Recombinant Bordetella bronchiseptica N- (5'-phosphoribosyl)anthranilate isomerase (trpF)

What is Bordetella bronchiseptica and why is it significant in respiratory infection research?

Bordetella bronchiseptica is a Gram-negative coccobacillus of the phylum Proteobacteria that causes acute and chronic respiratory infections in a variety of animals. It belongs to the "classical species" of Bordetella along with B. pertussis and B. parapertussis. The pathogen is transmitted through direct contact, respiratory aerosol droplets, or fomites .
Bordetella bronchiseptica initially adheres to ciliated epithelial cells in the nasopharynx through various protein adhesins, including filamentous hemagglutinin, pertactin, and fimbriae. These adhesins not only assist in adherence to epithelial cells but are also involved in attachment to immune effector cells . The significant animal health burden caused by B. bronchiseptica infections makes it an important research subject, particularly because currently there is no definitive vaccine to prevent these infections .

What is N-(5'-phosphoribosyl)anthranilate isomerase (trpF) and what role does it play in bacteria?

N-(5'-phosphoribosyl)anthranilate isomerase (trpF, also known as PRAI) is an enzyme involved in the tryptophan biosynthesis pathway. It catalyzes the conversion of N-(5'-phosphoribosyl)anthranilate to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) .
In many organisms, including some bacteria, trpF exists as part of a bifunctional enzyme called TrpCF, which combines both phosphoribosylanthranilate isomerase (PRAI) activity and indole-glycerol-phosphate synthase (IGPS) activity in a single protein . This bifunctionality is an important aspect of the enzyme's role in the metabolic pathway for tryptophan biosynthesis, which is essential for bacterial growth and survival when environmental tryptophan is limited.

What are the optimal expression systems for producing recombinant Bordetella bronchiseptica trpF protein?

For the expression of recombinant Bordetella bronchiseptica trpF protein, several expression systems can be employed, including Escherichia coli, yeast, baculovirus, and mammalian cell systems . The choice depends on the research objectives and required protein characteristics.
E. coli is often the preferred system for initial expression due to its rapid growth, high protein yields, and cost-effectiveness. For structural studies requiring properly folded protein, E. coli BL21(DE3) strains with pET-based vectors typically offer good results with IPTG induction.
Based on research with similar recombinant Bordetella proteins, the following parameters have proven effective:

• Induction at OD600 of 0.6-0.8

• IPTG concentration: 0.5-1.0 mM

• Post-induction incubation: 4-6 hours at 30°C or overnight at 18°C to minimize inclusion body formation
  If E. coli expression results in insoluble protein, alternative systems such as yeast (Pichia pastoris) or insect cells using baculovirus vectors might provide better solubility with more authentic post-translational modifications .

What cloning strategies have been successful for Bordetella bronchiseptica trpF gene isolation?

Successful cloning of Bordetella bronchiseptica genes, including those encoding enzymes like trpF, typically involves the following methodology:

• Genomic DNA extraction from Bordetella bronchiseptica using standard bacterial DNA isolation methods

• PCR amplification of the trpF gene using primers designed based on the published genome sequence

• Addition of appropriate restriction enzyme sites to the PCR primers for directional cloning

• Restriction digestion of the PCR product and target vector

• Ligation and transformation into a cloning strain of E. coli (e.g., DH5α)
  For studies involving bifunctional enzymes like TrpCF, researchers have successfully amplified the full-length gene as well as the individual domains to study the function of each component separately . When isolating the trpF portion, it's critical to carefully analyze the domain boundaries to ensure the recombinant protein retains its native activity.

What methods are most effective for purifying recombinant Bordetella bronchiseptica trpF protein?

For the purification of recombinant Bordetella bronchiseptica trpF protein, a multi-step purification process is typically employed:

• Affinity chromatography: If the recombinant protein is expressed with a His-tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the preferred initial step. The typical elution profile uses an imidazole gradient from 20-250 mM.

• Ion-exchange chromatography: Based on the theoretical pI of trpF, either cation or anion exchange can be used as a secondary purification step. For Bordetella proteins, anion exchange (Q-Sepharose) at pH 8.0 typically provides good separation from contaminants.

• Size-exclusion chromatography: As a final polishing step, gel filtration using Superdex 75 or Superdex 200 columns can remove aggregates and ensure a homogeneous protein preparation.
  The purification protocol should be optimized to maintain the enzymatic activity of trpF, often requiring the inclusion of stabilizing agents such as 10% glycerol and 1-5 mM DTT or β-mercaptoethanol in all buffers to maintain the protein in a reduced state .

How can the enzymatic activity of recombinant Bordetella bronchiseptica trpF be measured?

The enzymatic activity of recombinant Bordetella bronchiseptica trpF (PRAI) can be measured using spectrophotometric assays that monitor the conversion of N-(5'-phosphoribosyl)anthranilate (PRA) to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP).
A standard assay procedure includes:

• Preparation of the substrate: PRA can be enzymatically synthesized using anthranilate phosphoribosyltransferase (TrpD) with anthranilate and phosphoribosyl pyrophosphate (PRPP) as substrates.

• Assay conditions: The reaction is typically performed in a buffer containing 100 mM Tris-HCl (pH 7.5-8.0), 5 mM MgCl2, and 1 mM DTT at 25-37°C.

• Activity measurement: The disappearance of PRA (decrease in absorbance at 350 nm) or appearance of CdRP (increase in absorbance at 400 nm) can be monitored spectrophotometrically.
  For bifunctional TrpCF enzymes, it's important to design the assay to specifically measure the PRAI activity independent of the IGPS activity. This can be achieved by using purified PRA as the substrate without the addition of indole-3-glycerol phosphate (IGP) synthase substrates .

How can recombinant Bordetella bronchiseptica trpF be evaluated for potential vaccine applications?

Evaluating recombinant Bordetella bronchiseptica trpF for vaccine applications involves a systematic approach:

• Immunogenicity testing: Assess the protein's ability to elicit antibody responses in animal models. Research has shown that recombinant Bordetella proteins can induce high antibody titers when administered with appropriate adjuvants .

• Protection assessment: Challenge studies in appropriate animal models (often mice for initial studies) to evaluate protective efficacy. Protection ratios should be calculated by comparing survival rates between vaccinated and control groups .

• Immune response characterization:

  • Humoral immunity: Measure IgG subtypes (IgG1, IgG2a, etc.) to determine Th1/Th2 balance

  • Cell-mediated immunity: Evaluate cytokine profiles (IL-4, IL-5, IFN-γ) from stimulated splenocytes
    For comparative analysis, recombinant Bordetella proteins like outer membrane porin protein (PPP) and lipoprotein (PL) have shown protection ratios of 62.5% and 50%, respectively, compared to 12.5% for control vaccinations . These proteins induced both humoral and cell-mediated immune responses, with a predominantly Th2-type humoral immune response . Similar methodologies could be applied to evaluate trpF.

What adjuvants are most effective for enhancing immune responses to recombinant Bordetella bronchiseptica trpF?

The selection of appropriate adjuvants is critical for maximizing the immunogenicity of recombinant Bordetella proteins, including trpF:

• Aluminum-based adjuvants (Alum, Aluminum hydroxide): These adjuvants predominantly promote Th2-type responses and have been used successfully with Bordetella antigens. They enhance antibody production but may provide limited stimulation of cell-mediated immunity.

• Oil-in-water emulsions (MF59, AS03): These adjuvants can enhance both humoral and cellular responses and may be particularly useful for recombinant protein-based vaccines.

• TLR agonists (CpG oligonucleotides, MPL): These adjuvants can shift the immune response toward a Th1-type response, which may be beneficial for protection against intracellular phases of Bordetella infection.
  In studies with recombinant Bordetella proteins, Freund's adjuvant has been used in experimental models to evaluate initial immunogenicity, but alternative adjuvants approved for veterinary use would be required for practical vaccine applications . The choice of adjuvant should be guided by the desired type of immune response (Th1 vs. Th2) and the target animal species.

How does the structure of Bordetella bronchiseptica trpF compare to homologs in other bacterial species?

The structural analysis of bacterial trpF proteins reveals important conservation patterns and species-specific differences:
While specific structural data for Bordetella bronchiseptica trpF is limited in the provided search results, insights can be drawn from related structures. TrpF proteins typically adopt an α/β fold characteristic of the N-(5'-phosphoribosyl)anthranilate isomerase family. Studies on the bifunctional TrpCF from other bacteria provide valuable comparative information.
For example, in Corynebacterium glutamicum, the crystal structure of TrpCF shows conformational changes in loop regions upon substrate binding . Amino acid sequence analysis of TrpCF homologs reveals highly conserved residues at the substrate-binding site, with certain positions showing variability that affects enzymatic activity .
Through site-directed mutagenesis, researchers have identified key residues that can enhance enzymatic activity, such as the P294K variant in C. glutamicum TrpCF that demonstrated improved catalytic efficiency . Similar approaches could be applied to Bordetella bronchiseptica trpF to understand structure-function relationships.

What role might trpF play in Bordetella bronchiseptica pathogenesis and host adaptation?

The role of trpF in Bordetella bronchiseptica pathogenesis and host adaptation presents an intriguing research area:

• Metabolic requirements during infection: As a tryptophan biosynthesis enzyme, trpF may be crucial for bacterial survival in host environments where tryptophan is limited. The ability to synthesize tryptophan could be particularly important during specific stages of infection.

• Host adaptation patterns: Electrophoretic mobility studies of metabolic enzymes in 188 Bordetella strains have revealed distinct patterns of genetic diversity and host adaptation . B. bronchiseptica shows considerable genetic diversity compared to other Bordetella species, with different electrophoretic types (ETs) showing varying degrees of host specificity .

• Potential as a virulence factor: While not traditionally classified as a virulence factor, metabolic enzymes like trpF could contribute to pathogenesis by enabling bacterial persistence in nutrient-limited host environments.
  The genetic diversity observed among B. bronchiseptica isolates from different host species suggests that metabolic adaptations, possibly including variations in tryptophan metabolism enzymes like trpF, may play a role in the bacterium's ability to infect a wide range of hosts . Further research comparing trpF sequences from B. bronchiseptica strains isolated from different host species could provide insights into potential host-specific adaptations.

How do molecular evolution patterns of trpF compare with mobile genetic elements in Bordetella species?

The molecular evolution of metabolic genes like trpF and their relationship to mobile genetic elements in Bordetella species presents a complex picture:
Analysis of 188 Bordetella strains has revealed interesting patterns of genetic diversity and molecular evolution. While the search results don't specifically address trpF evolution, they provide insights into Bordetella genome dynamics:

• Insertion sequence (IS) elements distribution: IS elements like IS1001, IS1002, and IS481 show distinct distribution patterns among Bordetella species. IS1001 is present in all B. parapertussis strains and in 70 of 144 B. bronchiseptica strains, with copy numbers ranging from 1-7 in B. bronchiseptica .

• Genetic diversity correlation: Genotypic diversity, as estimated by restriction fragment length polymorphism (RFLP) analysis, varies considerably among Bordetella species. Human B. parapertussis isolates show limited diversity, while B. bronchiseptica exhibits the highest diversity .

• Phylogenetic clustering: Phylogenetic analysis based on electrophoretic types (ETs) reveals clustering patterns that correlate with the presence of certain IS elements. All IS1001-containing Bordetella isolates cluster within a related group of ETs .
  The evolutionary pattern of metabolic genes like trpF might reflect core genome evolution, while mobile genetic elements represent more recent genetic events. Comparative analysis of trpF sequence conservation versus IS element distribution could provide insights into the different evolutionary forces shaping Bordetella genomes.

What are the optimal conditions for measuring kinetic parameters of recombinant Bordetella bronchiseptica trpF?

Determining the kinetic parameters of recombinant Bordetella bronchiseptica trpF requires careful optimization of reaction conditions:

• Reaction buffer optimization:

  • pH range: 7.0-8.5 (typically optimal around pH 7.5)

  • Buffer system: Tris-HCl or HEPES

  • Essential components: 5-10 mM MgCl₂ as a cofactor

  • Reducing agent: 1-2 mM DTT or β-mercaptoethanol to maintain cysteine residues in reduced form

• Temperature optimization:

  • Standard assay temperature: 25-37°C

  • Temperature stability profile should be established to ensure enzyme stability during the assay period

• Substrate concentration range:

  • For Km determination: 0.1-10 times the estimated Km value

  • Typically, N-(5'-phosphoribosyl)anthranilate concentrations from 1-100 μM

• Enzyme concentration:

  • Should be in the linear range of activity response

  • Typically 10-100 nM purified enzyme
    Data analysis should employ appropriate enzyme kinetics models (Michaelis-Menten, Lineweaver-Burk, or non-linear regression) to determine Km, Vmax, kcat, and catalytic efficiency (kcat/Km) . For bifunctional enzymes like TrpCF, it's important to design assays that can distinguish the PRAI activity from the IGPS activity.

What techniques are available for analyzing the structural dynamics of Bordetella bronchiseptica trpF?

Several complementary techniques can be employed to analyze the structural dynamics of Bordetella bronchiseptica trpF:

• X-ray crystallography:

  • Can provide high-resolution static structures

  • Co-crystallization with substrate analogs or inhibitors can capture different conformational states

  • As demonstrated with TrpCF from Corynebacterium glutamicum, crystallographic analysis can reveal conformational changes in loop regions upon substrate binding

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Provides information about protein dynamics and solvent accessibility

  • Can identify regions that undergo conformational changes upon substrate binding

  • Particularly useful for mapping protein-protein or protein-ligand interfaces

• Molecular dynamics (MD) simulations:

  • Allow in silico exploration of protein flexibility and conformational changes

  • Can model the effects of mutations on protein dynamics

  • Useful for generating hypotheses about structural mechanisms

• Site-directed mutagenesis combined with activity assays:

  • Strategic mutations can probe the functional roles of specific residues

  • Similar to the P294K variant of C. glutamicum TrpCF that showed enhanced activity

  • Sequential mutations can map the substrate-binding site and catalytic residues These techniques, used in combination, can provide comprehensive insights into how the structure of trpF relates to its function, and how conformational dynamics may contribute to its catalytic mechanism.