Recombinant Brucella melitensis biotype 2 Urocanate hydratase (hutU), partial

What is Brucella melitensis and why study its urocanate hydratase (hutU)?

Brucella melitensis is a Gram-negative coccobacillus in the family Brucellaceae that causes brucellosis, a zoonotic bacterial disease. It primarily infects sheep and goats but can also affect camels, cattle, and humans, with B. melitensis responsible for approximately 70% of human brucellosis cases . Urocanate hydratase (hutU) is an enzyme in the histidine utilization pathway that catalyzes the conversion of urocanate to 4-imidazolone-5-propionate. Studying this enzyme provides insights into B. melitensis metabolism and potential virulence mechanisms, as metabolic pathways can contribute to pathogen survival in host environments. The enzyme may represent a target for therapeutic intervention or diagnostic development, particularly given the significant public health impact of B. melitensis as the most common cause of human brucellosis.

How does B. melitensis biotype 2 differ from other Brucella species and biotypes?

B. melitensis has three biovars (1-3) , with biotype 2 representing one distinct variant. Genomic analyses have revealed multiple differences between B. melitensis and other Brucella species, including specific deletions, inversions, and pseudogenes. B. melitensis and B. abortus share 57 common pseudogenes, while only 5 are shared between B. melitensis and B. suis . Compared to B. abortus and B. suis, B. melitensis has 16 complete genes, four gene segments, and a tRNA-Glu missing due to seven deletions . B. melitensis also harbors two small inversions in chromosome II not found in other species . These genomic differences contribute to host preference, virulence characteristics, and metabolic capabilities that distinguish biotype 2 from other variants.

What is the genetic structure of the hutU gene in B. melitensis and how conserved is it across biotypes?

While the search results don't provide specific information about the hutU gene structure in B. melitensis, genomic analyses of Brucella species indicate considerable genetic conservation with strategic variations. The hutU gene likely maintains core catalytic domains essential for urocanate hydratase function while potentially exhibiting biotype-specific variations in non-catalytic regions. Comparative genomic studies have revealed that B. melitensis has undergone specific deletion events through homologous recombination, particularly between highly similar transposase-encoding genes . This suggests that while the hutU gene's functional domains may be conserved, regulatory regions or non-essential portions could vary between biotypes. Full characterization would require targeted sequencing of the hutU locus across multiple isolates of biotype 2 and comparison with other biotypes.

What are the optimal expression systems for producing recombinant B. melitensis hutU protein?

For recombinant expression of B. melitensis proteins, E. coli expression systems have been successfully employed, as demonstrated with the Omp31 outer membrane protein . For hutU specifically, a methodological approach would include:

• Vector selection: pET or pGEX systems with T7 or tac promoters for high-level expression

• Host strain optimization: BL21(DE3), Rosetta, or Origami strains depending on codon usage and disulfide bond requirements

• Induction parameters: IPTG concentration (typically 0.5-1.0 mM), temperature (18-37°C), and duration (4-24 hours)

• Solubility enhancement: Co-expression with chaperones or fusion with solubility tags (MBP, SUMO, or TRX)

Expression conditions should be optimized through small-scale trials monitoring protein production via SDS-PAGE before scaling up. For hutU specifically, lower induction temperatures (18-25°C) may enhance proper folding of this metabolic enzyme.

What purification strategies are most effective for isolating recombinant hutU while maintaining enzymatic activity?

Effective purification of enzymatically active recombinant hutU requires a multi-step approach:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins for His-tagged hutU protein

• Intermediate purification: Ion exchange chromatography, with column selection based on hutU's theoretical isoelectric point

• Polishing: Size exclusion chromatography to remove aggregates and achieve high purity

• Activity preservation: Throughout purification, incorporate:

  • Buffer optimization with stabilizing agents (glycerol 10-20%, reducing agents)

  • Temperature control (4°C processing)

  • Protease inhibitor cocktails

  • Avoidance of harsh elution conditions

Functional assessment at each purification stage using urocanate conversion assays ensures retention of enzymatic activity. This methodological approach parallels successful strategies used for other Brucella recombinant proteins, such as Omp31, where protein functionality was maintained throughout purification processes .

How can researchers verify the structural integrity and enzymatic activity of purified recombinant hutU?

Verification of recombinant hutU structural integrity and activity requires complementary analytical approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure composition

  • Thermal shift assays to determine protein stability

  • Dynamic light scattering to evaluate monodispersity

  • Limited proteolysis to confirm proper folding

  • Mass spectrometry for intact mass analysis and peptide mapping

• Enzymatic activity verification:

  • Spectrophotometric assays monitoring urocanate decrease at 277 nm

  • Coupled enzyme assays tracking product formation

  • Isothermal titration calorimetry for substrate binding kinetics

  • HPLC or LC-MS for direct product identification

• Validation controls:

  • Site-directed mutagenesis of catalytic residues as negative controls

  • Comparison with commercially available urocanate hydratase (if available)

  • Activity testing under various pH and temperature conditions

This comprehensive approach ensures both structural and functional integrity of the recombinant protein, similar to methods employed for functional assessment of recombinant Brucella Omp31 .

How might hutU contribute to B. melitensis pathogenesis and host adaptation?

The hutU enzyme may contribute to B. melitensis pathogenesis through several mechanisms:

• Metabolic adaptation: Histidine catabolism via hutU could provide alternative carbon and nitrogen sources during intracellular infection, particularly in nutrient-limited phagocytic compartments.

• pH adaptation: The histidine utilization pathway may contribute to acid tolerance, which is crucial for survival in the acidic environment of macrophage phagosomes, similar to adaptation mechanisms in other intracellular pathogens.

• Immune modulation: Metabolites produced by hutU activity could potentially interfere with host immune signaling pathways, similar to how other bacterial metabolic products can modulate host responses.

• Niche colonization: The ability to utilize histidine might provide a competitive advantage in specific host tissues where this amino acid is available, contributing to tissue tropism.

Studying hutU in the context of B. melitensis virulence would require targeted gene knockout or expression modulation studies, followed by infection models to assess changes in bacterial persistence, replication rates, and host immune responses .

What is the potential of recombinant hutU as a diagnostic marker or vaccine candidate?

Recombinant hutU has several characteristics that make it potentially valuable as a diagnostic marker or vaccine component:

• Diagnostic applications:

  • Serological detection: Anti-hutU antibodies may serve as biomarkers of active infection

  • Antigen detection: Specific detection of hutU in clinical samples could indicate metabolically active bacteria

  • PCR targeting: The hutU gene sequence might offer specific detection targets for molecular diagnostics

• Vaccine potential:

  • Subunit vaccine: Purified recombinant hutU could elicit protective immune responses

  • Epitope mapping: Identification of immunodominant regions might inform peptide vaccine design

  • Carrier protein: hutU could serve as a carrier for other Brucella antigens

The approach used for Omp31 protein vaccination studies provides a methodological framework, where recombinant protein administered with adjuvant elicited protective immunity through both humoral and cell-mediated responses . Similar immunological assessment for hutU would include antibody profiling (IgG1/IgG2 ratios), cytokine production patterns (IL-2, IFN-γ), and T-cell subset analysis (CD4+/CD8+) .

How does genetic variation in hutU across different B. melitensis isolates correlate with virulence and host preference?

Genetic variation in hutU across B. melitensis isolates may contribute to differential virulence and host adaptation through:

• Sequence polymorphisms affecting:

  • Catalytic efficiency of the enzyme

  • Substrate specificity or binding parameters

  • Protein stability under different environmental conditions

  • Immunogenicity and immune evasion potential

• Methodological approach to investigate these correlations:

  • Comparative genomics: Sequence multiple hutU alleles from diverse isolates with different virulence profiles

  • Structure-function analysis: Model variant effects on protein structure and function

  • Recombinant protein studies: Express variant forms and compare enzymatic parameters

  • Infection models: Complement hutU mutants with variant alleles to assess virulence restoration

Genomic analyses of Brucella species have revealed specific deletions, inversions, and pseudogenes that contribute to host specificity and metabolic capabilities . Similar approaches could identify hutU variants associated with specific virulence phenotypes or host preferences.

What challenges are commonly encountered when working with recombinant B. melitensis proteins and how can they be addressed?

Common challenges with recombinant B. melitensis proteins include:

• Expression issues:

  • Low expression levels: Optimize codon usage, promoter strength, and induction parameters

  • Inclusion body formation: Lower expression temperature (16-25°C), co-express chaperones, or use solubility-enhancing tags

  • Protein toxicity: Use tightly regulated expression systems or specialized host strains

• Purification difficulties:

  • Insufficient purity: Develop multi-step purification strategies combining affinity, ion exchange, and size exclusion chromatography

  • Loss of activity: Optimize buffer conditions with stabilizing agents (glycerol, reducing agents) and avoid harsh elution conditions

  • Protein aggregation: Include low concentrations of detergents or arginine in buffers

• Methodological solutions from Brucella research:

  • Successful approaches have included fusion protein strategies, specialized induction protocols, and targeted purification methods as demonstrated with recombinant Omp31 protein

  • For membrane-associated proteins (potentially including hutU if membrane-associated), specific detergent screening may be necessary to maintain native structure

How can researchers differentiate between immune responses to hutU and other B. melitensis antigens in experimental studies?

Differentiating immune responses to hutU from other B. melitensis antigens requires several methodological approaches:

• Antigen-specific isolation techniques:

  • Protein-specific ELISA using highly purified recombinant hutU

  • Antigen-specific B-cell ELISpot assays

  • T-cell stimulation with defined hutU peptide pools

• Cross-reactivity assessment:

  • Competitive binding assays with related Brucella antigens

  • Absorption studies removing antibodies to common epitopes

  • Western blot analysis with various Brucella protein fractions

• Epitope mapping approaches:

  • Overlapping peptide libraries covering the hutU sequence

  • Phage display technology to identify specific binding regions

  • Structural epitope prediction and validation

Similar approaches have been successfully implemented with Omp31, where researchers precisely characterized the immune response by:

• Measuring specific antibody isotypes (IgG1, IgG2)

• Quantifying cytokine production (IL-2, IFN-γ, IL-10, IL-4)

• Assessing T-cell subset involvement through in vitro and in vivo depletion studies

What are the considerations for interpreting contradictory findings between in vitro enzymatic studies and in vivo infection models with hutU mutants?

When interpreting contradictory findings between in vitro enzymatic studies and in vivo infection models involving hutU, researchers should consider:

• Methodological factors:

  • Enzyme assay conditions may not reflect the intracellular environment

  • Growth media composition can affect metabolic pathway utilization

  • In vitro systems lack host immune pressures and environmental signals

• Biological complexities:

  • Metabolic redundancy may compensate for hutU deficiency in vivo

  • Host-specific factors might influence enzyme function differently than in vitro conditions

  • Temporal regulation of gene expression during different infection stages

• Analytical approaches to resolve contradictions:

  • Conditional mutants to control gene expression timing

  • In vivo expression studies (RNA-seq, proteomics) to confirm actual expression during infection

  • Metabolomic analysis to track metabolic changes in both systems

  • Complementation studies with variant alleles to identify critical features

• Integration framework:

  • Systems biology approaches integrating transcriptomic, proteomic, and metabolomic data

  • Mathematical modeling of metabolic networks to predict pathway importance under different conditions

  • Consideration of evolutionary conservation as an indicator of biological importance

Genomic analysis of Brucella species has revealed complex patterns of gene loss and adaptation that affect metabolic capabilities and host interactions , suggesting that the biological role of hutU may be similarly complex and context-dependent.

How might comparative analysis of hutU across Brucella species contribute to understanding host adaptation?

Comparative analysis of hutU across Brucella species offers valuable insights into host adaptation mechanisms:

• Evolutionary perspectives:

  • Sequence variation analysis to identify selection pressures on different domains

  • Phylogenetic mapping of hutU variants against host specificity patterns

  • Analysis of synonymous versus non-synonymous mutations to detect positive selection

• Functional implications:

  • Comparative enzymatic kinetics of hutU variants from different Brucella species

  • Structure-function correlations between amino acid changes and substrate specificity

  • Expression pattern differences across species in various infection models

• Methodological approaches:

  • Whole-genome sequencing and comparative genomics focusing on hutU and adjacent regulatory regions

  • Recombinant expression of variants for biochemical characterization

  • Complementation of knockout strains with cross-species hutU variants

Genomic analyses have already revealed specific deletions, inversions, and pseudogenes that distinguish B. melitensis from other Brucella species . Extending this approach to specifically focus on hutU and related metabolic pathways could reveal important adaptation mechanisms that contribute to host preference and tissue tropism.

What role might hutU play in interactions between B. melitensis and the host immune system?

The potential interactions between hutU and host immune responses could involve:

• Immunomodulatory effects:

  • Products of the urocanate hydratase reaction may influence immune cell function

  • Histidine metabolism might impact local microenvironments within infected cells

  • Enzymatic activity could affect pathogen-associated molecular pattern (PAMP) expression

• Recognition by immune surveillance:

  • hutU may contain epitopes recognized by T and B cells

  • Expression levels during infection might influence immunogenicity

  • Post-translational modifications could affect antigen processing and presentation

• Investigation methodologies:

  • Co-culture systems with immune cells and recombinant hutU

  • Cytokine profiling after exposure to enzyme or its metabolites

  • T-cell proliferation and activation assays with hutU-derived peptides

Studies with recombinant Omp31 demonstrated that protein vaccination elicited a robust T helper 1 (Th1) response mediated primarily by CD4+ T cells, with specific cytokine profiles (IL-2 and IFN-γ production) . Similar immunological characterization of hutU would be valuable to understand its potential immunomodulatory roles or vaccine applications.

How can structural biology approaches enhance understanding of hutU function and potential applications?

Structural biology approaches can significantly advance understanding of hutU through:

• Structure determination methods:

  • X-ray crystallography of purified recombinant hutU

  • Cryo-electron microscopy for larger complexes

  • NMR spectroscopy for dynamic regions and ligand interactions

  • Computational modeling and molecular dynamics simulations

• Functional insights from structure:

  • Identification of catalytic residues and substrate binding pockets

  • Elucidation of conformational changes during catalytic cycle

  • Understanding of oligomerization interfaces and regulatory sites

  • Mapping of surface-exposed regions for antibody binding

• Application-oriented structural analysis:

  • Structure-based drug design targeting hutU

  • Rational engineering of catalytic properties

  • Identification of immunodominant epitopes for vaccine development

  • Design of protein variants with enhanced stability for diagnostic applications

This multifaceted structural biology approach would complement genetic and biochemical studies, providing atomic-level insights into hutU function that could guide both fundamental research and application development.