Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA.

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with the ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.

Crystal Structure, Steady-State, and Pre-Steady-State Kinetics of Acinetobacter baumannii ATP Phosphoribosyltransferase.

The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat when Mg2+ was replaced by Mn2+, and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.

A comprehensive profiling of quorum quenching by bacterial pigments identifies quorum sensing inhibition and antibiofilm action of prodigiosin against Acinetobacter baumannii.

Bacterial pigments represent a diverse group of secondary metabolites, which confer fitness advantages to the producers while residing in communities. The bioactive potential of such metabolites, including antimicrobial, anticancer, and immunomodulation, are being explored. Reckoning that a majority of such pigments are produced in response to quorum sensing (QS) mediated expression of biosynthetic gene clusters and, in turn, influence cell-cell communication, systemic profiling of the pigments for possible impact on QS appears crucial. A systemic screening of bacterial pigments for QS-inhibition combined with exploration of antibiofilm and antimicrobial action against Acinetobacter baumannii might offer viable alternatives to combat the priority pathogen. Major bacterial pigments are classified (clustered) based on their physicochemical properties, and representatives of the clusters are screened for QS inhibition. The screen highlighted prodigiosin as a potent quorum quencher, although its production from Serratia marcescens appeared to be QS-independent. In silico analysis indicated potential interactions between AbaI and AbaR, two major QS regulators in A. baumannii, and prodigiosin, which impaired biofilm formation, a major QS-dependent process in the bacteria. Prodigiosin augmented antibiotic action of ciprofloxacin against A. baumannii biofilms. Cell viability analysis revealed prodigiosin to be modestly cytotoxic against HEK293, a non-cancer human cell line. While developing dual-species biofilm, prodigiosin producer S. marcescens significantly impaired the fitness of A. baumannii. Enhanced susceptibility of A. baumannii toward colistin was also noted while growing in co-culture with S. marcescens. Antibiotic resistant isolates demonstrated varied responsiveness against prodigiosin, with two resistant strains demonstrating possible collateral sensitivity. Collectively, the results underpin the prospect of a prodigiosin-based therapeutic strategy in combating A. baumannii infection.

The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection.

IMPORTANCE: As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Sulbactam/Durlobactam: First Approval.

Sulbactam/durlobactam (XACDURO®), is a co-packaged antibacterial product that has been developed by Entasis Therapeutics Inc. for the treatment of infections caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Coadministration of durlobactam (a ß-lactamase inhibitor with potent activity against a broad range of serine ß-lactamases) with sulbactam (an established class A ß-lactamase inhibitor with antibacterial activity against A. baumannii) prevents sulbactam degradation by ABC-produced ß-lactamases. In May 2023, sulbactam/durlobactam was approved in the USA for use in patients 18 years of age and older for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia (HABP/VABP) caused by susceptible isolates of ABC. This article summarizes the milestones in the development of sulbactam/durlobactam leading to this first approval for the treatment of infections caused by ABC.

Genomic and phenotypic features of Acinetobacter baumannii isolated from oil reservoirs reveal a novel subspecies specialized in degrading hazardous hydrocarbons.

The genus Acinetobacter encompasses biotechnologically relevant species and nosocomial pathogens. In this study, nine isolates recovered from different oil reservoir samples showed the ability to grow with petroleum as the only carbon source and possessed the ability to emulsify kerosene. The whole genomes of the nine strains were sequenced and analyzed. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all strains were compared to the reference strains, and the results were below the reference values (<97.88 and 82, respectively), suggesting that the isolates belong to a new subspecies of Acinetobacter baumannii. The name Acinetobacter baumannii oleum ficedula is proposed. A comparison of the whole genome repertoire of 290 Acinetobacter species indicated that the strains in this study resemble non-pathogenic Acinetobacter strains. However, the new isolates resemble A. baumannii when comparing virulence factors. The isolates in this study carry many genes involved in hydrocarbon degradation, indicating the potential to degrade most toxic compounds listed by environmental regulatory agencies such as ATSDR, EPA, and CONAMA. In addition, despite the absence of known biosurfactant or bioemulsifier genes, the strains showed emulsifying activity, suggesting the presence of new pathways or genes related to this process. This study investigated the genomic, phenotypic, and biochemical features of the novel environmental subspecies A. baumannii oleum ficedula, revealing their potential to degrade hydrocarbons and to produce biosurfactants or bioemulsifiers. Applying these environmental subspecies in bioaugmentation strategies sheds light on future approaches to bioremediation. The study shows the importance of genomic analysis of environmental strains and their inclusion in metabolic pathways databases, highlighting unique enzymes/alternative pathways for consuming hazardous hydrocarbons.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

Contribution of Iron-Transport Systems and ß-Lactamases to Cefiderocol Resistance in Clinical Isolates of Acinetobacter baumannii Endemic to New York City.

The development of resistance to cefiderocol among multidrug resistant Acinetobacter baumannii has been attributed to downregulation in iron transport systems and a variety of ß-lactamases. However, the precise contribution of each in clinical isolates remains to be determined. Sixteen clinical isolates with varying degrees of cefiderocol resistance were investigated. Susceptibility testing was performed with and without the presence of iron and avibactam. Expression of 10 iron transport systems and blaADC and blaOXA-51-type were analyzed by real time RT-PCR. The acquisition of a variety of ß-lactamases was also determined. In 2 isolates the impact of silencing the blaADC gene was achieved using a target specific group II intron. For most resistant isolates, MICS for cefiderocol were similar with or without the presence of iron, and there was an overall decrease in expression of receptors (including pirA and piuA) involved in ferric uptake. However, expression of the ferrous uptake system (faoA) persisted. The addition of avibactam (4 µg/mL) lowered most cefiderocol MICs to 2 to 4 µg/mL. Most isolates possessed ADC-25 or ADC-33. Cefiderocol resistance correlated with over-expression of blaADC; silencing of this ß-lactamase resulted in a ≥ 8-fold decrease in cefiderocol MICs. Over-expression of specific blaADC subtypes, in a background of generalized repression of ferric uptake systems, were consistent features in clinical isolates of cefiderocol-resistant A. baumannii.

Acinetobacter baumannii outer membrane protein A induces autophagy in bone marrow-derived dendritic cells involving the PI3K/mTOR pathway.

BACKGROUND: Outer membrane protein A (OmpA) is the major virulence factor of Acinetobacter baumannii and plays a wide role in the pathogenesis and antimicrobial resistance of A. baumannii. Dendritic cells (DCs) are the most effective antigen-presenting cells and play a crucial role in regulating the immune response to multiple antigens and immune sentries. We aimed to study the role and molecular mechanisms of OmpA-induced mouse bone marrow-derived dendritic cells (BMDCs) autophagy in the immune response of A. baumannii. METHODS: First, purified A. baumannii OmpA was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. OmpA effect on BMDCs viability was evaluated by MTT assay. BMDCs were pretreated with autophagy inhibitor chloroquine or transfected with overexpression plasmids (oe-NC or oe-PI3K). Then BMDCs apoptosis, inflammatory cytokines, protein kinase B (PI3K)/mammalian target of rapamycin (mTOR) pathway, and autophagy-related factors levels were evaluated. RESULTS: SDS-PAGE and western blot verified the successful purification of OmpA. BMDCs viability repressed gradually with the increase of OmpA concentration. OmpA treatment of BMDCs led to apoptosis and inflammation in BMDCs. OmpA caused incomplete autophagy in BMDCs, and light chain 3 (LC3), Beclin1, P62, and LC3II/I levels were significantly elevated with the increase of the time and concentration of OmpA treatment. Chloroquine reversed OmpA effects on autophagy in BMDCs, that was, LC3, Beclin1, and LC3II/I levels were reduced, while P62 level was elevated. Furthermore, chloroquine reversed OmpA effects on apoptosis and inflammation in BMDCs. PI3K/mTOR pathway-related factor expression was affected by OmpA treatment of BMDCs. After overexpression of PI3K, these effects were reversed. CONCLUSIONS: A. baumannii OmpA induced autophagy in BMDCs involving the PI3K/mTOR pathway. Our study may provide a novel therapeutic target and theoretical basis for treating infections caused by A. baumannii.

Megalochelin, a Tridecapeptide Siderophore from a Talented Streptomycete.

Streptomycetes are bacteria known for their extraordinary biosynthetic capabilities. Herein, we describe the genome and metabolome of a particularly talented strain, Streptomyces ID71268. Its 8.4-Mbp genome harbors 32 bioinformatically predicted biosynthetic gene clusters (BGCs), out of which 10 are expressed under a single experimental condition. In addition to five families of known metabolites with previously assigned BGCs (nigericin, azalomycin F, ectoine, SF2766, and piericidin), we were able to predict BGCs for three additional metabolites: streptochlorin, serpetene, and marinomycin. The strain also produced two families of presumably novel metabolites, one of which was associated with growth inhibitory activity against the human opportunistic pathogen Acinetobacter baumannii in an iron-dependent manner. Bioassay-guided fractionation, followed by extensive liquid chromatography-mass spectrometry (LC-MS) and NMR analyses, established that the molecule responsible for the observed antibacterial activity is an unusual tridecapeptide siderophore with a ring-and-tail structure: the heptapeptide ring is formed through a C-C bond between a 2,3-dihydroxybenzoate (DHB) cap on Gly1 and the imidazole moiety of His7, while the hexapeptide tail is sufficient for binding iron. This molecule, named megalochelin, is the largest known siderophore. The megalochelin BGC encodes a 13-module nonribosomal peptide synthetase for the synthesis of the tridecapeptide, and a copper-dependent oxidase, likely responsible for the DHB-imidazole cross-link, whereas the genes for synthesis of the DHB starter unit are apparently specified in trans by a different BGC. Our results suggest that prolific producers of specialized metabolites may conceal hidden treasures within a background of known compounds.

Identification of a Novel LysR Family Transcriptional Regulator Controlling Acquisition of Sulfur Sources in Acinetobacter baumannii.

l-cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in Escherichia coli. However, the regulation of sulfur metabolism in Acinetobacter baumannii is only partly understood, with the LysR-type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 (cbl), in a highly multidrug resistant and virulent isolate of A. baumannii. We have shown that Cbl is involved in controlling expression of the genes required for uptake and reduction of various sulfur sources in A. baumannii. Collectively, we have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in A. baumannii.

Interactions between host epithelial cells and Acinetobacter baumannii promote the emergence of highly antibiotic resistant and highly mucoid strains.

Acinetobacter baumannii is an important nosocomial pathogen. Upon colonizing a host, A. baumannii are subjected to selective pressure by immune defenses as they adapt to the host environment. However, the mechanism of this pathoadaptation is unknown. Here, we established an in vitro system to evolve A. baumannii driven by the continuous selective pressure exerted by epithelial cells, and we used a combination of experimental evolution, phenotypic characterization and multi-omics analysis to address the underlying mechanism. When continuously exposed to selective pressure by pulmonary epithelial cells, A. baumannii showed ptk mutation-mediated mucoid conversion (reduced adhesion and increased anti-phagocytic ability) by enhancement of capsular exopolysaccharide chain length; rsmG mutation-mediated deficiency of 7-methylguanosine modification in the 524th nucleotide of 16S rRNA, which increased ribosome translation efficiency; and rnaseI mutation-mediated changes in outer membrane permeability and efflux pump expression. Together, these mutations altered susceptibility to a variety of antimicrobial agents, including the novel antibiotic cefiderocol, by regulating siderophore and siderophore-receptor biosynthesis. In conclusion, pulmonary epithelial cells modulate A. baumannii pathoadaptation, implicating the host-microbe interaction in the survival and persistence of A. baumannii.

Lipocalin-2 is an essential component of the innate immune response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii.

Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central l-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of l-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of l-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of l-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of l-Ser, DHB, and l-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (l-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.

Discovery of the Lead Molecules Targeting the First Step of the Histidine Biosynthesis Pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant, opportunistic, nosocomial pathogen for which a new line of treatments is desperately needed. We have targeted the enzyme of the first step of the histidine biosynthesis pathway, viz., ATP-phosphoribosyltransferase (ATP-PRT). The three-dimensional structure of ATP-PRT was predicted on the template of the known three-dimensional structure of ATP-PRT from Psychrobacter arcticus (PaATPPRT) using a homology modeling approach. High-throughput virtual screening (HTVS) of the antibacterial library of Life Chemicals Inc., Ontario, Canada was carried out followed by molecular dynamics simulations of the top hit compounds. In silico results were then biochemically validated using surface plasmon resonance spectroscopy. We found that two compounds, namely, F0843-0019 and F0608-0626, were binding with micromolar affinities to the ATP-phosphoribosyltransferase from Acinetobacter baumannii (AbATPPRT). Both of these compounds were binding in the same way as AMP in PaATPPRT, and the important residues of the active site, viz., Val4, Ser72, Thr76, Tyr77, Glu95, Lys134, Val136, and Tyr156, were also interacting via hydrogen bonds. The calculated binding energies of these compounds were -10.5 kcal/mol and -11.1 kcal/mol, respectively. These two compounds can be used as the potential lead molecules for designing antibacterial compounds in the future, and this information will help in drug discovery programs against Acinetobacter worldwide.

Outer Membrane Vesicles of Acinetobacter baumannii DS002 Are Selectively Enriched with TonB-Dependent Transporters and Play a Key Role in Iron Acquisition.

Outer membrane vesicles (OMVs) of Acinetobacter baumannii DS002 carry proteins which perform selective biological functions. The proteins involved in cell wall/membrane biogenesis and inorganic ion transport and metabolism occupied a significant portion of the 302 proteins associated with OMVs. Interestingly, the TonB-dependent transporters (TonRs), linked to the active transport of nutrients across the energy-deprived outer membrane, are predominant among proteins involved in inorganic ion transport and metabolism. The OMVs of DS002 contain TonRs capable of transporting iron complexed to catecholate, hydroximate, and mixed types of siderophores. Consistent with this observation, the OMVs were firmly bound to ferric-enterobactin (55Fe-Ent) and successfully transported iron into A. baumannii DS002 cells grown under iron-limiting conditions. In addition to the TonRs, OMVs also carry proteins known to promote pathogenesis, immune evasion, and biofilm formation. Our findings provide conclusive evidence for the role of OMVs in the transport of nutrients such as iron and show the presence of proteins with proven roles in pathogenicity and immune response. IMPORTANCE TonB-dependent transporters (TonRs) play a crucial role in transporting nutrients such as iron, nickel, copper, and complex carbohydrates across the energy-deprived outer membrane. Due to their unique structural features, TonRs capture nutrients in an energy-independent manner and transport them across the outer membrane by harvesting energy derived from the inner membrane-localized Ton-complex. In this study, we report the presence of TonRs capable of transporting various nutrients in OMVs and demonstrate their role in capturing and transporting ferric iron complexed with enterobactin into A. baumannii DS002 cells. The OMV-associated TonRs appear to play a critical role in the survival of A. baumannii, listed as a priority pathogen, under nutrient-deprived conditions.

The oxidative stress and metabolic response of Acinetobacter baumannii for aPDT multiple photosensitization.

The use of antimicrobial photodynamic inactivation as a non-antibiotic alternative method to inactivate Acinetobacter baumannii was described in response to the ever-growing problem of antibiotic resistance. It was found that irradiation of the bacterial suspension for 10 min reduced the number of viable cells by approximately 99% and this energy fluence was considered to be sub-lethal phototherapy. The lethal dose of laser light (cell mortality about 99.9%) was 9.54 J cm-2, which corresponds to 30 min of irradiation. After a 15-fold phototherapy cycle, the tolerance to aPDT decreased, resulting in a decrease in the number of viable cells by 2.15 and 3.23 log10 CFU/ml units with the use of sub-lethal and lethal light doses, respectively. Multiple photosensitizations decreased the biofilm formation efficiency by 25 ± 1% and 35 ± 1%, respectively. No changes in antibiotic resistance were observed, whereas the cells were more sensitive to hydrogen peroxide. Metabolomic changes after multiple photosensitization were studied and 1H NMR measurements were used in statistical and multivariate data analysis. Many significant changes in the levels of the metabolites were detected demonstrating the response of A. baumannii to oxidative stress.

The choline dehydrogenase BetA of Acinetobacter baumannii: a flavoprotein responsible for osmotic stress protection.

Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.

Genome sequencing and molecular characterisation of XDR Acinetobacter baumannii reveal complexities in resistance: Novel combination of sulbactam-durlobactam holds promise for therapeutic intervention.

Emerging nosocomial strains of Acinetobacter baumannii are of recent concern as they are expressing extensive drug resistance (XDR). Using whole-genome sequencing and molecular characterisation analysis, the current study reveals the presence of carbapenemase genes in 92.86% of studied Indian isolates. These included blaOXA-51 , blaOXA-23 , blaOXA-58 , and blaNDM genes, with over a third expressing dual carbapenemase genes. As per the MLST scheme, IC2Oxf /CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of these carbapenemase genes resulted in sulbactam (SUL) resistance (MIC: 16-256 µg/ml) in all of the studied isolates. The efficacy of durlobactam (DUR), a novel ß-lactamase inhibitor that also inhibits PBP2 was assessed through in silico intermolecular interaction analysis. Several nonsynonymous single nucleotide polymorphisms were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein backbone dynamics in active-site motifs of wild-type and mutants, which correlated with negligible binding energy fluctuations for the PBP3-SUL (-5.85 ± 0.04 kcal/mol) and PBP2-DUR (-5.16 ± 0.66 kcal/mol) complexes. Furthermore, higher binding affinities and low inhibition constants were noted in OXA23-DUR (-7.36 kcal/mol; 4.01 µM), OXA58-DUR (-6.44 kcal/mol; 19.07 µM), and NDM-DUR (-6.82 kcal/mol; 10.01 µM) complexes when compared with the conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR with carbapenemases can possibly restore SUL activity against both PBP3WT and PBP3MTs . The study establishes the efficacy of the novel SUL-DUR combination as a successful treatment strategy in combating emerging XDR strains of A. baumannii.