Recombinant Brucella abortus Urease accessory protein UreE 1 (ureE1)

What is the primary function of Urease accessory protein UreE 1 in Brucella abortus?

UreE1 serves as a critical component in the urease metallocenter assembly pathway in Brucella abortus. The protein specifically binds nickel and functions as a nickel donor during the assembly process of the urease enzyme complex. This metallocenter assembly is essential for the catalytic activity of urease, which hydrolyzes urea to ammonia and carbon dioxide . The functional urease enzyme enables B. abortus to neutralize acidic environments through ammonia production, which plays a significant role in bacterial survival during host infection, particularly during passage through the stomach's acidic environment .

The nickel-binding capability of UreE1 represents a specialized adaptation for ensuring proper incorporation of the metal cofactor required for urease activity. Without functional UreE1, the assembly of active urease would be compromised, significantly reducing bacterial virulence and survival potential within hosts.

How does UreE1 interact with other proteins in the urease operon?

UreE1 operates within a sophisticated protein network within the urease operon, coordinating with both structural and accessory proteins to ensure proper urease enzyme assembly. Based on protein interaction data, UreE1 demonstrates particularly strong functional partnerships with UreF (0.991 confidence score) and works in concert with other accessory proteins including UreG and UreD . These interactions form part of a hierarchical assembly process for the incorporation of nickel into the urease metallocenter.

The assembly pathway likely involves:

• Initial formation of a UreD-UreF-UreG complex that interacts with the urease apoprotein

• UreE1 delivery of nickel ions to this complex, particularly through interaction with UreG

• GTP hydrolysis by UreG to provide energy for the nickel incorporation process

• Completion of metallocenter assembly, resulting in an active urease enzyme

These proteins work collectively within the ure1 gene cluster, which has been shown to be the primary active urease operon in B. abortus strain 2308, as opposed to the apparently inactive ure2 cluster also present in the genome .

What are the most effective methods for expressing recombinant UreE1 protein?

Expression of recombinant UreE1 requires careful optimization of multiple parameters to ensure proper protein folding and functionality. Based on successful recombinant protein expression strategies for other Brucella proteins, the following methodological approach is recommended:

The urease accessory protein gene should be PCR-amplified from B. abortus genomic DNA using high-fidelity polymerase and specific primers designed based on the genomic sequence. Following successful cloning and transformation, expression conditions should be optimized through small-scale test expressions before scaling up production . Protein verification through SDS-PAGE, Western blotting, and mass spectrometry is essential before proceeding to functional studies.

How can researchers verify the functionality of recombinant UreE1 protein?

Verification of recombinant UreE1 functionality requires multiple complementary approaches that assess both its structural integrity and functional capabilities:

• Nickel-binding assays: Isothermal titration calorimetry (ITC) or equilibrium dialysis to quantify nickel binding affinity and stoichiometry, which are essential properties of functional UreE1.

• Protein-protein interaction studies: Pull-down assays, yeast two-hybrid systems, or surface plasmon resonance (SPR) to verify interactions with other urease accessory proteins, particularly UreG .

• Complementation assays: Introduction of recombinant UreE1 into ureE1-deficient B. abortus mutants should restore urease activity if the protein is functional. Urease activity can be measured using phenol-hypochlorite assays or pH-indicator methods .

• Structural analysis: Circular dichroism (CD) spectroscopy to assess secondary structure elements, and more advanced techniques like X-ray crystallography or NMR for tertiary structure determination.

• Immunoreactivity testing: The recombinant protein should react with Brucella-positive serum but not with Brucella-negative serum, similar to the testing performed for other recombinant Brucella proteins .

A comprehensive functionality assessment would combine these approaches to ensure that the recombinant UreE1 maintains both structural and functional properties comparable to the native protein.

How does UreE1 contribute to the virulence of Brucella abortus during infection?

UreE1's contribution to B. abortus virulence is primarily indirect, through its essential role in urease functionality. Experimental evidence demonstrates that urease activity significantly enhances bacterial survival under acidic conditions in the presence of urea, which directly relates to pathogenesis during oral infection routes .

The mechanistic pathway appears to be:

• UreE1 facilitates nickel incorporation into the urease metallocenter

• Functional urease catalyzes urea hydrolysis, producing ammonia

• Ammonia neutralizes the acidic environment around the bacteria

• This neutralization protects B. abortus during passage through the stomach

• Protected bacteria successfully reach the intestinal mucosa for invasion

This survival advantage is particularly significant considering that the oral route represents a major infection pathway in human brucellosis. Experiments with urease-deficient mutants show they are killed more efficiently than urease-producing strains during transit through the stomach . While UreE1 itself is not directly involved in host-pathogen interactions, its role in ensuring urease functionality makes it an indirect but critical virulence factor for B. abortus.

Can recombinant UreE1 be used as a component in subunit vaccines against brucellosis?

Based on research with other recombinant Brucella proteins, UreE1 shows potential as a component in subunit vaccine formulations, particularly as part of a combined approach. Studies with recombinant protein vaccines have demonstrated that combinatorial approaches often provide superior protection compared to single-antigen formulations .

Table: Predicted immune responses to UreE1 compared with other recombinant B. abortus proteins

To evaluate UreE1's vaccine potential, researchers should:

• Assess immunoreactivity with Brucella-positive serum

• Measure T-helper cell response profiles, with particular focus on Th1 responses (IFN-γ, IL-2) critical for intracellular pathogen clearance

• Determine protection levels against virulent B. abortus challenge in animal models

• Compare efficacy to established vaccines like RB51

• Consider combining UreE1 with other proven immunogenic proteins for enhanced protection

A T-helper-1-dominated immune response would be particularly desirable, as this has been associated with superior protection against brucellosis in previous studies with other recombinant proteins .

What experimental design approaches are most suitable for studying UreE1 function in vivo?

Robust experimental design for investigating UreE1 function in vivo requires careful consideration of multiple design elements:

For in vivo studies, a between-subjects design with a randomized block approach offers the most rigorous assessment . Animal models should be carefully selected based on their ability to replicate human brucellosis pathogenesis. BALB/c mice represent a well-established model, as they have been successfully used to evaluate other recombinant Brucella vaccines .

The experimental workflow should incorporate:

• Generation of precise UreE1 knockout mutants using gene replacement techniques

• Complementation with functional UreE1 to confirm phenotype specificity

• Challenge experiments with varying infectious doses and routes

• Comprehensive assessment of bacterial loads in multiple tissues over time

• Statistical analysis with appropriate power calculations and significance testing

This methodology aligns with established principles for controlled experiments, which require systematic manipulation of independent variables, precise measurement of dependent variables, and control of potential confounding factors .

What are the best approaches for analyzing data related to UreE1 expression and activity?

• Data preparation and cleaning:

  • Verification of data completeness and accuracy

  • Identification and appropriate handling of outliers

  • Assessment of data distribution and normality

  • Checking for sufficient observations to power statistical analyses

• Visualization strategies:

  • Enzyme activity data plotted against relevant variables (time, pH, substrate concentration)

  • Box plots or violin plots for comparing activity levels between wild-type and mutant strains

  • Heat maps for visualizing protein interaction networks

  • Time-course plots for in vivo infection studies

• Statistical analysis approaches:

  • ANOVA or t-tests for comparing means between experimental groups

  • Non-parametric alternatives when assumptions are violated

  • Regression analysis for identifying relationships between variables

  • Multiple comparisons correction (e.g., Bonferroni, FDR) to control false discovery rate

• Specialized analyses for protein function:

  • Enzyme kinetics modeling (Michaelis-Menten, Lineweaver-Burk plots)

  • Protein-protein interaction network analysis

  • Structure-function relationship assessments

  • Comparative analysis with homologous proteins from other species

How do researchers address the presence of two urease clusters (ure1 and ure2) when studying UreE1 function?

The presence of two urease gene clusters in Brucella species presents a significant experimental challenge that must be carefully addressed in UreE1 research:

To address this complexity, researchers should:

• Utilize specific genetic tools: Design primers and probes that selectively target ureE1 versus any homologous gene in the ure2 cluster. Sequence alignment analyses can identify unique regions for specific targeting.

• Generate precise knockout mutants: Create deletion mutants that specifically target ureE1 without affecting other genes in the operon or potential homologs in the ure2 cluster. Complementation studies with the wild-type gene should restore function if the mutation is specific.

• Consider conditional expression: Investigate whether the ure2 cluster might be activated under specific environmental conditions not typically encountered in laboratory settings. This could explain the maintenance of two clusters despite apparent inactivity of one.

• Perform comparative genomics: Analyze the conservation and evolution of both urease clusters across Brucella species and related bacteria to understand their relative importance and functional divergence.

These strategies help disambiguate the specific contribution of UreE1 from the ure1 cluster, which has been identified as essential for urease activity in B. abortus strain 2308 .

What challenges arise when attempting to use UreE1 in recombinant vaccine development?

Development of UreE1-based recombinant vaccines faces several technical and biological challenges that researchers must systematically address:

• Protein stability and folding issues: Recombinant UreE1 may not always maintain its native conformation when expressed in heterologous systems. Strategies to overcome this include co-expression with molecular chaperones, optimization of purification protocols, and verification of functional activity prior to immunization studies.

• Immune response optimization: The ideal vaccine candidate should induce a strong Th1-type immune response characterized by IFN-γ and IL-2 production with limited IL-10, similar to what has been observed with other recombinant B. abortus proteins . Adjuvant selection becomes critical for directing the appropriate immune response profile.

• Comparative efficacy assessment: Any new UreE1-based vaccine must be evaluated against established vaccines like RB51, which despite limitations, provides a benchmark for protection. Studies with other recombinant vaccines have shown promising results using various approaches, including incorporating apoptotic proteins to enhance immune responses .

• Cross-protection considerations: Brucellosis can be caused by multiple Brucella species, necessitating evaluation of cross-protection. While UreE1 is conserved across species, sequence variations may affect immunogenicity and protection breadth.

• Delivery system development: Effective presentation of UreE1 to the immune system may require specialized delivery systems. Options include liposomes, nanoparticles, or viral vectors that can enhance antigen presentation and stability.

Addressing these challenges requires iterative experimental approaches with careful comparative analyses between different formulations and delivery systems. The promising results obtained with other recombinant Brucella proteins suggest that these challenges can be overcome with systematic investigation .