Recombinant Acinetobacter sp. Urease accessory protein UreE (ureE)

What is the role of UreE in the urease system of Acinetobacter species?

UreE functions as a metallochaperone that delivers nickel ions to the urease apoenzyme during its maturation. In Acinetobacter, the urease system plays a critical role in nitrogen metabolism by catalyzing the hydrolysis of urea to ammonia and carbon dioxide. This system comprises multiple components, including the structural proteins UreA, UreB, and UreC, along with accessory proteins UreD, UreE, UreF, and UreG that facilitate the assembly of the nickel-containing active site . UreE is particularly important as it coordinates with UreG in metal ion trafficking to ensure proper enzyme assembly and function in various environmental conditions.

How does urease activity contribute to Acinetobacter baumannii pathogenesis?

Urease activity in A. baumannii is intricately linked to pathogenesis through several mechanisms:

• pH modulation: The production of ammonia from urea raises the local pH, creating favorable conditions for bacterial growth in acidic environments.

• Nutrient acquisition: Urease facilitates nitrogen acquisition in nitrogen-limited environments.

• Metal homeostasis: The urease system coordinates with metal acquisition systems, particularly for nickel and manganese, which are essential for bacterial survival during infection .

• Host immune evasion: Urease activity helps bacteria resist calprotectin-mediated nutritional immunity by facilitating alternative metal acquisition pathways .

Research shows that A. baumannii coordinates urea metabolism with metal import to overcome host-imposed metal limitation during infection. When exposed to calprotectin (a host antimicrobial protein that sequesters manganese and zinc), A. baumannii upregulates urease expression alongside NRAMP family transporters to facilitate bacterial growth and survival .

What expression systems are most effective for producing recombinant urease proteins from Acinetobacter?

Based on current research methodologies, the most effective expression systems include:

For UreE specifically, E. coli systems using pET vectors with N-terminal His-tags have been successfully employed for related urease accessory proteins in Acinetobacter . The addition of nickel supplementation (25-50 μM NiCl₂) during expression can improve the stability and functionality of the recombinant UreE protein.

What are the optimal conditions for inducing expression of recombinant Acinetobacter UreE in E. coli?

Based on methodologies used for similar Acinetobacter recombinant proteins:

• Bacterial strain: BL21(DE3) or derivatives lacking proteases

• Induction parameters:

  • Temperature: 30°C (preferred over 37°C to enhance solubility)

  • IPTG concentration: 0.5-1.0 mM

  • Induction time: 2-4 hours

• Media supplements:

  • Metal ions: 25-50 μM NiCl₂ to stabilize the metalloprotein

  • Glucose: 0.5% to control basal expression

• Culture conditions:

  • Aeration: High (>200 rpm) to maintain growth and prevent inclusion body formation

  • OD₆₀₀ at induction: 0.6-0.8 for optimal balance between cell density and expression

Similar protocols used for recombinant urease proteins from Acinetobacter demonstrate that these conditions typically yield 15-20 mg of purified protein per liter of culture .

What purification strategy yields the highest purity and activity for recombinant UreE?

A multi-step purification protocol is recommended for optimal results:

• Initial capture: Nickel-affinity chromatography using His-tagged UreE

  • Buffer composition: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol

  • Imidazole gradient: 20-250 mM for elution

  • Expected purity: >80%

• Secondary purification: Size exclusion chromatography

  • Column: Superdex 75 or 200

  • Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl

  • Expected purity: >95%

• Metal content verification:

  • ICP-MS to confirm nickel incorporation

  • Metal:protein ratio should approach 1:1 for fully functional UreE

This approach is similar to techniques used for other Acinetobacter recombinant proteins where >85% purity was achieved through affinity chromatography .

How can researchers assess the functional activity of recombinant UreE?

Multiple complementary approaches can be employed to verify UreE functionality:

• Metal binding assays:

  • Isothermal titration calorimetry (ITC) to determine nickel binding affinity

  • Circular dichroism (CD) to monitor structural changes upon metal binding

• Protein-protein interaction studies:

  • Pull-down assays with other urease accessory proteins (particularly UreG)

  • Surface plasmon resonance (SPR) to quantify binding kinetics

• Functional complementation:

  • Transform UreE-deficient bacterial strains with recombinant UreE

  • Measure restoration of urease activity using phenol-hypochlorite assay

• In vitro reconstitution:

  • Combine purified urease apoenzyme with accessory proteins

  • Monitor nickel incorporation and activation

These methodologies are consistent with characterization approaches used for other recombinant proteins from Acinetobacter, such as acid phosphatase and superoxide dismutases .

What genetic tools are available for manipulating urease genes in Acinetobacter species?

Current genetic manipulation approaches applicable to urease genes include:

• CRISPR-Cas systems:

  • Modified CRISPR-Cas9 systems have been developed specifically for Acinetobacter

  • The exogenous recombination system enhances efficiency in clinical isolates

  • Point mutations can be introduced without donor templates using cytidine base-editing

• Homologous recombination strategies:

  • Single-step homologous recombination allows for scarless deletions

  • FLP-FRT systems enable creation of unmarked deletions

  • RecET recombinase systems can be exploited for genetic manipulations

• Transposon mutagenesis:

  • Mini-Tn5 and Tn5-derived systems work effectively in Acinetobacter

  • Can be used for random mutagenesis screening to identify urease-related phenotypes

Researchers should consider strain compatibility and antibiotic resistance profiles when selecting genetic tools, as methods optimized for laboratory strains may require adaptation for clinical isolates due to their high antibiotic resistance .

How can researchers overcome challenges in expressing recombinant Acinetobacter proteins that form inclusion bodies?

When facing inclusion body formation with recombinant UreE, researchers can implement:

• Expression optimization:

  • Reduce expression temperature to 16-25°C

  • Lower IPTG concentration to 0.1-0.2 mM

  • Use richer media (e.g., Terrific Broth) to support proper folding

• Solubility enhancement:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use fusion partners (MBP, SUMO, Thioredoxin) to increase solubility

  • Add low concentrations of non-denaturing detergents (0.05% Triton X-100)

• Refolding strategies if inclusion bodies persist:

  • Solubilize in 8M urea or 6M guanidine hydrochloride

  • Perform stepwise dialysis with decreasing denaturant concentration

  • Add metal cofactors (Ni²⁺) during refolding to stabilize structure

This approach is similar to that used for other challenging Acinetobacter proteins, where truncated versions (e.g., removing signal peptides) helped achieve soluble expression, as demonstrated with AV3SodC-p .

How does the structure of UreE compare with other metal chaperones in Acinetobacter species?

UreE shares structural features with other metal chaperones in Acinetobacter:

• Metal-binding domains:

  • UreE typically contains histidine-rich C-terminal regions for nickel binding

  • This differs from copper chaperones like AV3SodC, which use cysteine residues for metal coordination

• Oligomeric state:

  • UreE typically forms homodimers similar to other urease accessory proteins

  • This contrasts with AV3SodB (iron-binding), which forms tetramers

• Domain organization:

  • Single domain structure with conserved metal-binding motifs

  • Lacks the signal peptides found in periplasmic metal-binding proteins

Structural analysis based on homology modeling suggests that UreE from Acinetobacter shares approximately 35-40% sequence identity with well-characterized UreE proteins from other bacteria, while maintaining the conserved nickel-binding residues essential for function.

What structural features of UreE are critical for its interaction with other urease accessory proteins?

Key structural features important for UreE functionality include:

Research on related urease accessory proteins suggests that mutations in these regions significantly impact protein function and stability, similar to findings with other metal-binding proteins in Acinetobacter .

How can researchers assess the contribution of UreE to urease activity in Acinetobacter cell models?

To evaluate UreE's contribution to urease function in cellular contexts:

• Gene knockout and complementation:

  • Generate ΔureE mutants using CRISPR-Cas9 or homologous recombination

  • Complement with wild-type or mutant variants of UreE

  • Measure changes in urease activity using phenol-hypochlorite assays

• Protein-protein interaction studies:

  • Bacterial two-hybrid systems to map interaction networks

  • Co-immunoprecipitation to identify protein complexes in vivo

  • Fluorescence microscopy with tagged proteins to visualize localization

• Metal homeostasis analysis:

  • ICP-MS to quantify intracellular nickel levels

  • Metal-sensitive fluorescent probes to track metal distribution

  • Transcriptomics to identify compensatory responses

Studies with other metal-related systems in Acinetobacter demonstrate that combining these approaches provides comprehensive insights into protein function within cellular pathways .

What role does UreE play in Acinetobacter's response to metal limitation during infection?

UreE likely contributes to metal homeostasis during infection through:

• Nickel acquisition and trafficking:

  • Ensures efficient nickel utilization under limited conditions

  • Prioritizes essential nickel-requiring enzymes during scarcity

• Integration with other metal acquisition systems:

  • Coordinates with NRAMP family transporters that import manganese

  • Functions within networks that respond to calprotectin-mediated metal sequestration

• Impact on virulence:

  • Supports urease activity needed for pH modulation and ammonia production

  • Contributes to metabolic flexibility during infection

Research on related systems shows that A. baumannii coordinates urea metabolism with metal import systems to overcome calprotectin-mediated nutritional immunity, suggesting UreE plays a key role in this adaptive response .

What are the methodological approaches to study UreE's role in multi-protein urease maturation complexes?

Advanced techniques to investigate UreE within urease maturation complexes include:

• Structural biology approaches:

  • Cryo-electron microscopy of urease accessory protein complexes

  • Cross-linking mass spectrometry to map protein interfaces

  • Hydrogen-deuterium exchange to identify dynamic regions

• Real-time monitoring of complex formation:

  • FRET-based assays to track protein-protein interactions

  • Single-molecule tracking to observe complex dynamics

  • Native mass spectrometry to determine complex stoichiometry

• Functional reconstitution:

  • In vitro assembly of urease maturation complexes with purified components

  • Step-wise addition of accessory proteins to determine assembly order

  • Monitoring nickel incorporation using spectroscopic methods

These approaches have been successfully applied to other multi-protein complexes in Acinetobacter, revealing critical insights into their assembly and function .

How can researchers address the challenge of studying metal transfer kinetics between UreE and UreG?

To investigate the metal transfer mechanism between UreE and UreG:

• Direct measurement techniques:

  • Stopped-flow spectroscopy with metal-dependent fluorescent probes

  • Rapid-freeze quench coupled with EPR spectroscopy

  • ITC for thermodynamic parameters of metal transfer

• Computational approaches:

  • Molecular dynamics simulations of the UreE-UreG interface

  • QM/MM calculations to model the energetics of metal transfer

  • Protein-protein docking to predict interaction conformations

• Mutation analysis:

  • Systematic mutation of metal-coordinating residues

  • Creation of trapped intermediates through strategic mutations

  • Temperature-sensitive mutants to capture transitional states

These methodologies build upon approaches used to study metal transfer in other systems, such as the characterization of superoxide dismutases in Acinetobacter, which revealed critical insights into metal coordination and transfer mechanisms .

What is the potential of targeting UreE as an antimicrobial strategy against Acinetobacter infections?

The targeting of UreE presents several therapeutic possibilities:

• Rational drug design approaches:

  • Structure-based design of inhibitors that disrupt UreE-metal binding

  • Peptidomimetics that interfere with UreE-UreG interactions

  • Allosteric inhibitors that prevent conformational changes

• Therapeutic potential:

  • Reduced bacterial persistence under metal-limited conditions

  • Decreased virulence through impaired urease activity

  • Synergistic effects with conventional antibiotics

• Therapeutic advantages:

  • Novel target not addressed by current antibiotics

  • Potential to overcome existing resistance mechanisms

  • Specificity for bacterial systems over human counterparts

Recent research suggests that disrupting metal acquisition and utilization systems represents a promising strategy against antibiotic-resistant Acinetobacter strains, as demonstrated by studies on iron and manganese acquisition systems .

How might recombinant UreE be utilized in developing vaccines against Acinetobacter infections?

Potential applications of recombinant UreE in vaccine development include:

• As a direct antigen:

  • Conservation across Acinetobacter strains suggests broad protection

  • Metal-binding properties may enhance immunogenicity

  • Potential to generate neutralizing antibodies that disrupt function

• As a carrier protein for other antigens:

  • Fusion with hypervariable regions of other Acinetobacter proteins

  • Similar to the FliC-Omp22 approach that demonstrated enhanced protection

  • Potential to elicit both Th1 and Th2 immune responses

• Within multi-component vaccines:

  • Combination with other urease components for synergistic immunity

  • Inclusion in outer membrane vesicle-based vaccines

  • Co-administration with adjuvants targeting specific immune pathways

Research on recombinant protein vaccines for Acinetobacter has shown promise, particularly when antigens are selected based on their essential roles in pathogenesis and structural conservation across clinical isolates .