Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+.

Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Conformational flexibility in carbapenem hydrolysis drives substrate specificity of the class D carbapenemase OXA-24/40.

The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D ß-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D ß-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure-function relationship between the carbapenem-hydrolyzing class D ß-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D ß-lactamases.

Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

A naturally inspired antibiotic to target multidrug-resistant pathogens.

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin3. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens4. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.

Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility.

Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res 68:5318-5325, 2008) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.

OXA-23 ß-Lactamase Overexpression in Acinetobacter baumannii Drives Physiological Changes Resulting in New Genetic Vulnerabilities.

ß-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one ß-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 ß-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA ß-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 ß-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 ß-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, d-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA ß-lactamase-overexpressing Enterobacteriaceae, while having no impact on the normal flora. IMPORTANCE Acinetobacter baumannii has become a serious pathogen in both hospital and community settings. The ß-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of ß-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multidrug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 ß-lactamase leads to significant collateral changes, where peptidoglycan structure is altered. We have identified genes that become selectively essential in OXA-23-expressing strains and confirmed the relationship between altered peptidoglycan and OXA-23 expression by demonstrating that OXA-23 overexpression sensitizes cells to genetic and chemical inhibition of peptidoglycan synthesis. This work paves the way for the identification of new antimicrobial targets, where inhibitors would selectively kill ß-lactamase-expressing strains.

A New Class of Cell Wall-Recycling l,d-Carboxypeptidase Determines ß-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii.

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.

Evaluation antibiotic resistance and presence of blaOXA-51, blaOXA-58 and blaOXA-23 genes in Acinetobacter baumannii strains via multiplex PCR.

The resistance of Acinetobacter baumannii to most antibiotics is increasing. The presence of metallo-beta-lactamase and carbapenemase enzymes has led to the resistance of these bacteria to carbapenems as one of the major classes of broad-spectrum antibiotics and has raised concerns in human societies. This research evaluated the presence of blaOXA-51, blaOXA-58 and blaOXA-23 genes in A. baumannii strains during a 12 months period. One hundred strains were isolated from the patients hospitalized in ICU of Ali Asghar and Shahid Rajaee trauma hospitals in Shiraz. Bacterial identity was determined by biochemical tests and antibiotic resistance was determined by disk diffusion method. The isolated strains were then evaluated in terms of carrying blaOXA-23, blaOXA-51 and blaOXA-58 genes, using the multiplex PCR method. The results showed that A. baumannii was resistant to carbapenems but most strains were susceptible to tigecyclin and colistin. The majority of strains carried the blaOXA-23 and blaOXA-51 genes, but very few carried the bla OXA-58 gene. The results revealed that the antibiotic resistance of A. baumannii is increasing, which causes a more outbreak of this organism.

Comparative study of phenotypic-based detection assays for carbapenemase-producing Acinetobacter baumannii with a proposed algorithm in resource-limited settings.

The increasing incidence of carbapenem resistance in Acinetobacter baumannii is a critical concern worldwide owing to the limitations of therapeutic alternatives. The most important carbapenem resistance mechanism for A. baumannii is the enzymatic hydrolysis mediated by carbapenemases, mostly OXA-type carbapenemases (class D) and, to a lesser extent, metallo-ß-lactamases (class B). Therefore, early and accurate detection of carbapenemase-producing A. baumannii is required to achieve the therapeutic efficacy of such infections. Many methods for carbapenemase detection have been proposed as effective tests for A. baumannii; however, none of them are officially recommended. In this study, three carbapenemase detection methods, namely, CarbaAcineto NP test, modified carbapenem inactivation method (mCIM), and simplified carbapenem inactivation method (sCIM) were evaluated for phenotypic detection of clinically isolated A. baumannii. The MICs of imipenem, meropenem, and doripenem were determined for 123 clinically isolated A. baumannii strains before performing three phenotypic detections. The overall sensitivity and specificity values were 89.09%/100% for the carbAcineto NP test, 71.82%/100% for sCIM, and 32.73%/33.13% for mCIM. CarbAcineto NP test and sCIM performed excellently (100% sensitivity) when both Class B and Class D carbapenemases were present in the same isolate. Based on the results, the combined detection method of sCIM and CarbAcineto NP test was proposed to detect carbapenemase-producing A. baumannii rather than a single assay, significantly increasing the sensitivity of detection to 98.18%. The proposed algorithm was more reliable and cost-effective than the CarbAcineto NP test alone. It can be easily applied in routine microbiology laboratories for developing countries with limited resources.

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii.

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.

Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence.

Most bacteria possess alcohol dehydrogenase (ADH) genes (Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD+-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.

Homeoviscous Adaptation of the Acinetobacter baumannii Outer Membrane: Alteration of Lipooligosaccharide Structure during Cold Stress.

To maintain optimal membrane dynamics, cells from all domains of life must acclimate to various environmental signals in a process referred to as homeoviscous adaptation. Alteration of the lipid composition is critical for maintaining membrane fluidity, permeability of the lipid bilayer, and protein function under diverse conditions. It is well documented, for example, that glycerophospholipid content varies substantially in both Gram-negative and Gram-positive bacteria with changes in growth temperature. However, in the case of Gram-negative bacteria, far less is known concerning structural changes in lipopolysaccharide (LPS) or lipooligosaccharide (LOS) during temperature shifts. LPS/LOS is anchored at the cell surface by the highly conserved lipid A domain and localized in the outer leaflet of the outer membrane. Here, we identified a novel acyltransferase, termed LpxS, involved in the synthesis of the lipid A domain of Acinetobacter baumannii. A. baumannii is a significant, multidrug-resistant, opportunistic pathogen that is particularly difficult to clear from health care settings because of its ability to survive under diverse conditions. LpxS transfers an octanoate (C8:0) fatty acid, the shortest known secondary acyl chain reported to date, replacing a C12:0 fatty acid at the 2' position of lipid A. Expression of LpxS was highly upregulated under cold conditions and likely increases membrane fluidity. Furthermore, incorporation of a C8:0 acyl chain under cold conditions increased the effectiveness of the outer membrane permeability barrier. LpxS orthologs are found in several Acinetobacter species and may represent a common mechanism for adaptation to cold temperatures in these organisms. IMPORTANCE To maintain cellular fitness, the composition of biological membranes must change in response to shifts in temperature or other stresses. This process, known as homeoviscous adaptation, allows for maintenance of optimal fluidity and membrane permeability. Here, we describe an enzyme that alters the fatty acid content of A. baumannii LOS, a major structural feature and key component of the bacterial outer membrane. Although much is known regarding how glycerophospholipids are altered during temperature shifts, our understanding of LOS or LPS alterations under these conditions is lacking. Our work identifies a cold adaptation mechanism in A. baumannii, a highly adaptable and multidrug-resistant pathogen.

Crystal structure of the cofactor-free form of thioredoxin reductase from Acinetobacter baumannii.

Thioredoxin reductase (TrxR) is a central component in the thioredoxin system by involving in catalyzing the reduction of thioredoxin, which is critical for organism survival. Because this system is essential, it is a promising target for novel antimicrobial agents. Herein, we solved the 1.9 Å high-resolution structure of TrxR from Acinetobacter baumannii Thioredoxin reductase (AbTrxR), which is a Gram-negative, pathogenic bacterium and a drug-resistant superbug. AbTrxR was cofactor-free and formed a dimer in solution. AbTrxR contained a longer dimerization loop2 and a shorter ß7 -ß8 connecting loop than other TrxRs. AbTrxR cofactor-free form exhibited a flavin-oxidizing (FO) conformation, whose NADPH domain was located close to the dimeric interface. This structural information might be helpful for development of new antibiotic agents targeting superbugs.

Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii.

Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 µM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.

Molecular typing, biofilm production, and detection of carbapenemase genes in multidrug-resistant Acinetobacter baumannii isolated from different infection sites using ERIC-PCR in Hamadan, west of Iran.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that can cause several kinds of nosocomial infections. Increasing antibiotic resistance as well as identifying genetic diversity and factors associated with pathogenicity and prevalence of this bacterium is important. The aim of this study was the investigation of molecular typing, biofilm production, and detection of carbapenemase genes in multidrug-resistant Acinetobacter baumannii isolated from different infection sites using ERIC-PCR in Iran. METHODS: Forty isolates of A. baumannii were obtained from various wards of the central hospital, in the west of Iran. Phenotypic identification and genetic diversity, biofilm production assay, and detection of Carbapenemase genes carried out. RESULTS: Tracheal samples 26 (61.9 %) are the most frequent isolates, and 95 % of isolates were identified as MDR. 32.5 % of all A. baumannii strains were capable to form a strong biofilm. It was founded that antimicrobial resistance patterns had a significant relationship with strong biofilm formation (P = 0.001). Most frequencies of the studied genes were in the order of VIM (81 %), SPM (45.2 %), and IMP (35.7 %) genes. The VIM gene was the most frequent in all isolates which were significant (P = 0.006). 14 different ERIC-types were observed including 7 common types and 7 unique or single types. F type is the largest common type consisting of nine isolates and B, D, and E types contain two isolates separately. CONCLUSIONS: ERIC-PCR technique was used to genetically classify A. baumannii isolates as one of the most common microorganisms in nosocomial infections.

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and ß-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy.

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with ß-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca2+ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α1α2 loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α2 and α3-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α2-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein-protein interactions.

Recombinant Production and Characterization of an Extracellular Subtilisin-Like Serine Protease from Acinetobacter baumannii of Fermented Food Origin.

Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 ß-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/ß motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.

Structural comparison of Acinetobacter baumannii ß-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis.

Some Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the ß-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the ß-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure-function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host-pathogen interaction mechanisms and novel antibiotics discovery.

Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of blaNDM-1, blaOXA-66, blaOXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including blaNDM-1 Both the isolates carried blaNDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6')-Ib, aadA1, and aph(3')-VI], ß-lactams (blaCTX-M-15, blaOXA-9, and blaTEM-1A), fluoroquinolones [aac(6')-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of blaOXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (blaNDM-1, blaOXA-72, blaCTX-M-14b, and blaCTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.