaction. Its practicability was verified with a wide range of monosaccharide glycosyl acceptors and the first, one-pot synthesis of the challenging pentasaccharide repeating unit of an Acinetobacter baumannii K47 capsular polysaccharide.

Molecular interactions between two different classes of β-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A β-lactamase blaGES-11 and Class D β-lactamase Oxa23, responsible for hydrolysis of different types of β-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both β-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of β-lactamase enzymes in the periplasmic space.

The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional 1 H and 13 C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy-d-glucose (d-QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline β-elimination (NaOH-NaBH 4 ) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy-d-galacturonic acid residue (d-GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)-α-d-GalpNAcA6dAla-(1→4)-α-d-GalpNAcA-(1→3)-β-d-QuipNAc4NAc-(1→ . The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as d-QuipNAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one d-GalpNAcA residue is modified by a d-alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes d-alanine-d-alanine ligases, and thus was renamed Alt1 (alanine transferase). Alt1 represents a novel family that amidate the carboxyl group of d-GalpNAcA or d-GalpA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The herbicide, propanil, has been extensively applied in weed control, which causes serious environmental pollution. Acinetobacter baumannii DT isolated from soil has been used to determine the degradation rates of propanil and 3,4-dichloroaniline by freely suspended and biofilm cells. The results showed that the bacterial isolate could utilize both compounds as sole carbon and nitrogen sources. Edwards's model could be fitted well to the degradation kinetics of propanil, with the maximum degradation of 0.027 ± 0.003 mM h-1. The investigation of the degradation pathway showed that A. baumannii DT transformed propanil to 3,4-dichloroaniline before being completely degraded via the ortho-cleavage pathway. In addition, A. baumannii DT showed high tolerance to butachlor, a herbicide usually mixed with propanil to enhance weed control. The presence of propanil and butachlor in the liquid media increased the cell surface hydrophobicity and biofilm formation. Moreover, the biofilm reactor showed increased degradation rates of propanil and butachlor and high tolerance of bacteria to these chemicals. The obtained results showed that A. baumannii DT has a high potential in the degradation of propanil., (© FEMS 2020.)