Recombinant Bartonella quintana Serine hydroxymethyltransferase (glyA)

What is the functional role of Serine hydroxymethyltransferase (GlyA) in Bartonella quintana?

Serine hydroxymethyltransferase (GlyA) in B. quintana is a pyridoxal phosphate (PLP)-dependent enzyme primarily involved in the reversible interconversion of serine and glycine using tetrahydrofolate as the one-carbon carrier. Beyond this canonical function, GlyA exhibits remarkably broad reaction specificity, catalyzing additional side reactions typical for PLP-dependent enzymes, including decarboxylation, transamination, and retroaldol cleavage .

Particularly significant is its potential alanine racemase co-activity, which has been demonstrated in vitro for GlyA from E. coli . In organisms like Chlamydiaceae that lack dedicated alanine racemases, GlyA has been shown to substitute for this function by facilitating the racemization of alanine both in vivo and in vitro . Given the evolutionary relationships between bacterial species, this suggests GlyA may serve a similar multifunctional role in B. quintana metabolism.

How does Bartonella quintana GlyA differ from orthologous proteins in related Bartonella species?

B. quintana and B. henselae show significant differences in various enzyme systems despite their phylogenetic proximity. While specific comparative data for GlyA is limited in the provided search results, the pattern of gene decay observed in B. quintana compared to B. henselae for several enzymes may offer insights into potential differences in GlyA function or regulation.

For instance, B. quintana has experienced evolutionary decay of several tRNA modification genes (ttcA, trmFO, and trmL) that remain intact in B. henselae . This pattern of reductive genome evolution in B. quintana could potentially impact GlyA function either directly through sequence variations or indirectly through metabolic network alterations. The loss of specific modifications in B. quintana resembles observations in other organisms with minimal genomes, suggesting adaptation to its host-restricted lifestyle .

Why is recombinant production of B. quintana GlyA important for research?

Recombinant production of B. quintana GlyA is essential for several reasons:

• Difficulty in culturing: B. quintana is fastidious and challenging to culture in laboratory conditions , making native protein isolation impractical for most research applications.

• Functional characterization: Recombinant production allows for detailed enzymological characterization, including kinetic parameters, substrate specificity, and potential moonlighting functions.

• Structural studies: Purified recombinant protein enables crystallography and other structural analyses to understand catalytic mechanisms.

• Immunological research: Purified GlyA can be used to study host immune responses to B. quintana infection, as proteins from this pathogen often serve as important antigens recognized by the human immune system during infection .

• Drug target validation: As a metabolic enzyme potentially essential for bacterial survival, recombinant GlyA facilitates screening of inhibitors that could serve as novel antimicrobial compounds.

How might the multifunctional properties of GlyA contribute to B. quintana pathogenesis and host adaptation?

The multifunctional properties of GlyA may contribute significantly to B. quintana pathogenesis through several mechanisms:

First, if B. quintana GlyA exhibits alanine racemase activity similar to that demonstrated in other bacteria , it could be crucial for providing D-alanine necessary for peptidoglycan synthesis, especially if B. quintana lacks dedicated alanine racemases. This would make GlyA essential for cell wall integrity and bacterial survival during infection.

Second, the canonical function of GlyA in one-carbon metabolism links it to nucleotide synthesis, amino acid metabolism, and methylation reactions - all critical for bacterial adaptation to changing host environments. The serine-glycine interconversion pathway intersects with numerous metabolic networks that influence bacterial fitness during infection.

Third, the potential moonlighting functions of GlyA could play roles in stress responses within the host environment. For instance, if GlyA participates in unexpected protein-protein interactions or has non-canonical substrates, these activities might contribute to bacterial persistence during immune pressures or antibiotic exposure.

Given that B. quintana has undergone genome reduction compared to related species , multifunctional enzymes like GlyA may be particularly important for maintaining metabolic versatility with a minimal genome.

What are the implications of the B. quintana gene transfer agent (BaGTA) for evolution of the glyA gene?

The Bartonella gene transfer agent (BaGTA) mediates highly efficient horizontal gene transfer (HGT) and could significantly impact the evolution of genes like glyA through several mechanisms:

BaGTA facilitates high-frequency genome-wide recombination among Bartonella species , potentially allowing for exchange of glyA variants with adaptive mutations. This system provides a mechanism for rapid adaptation to new environments or hosts, which may be reflected in glyA sequence diversity across Bartonella species.

Research has shown that BaGTA preferentially transfers DNA from a ROR (run-off replication) module, with transfer efficiency correlating directly with the distance to ROR . If glyA is positioned advantageously relative to this replication origin, it might experience higher rates of transfer and recombination.

BaGTA may also counter Muller's ratchet (the accumulation of deleterious mutations in clonal populations) , potentially maintaining glyA functionality even under conditions that would otherwise favor mutational decay. This could be particularly important for preserving essential functions of multifunctional enzymes like GlyA during evolutionary streamlining of the B. quintana genome.

Analysis of glyA sequence conservation and variability across Bartonella isolates using phylogenetic methods similar to those employed for the citrate synthase gene (gltA) could reveal patterns of selection and recombination influenced by the BaGTA system.

How does the loss of specific tRNA modification genes in B. quintana potentially affect GlyA expression and function?

The documented decay of several tRNA modification genes in B. quintana compared to B. henselae (ttcA, trmFO, and trmL) could have significant implications for GlyA expression and function:

Translational efficiency: The loss of tRNA modifications affects translational efficiency and accuracy. Specifically, the absence of s²C32 (due to ttcA decay) and the reduction of m⁵U levels by approximately 80% in B. quintana may alter the translation rate of glyA mRNA depending on its codon usage.

Codon-specific effects: The loss of Cm methylation in tRNA^Leu^CAA reduces the efficiency of codon-wobble base interaction . If the glyA gene contains codons dependent on these modified tRNAs, its expression could be differentially affected compared to other genes.

Translational accuracy: The loss of both Cm and s²C modifications in B. quintana is expected to reduce translation accuracy , potentially leading to increased mistranslation of GlyA. This could impact enzyme folding, stability, or function.

Adaptive significance: The pattern of tRNA modification loss in B. quintana resembles what is observed in other organisms with minimal genomes , suggesting it may be part of an adaptive strategy. This could indicate that GlyA function remains essential despite translational alterations, possibly through compensatory mechanisms.

Further research examining codon usage in the B. quintana glyA gene relative to the modified tRNA population would provide insights into how these translational adaptations might specifically affect GlyA production and function.

What are the optimal expression systems and conditions for producing functional recombinant B. quintana GlyA?

Based on established methods for recombinant Bartonella proteins, the following approach is recommended for B. quintana GlyA expression:

Expression system selection:

• E. coli expression using pMX vector systems has been successful for other Bartonella proteins, with the protein expressed in-frame with glutathione S-transferase (GST) for simplified purification .

• BL21(DE3) or DH5α E. coli strains are suitable hosts, with DH5α showing good results for other Bartonella proteins .

Expression conditions optimization:

• Induce protein expression when bacterial cultures reach log phase (OD₆₀₀ of 0.6-0.8).

• Use IPTG at a final concentration of 1 nM for induction , though optimization between 0.1-1.0 mM may be necessary.

• Express at lower temperatures (16-25°C) to enhance proper folding of the PLP-dependent enzyme.

• Include 50-100 μM pyridoxal 5'-phosphate in the growth medium to ensure cofactor incorporation.

Purification strategy:

• Affinity chromatography using glutathione Sepharose 4B columns for GST-tagged protein .

• Release the protein from GST by thrombin cleavage.

• Further purify using ion exchange chromatography if needed.

• Concentrate the purified protein using centrifugal filter columns (10 kDa cutoff).

• Determine protein concentration by Bradford protein assay .

Storage considerations:

• Store purified GlyA with 5-50% glycerol at -20°C/-80°C for long-term storage.

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week .

What are the most reliable methods for confirming the identity and purity of recombinant B. quintana GlyA?

A comprehensive approach to confirm identity and purity of recombinant B. quintana GlyA should include:

Protein identity confirmation:

• SDS-PAGE analysis: Verify expected molecular weight (typically ~45-50 kDa for bacterial GlyA monomers).

• Mass spectrometry analysis:

  • Peptide mass fingerprinting (PMF) to match observed peptide masses with theoretical masses from the B. quintana GlyA sequence .

  • LC-MS/MS sequencing of tryptic peptides for definitive sequence confirmation.

• Western blotting: Using anti-GlyA antibodies if available, or anti-His/GST antibodies if the recombinant protein contains these tags.

• N-terminal sequencing: Edman degradation to confirm the first 5-10 amino acids match the expected sequence.

Purity assessment:

• SDS-PAGE: Aim for >85% purity as assessed by densitometry of Coomassie-stained gels .

• Size exclusion chromatography: To assess aggregation state and homogeneity.

• Dynamic light scattering: To evaluate size distribution and potential aggregation.

Functional verification:

• Spectroscopic analysis: Absorption spectrum to confirm PLP cofactor binding (characteristic peak at ~410-430 nm).

• Enzymatic activity assay: Measure serine hydroxymethyltransferase activity using established spectrophotometric assays.

• Circular dichroism: To assess secondary structure content and proper folding.

What are the challenges in designing specific primers for PCR amplification of the B. quintana glyA gene?

Designing specific primers for PCR amplification of the B. quintana glyA gene presents several challenges:

Sequence conservation challenges:

• GlyA is a highly conserved enzyme across bacterial species, making it difficult to design primers that specifically amplify only the B. quintana homolog.

• Sequence alignment with close relatives (especially B. henselae) is essential to identify unique regions for B. quintana-specific amplification.

Primer design strategy:

• Flanking regions approach: Design primers targeting conserved flanking regions of the glyA gene, similar to approaches used for amplification of Bartonella species-specific genes :

  • Forward primer targeting upstream conserved sequences

  • Reverse primer targeting downstream conserved sequences

  • Example primer design approach: identify conserved sequences in untranslated regions or neighboring genes as done for p26 amplification in Bartonella species

• Species-specific approach: For distinguishing B. quintana from other Bartonella species:

  • Target polymorphic regions within the glyA gene

  • Design internal primers that bind only to B. quintana-specific sequences

  • Consider nested PCR approaches for increased specificity

PCR optimization considerations:

• Optimal amplification conditions similar to those used for gltA gene: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30s, 52-55°C for 30s, and 72°C for 45s .

• Use high-fidelity DNA polymerase such as HotStarTaq for accurate amplification .

• GC content of Bartonella DNA may require addition of DMSO or other PCR enhancers.

Validation requirements:

• Sequence verification of amplicons is essential to confirm specificity.

• Include controls from related Bartonella species (especially B. henselae) to confirm specificity.

• Consider development of real-time PCR assays with species-specific probes for increased sensitivity and specificity, similar to approaches used for other Bartonella targets .

What assays can be employed to characterize the enzymatic activities of recombinant B. quintana GlyA?

Multiple assays can be employed to characterize the diverse enzymatic activities of recombinant B. quintana GlyA:

Canonical serine hydroxymethyltransferase activity:

• Spectrophotometric coupled assay: Measure the rate of tetrahydrofolate-dependent conversion of serine to glycine by coupling to NADH oxidation through appropriate secondary enzymes.

• Radiometric assay: Using [³H]-serine or [¹⁴C]-serine to quantify formation of labeled glycine and formaldehyde.

• HPLC-based assay: Separating and quantifying substrate and product concentrations over time.

Alanine racemase activity:

• D-amino acid oxidase coupled assay: Measuring the formation of D-alanine from L-alanine by coupling to H₂O₂ production and detection with horseradish peroxidase.

• Circular dichroism: Monitoring changes in optical rotation during racemization.

• HPLC with chiral columns: For separation and quantification of D- and L-alanine.

Other PLP-dependent side reactions:

• Transamination activity: Using spectrophotometric assays to monitor transfer of amino groups between amino acids and keto acids.

• Decarboxylation activity: Measuring CO₂ release from amino acid substrates.

• Retroaldol cleavage: Monitoring formation of specific products via HPLC or coupled spectrophotometric assays.

Cofactor binding and enzyme kinetics:

• Spectroscopic analysis: Measuring PLP binding through changes in absorption spectrum (typically 410-430 nm).

• Enzyme kinetics: Determination of Km, Vmax, kcat, and kcat/Km for various substrates under different conditions.

• Inhibition studies: Characterizing response to known SHMT inhibitors like methotrexate analogs.

Structural and biophysical characterization:

• Thermal shift assays: To assess protein stability and effects of ligands on protein folding.

• Isothermal titration calorimetry: For precise measurement of substrate binding thermodynamics.

• Hydrogen-deuterium exchange mass spectrometry: To identify conformational changes upon substrate binding.

These assays should be performed under physiologically relevant conditions, considering the intracellular environment of B. quintana during host infection.

How can the potential moonlighting functions of B. quintana GlyA be identified and characterized?

Identifying and characterizing potential moonlighting functions of B. quintana GlyA requires a multifaceted approach:

Bioinformatic analysis:

• Comparative sequence analysis with GlyA enzymes known to have moonlighting functions

• Structural modeling to identify potential binding sites for non-canonical interactions

• Analysis of surface-exposed residues that might mediate protein-protein interactions

• Scanning for sequence motifs associated with specific alternative functions

Protein-protein interaction studies:

• Yeast two-hybrid screening against a B. quintana protein library

• Pull-down assays using recombinant GlyA as bait

• Cross-linking studies coupled with mass spectrometry, similar to approaches used to identify BadA interactions with fibronectin

• Surface plasmon resonance to characterize binding kinetics with potential partners

Functional screening approaches:

• Complementation studies in bacterial mutants lacking specific enzymatic activities

• Activity-based protein profiling to identify non-canonical substrates

• Metabolomic analysis comparing wild-type and GlyA-depleted B. quintana to identify unexpected metabolic changes

• In vitro screening of recombinant GlyA against substrate libraries

Cellular localization studies:

• Immunofluorescence microscopy to determine if GlyA localizes to unexpected cellular compartments during infection

• Subcellular fractionation coupled with Western blotting to identify GlyA in membrane fractions or secreted components

• Analysis of surface proteome to determine if GlyA is exposed on the bacterial surface

In vivo relevance studies:

• Gene knockout or knockdown studies with phenotypic characterization beyond expected metabolic effects

• Site-directed mutagenesis to separate canonical and moonlighting functions

• Infection models comparing wild-type and GlyA-mutant strains

This approach has successfully identified moonlighting functions in other bacterial enzymes and could reveal unexpected roles for GlyA in B. quintana pathogenesis or survival.

How does the presence or absence of PLP cofactor affect the stability and activity of recombinant B. quintana GlyA?

As a PLP (pyridoxal 5'-phosphate)-dependent enzyme, the cofactor plays a critical role in both the stability and catalytic activity of GlyA:

Structural and stability effects:

• PLP forms a Schiff base with a conserved lysine residue in the active site, creating an internal aldimine that stabilizes the enzyme's tertiary structure.

• Apo-GlyA (without PLP) typically exhibits reduced thermal stability compared to the holo-enzyme (with PLP).

• PLP binding induces conformational changes that can be monitored through spectroscopic methods:

  • Holo-GlyA shows characteristic absorption peaks at approximately 410-430 nm

  • Changes in circular dichroism spectra reflect secondary structure stabilization

Catalytic implications:

• PLP is essential for all known catalytic activities of GlyA, including:

  • Canonical serine/glycine interconversion

  • Alanine racemase activity

  • Other side reactions (transamination, decarboxylation)

• The cofactor participates directly in catalysis by:

  • Stabilizing carbanion intermediates

  • Facilitating proton abstraction from substrate α-carbon

  • Enabling electron delocalization through its conjugated π system

Experimental considerations for recombinant production:

• Expression in PLP-supplemented media (50-100 μM) increases the proportion of correctly folded, active enzyme.

• Purification buffers should contain 10-20 μM PLP to maintain cofactor saturation.

• Storage conditions should protect the PLP from light degradation (amber vials, protection from light).

• For activity assays, pre-incubation with excess PLP followed by gel filtration ensures cofactor saturation.

Quantitative measurements:

• The PLP occupancy can be determined by:

  • Releasing bound PLP with base treatment and measuring fluorescence

  • Comparing absorption at 410-430 nm before and after reconstitution with excess PLP

• Kinetic parameters often show significant differences between partially and fully PLP-saturated enzyme.

Understanding the relationship between PLP binding and GlyA function is crucial for accurately characterizing the enzyme's various activities and for designing potential inhibitors for therapeutic applications.

What is the potential of B. quintana GlyA as a diagnostic marker for Bartonella infections?

The potential of B. quintana GlyA as a diagnostic marker for Bartonella infections can be evaluated from several perspectives:

Advantages as a diagnostic target:

• GlyA is likely a constitutively expressed metabolic enzyme, ensuring consistent presence during infection.

• As an essential enzyme, its sequence may be more conserved than surface antigens that undergo selective pressure from the immune system.

• If B. quintana GlyA has unique epitopes compared to human SHMT or other bacterial homologs, it could enable specific detection.

Serological diagnostic applications:

• Analysis of immunoreactive B. quintana proteins has identified multiple antigens recognized by patient sera during infection . While GlyA was not specifically mentioned among the immunodominant antigens in the search results, recombinant GlyA could be screened for immunoreactivity with:

  • Patient sera from confirmed B. quintana infections

  • Control sera from healthy individuals and those with other bacterial infections

  • Comparative analysis with sera reactive to B. henselae

Molecular diagnostic applications:

• PCR-based detection targeting glyA could complement existing molecular diagnostics that target the citrate synthase gene (gltA) or 16S rRNA .

• Species-specific regions within the glyA gene could enable differentiation between B. quintana and other Bartonella species.

• Quantitative PCR targeting glyA could potentially assess bacterial load in clinical samples.

Technical considerations for diagnostic development:

• Sensitivity and specificity would need to be compared with existing diagnostic methods, such as the semiquantitative PCR-based enzyme immunoassay for B. quintana detection , which has demonstrated sensitivity equivalent to 5 CFU per reaction.

• Integration into multiplexed detection systems could enhance diagnostic value.

• Validation with diverse clinical samples would be necessary to establish diagnostic performance characteristics.

While more research is needed to fully evaluate GlyA's diagnostic potential, its essential metabolic role and potential species-specific characteristics make it a candidate worth investigating for B. quintana detection.

How might GlyA function as a potential drug target for treating Bartonella quintana infections?

GlyA represents a promising drug target for treating B. quintana infections for several reasons:

Target validation considerations:

• As an essential metabolic enzyme involved in one-carbon metabolism, inhibition of GlyA would likely impair bacterial growth and survival.

• If B. quintana GlyA indeed possesses alanine racemase activity in the absence of dedicated alanine racemases , it would be even more critical for bacterial viability by providing D-alanine necessary for peptidoglycan synthesis.

• The potential multifunctional nature of GlyA in B. quintana makes it an attractive target, as inhibition could disrupt multiple metabolic pathways simultaneously.

Structural and biochemical targeting strategy:

• The active site of GlyA contains the PLP cofactor, which offers a unique binding pocket for the development of competitive inhibitors.

• Crystal structures of bacterial GlyA enzymes reveal several targetable sites:

  • The PLP binding pocket

  • The substrate binding site

  • Allosteric sites that regulate enzyme function

  • The intersubunit interfaces (as GlyA typically functions as a dimer or tetramer)

Drug development approaches:

• Structure-based design: Using homology models of B. quintana GlyA based on crystal structures of related bacterial SHMTs to design specific inhibitors.

• High-throughput screening: Testing chemical libraries against recombinant B. quintana GlyA to identify lead compounds.

• Fragment-based drug discovery: Building inhibitors by linking smaller molecules that bind to different regions of the enzyme.

• Repurposing existing SHMT inhibitors: Evaluating antifolate drugs or other known inhibitors of SHMT for activity against B. quintana GlyA.

Selectivity considerations:

• Human cells express both cytosolic and mitochondrial SHMT isoforms, necessitating selective targeting of bacterial GlyA.

• Potential structural differences between bacterial and human enzymes could be exploited for selective inhibition.

• Bacterial-specific moonlighting functions of GlyA could offer unique targeting opportunities not present in the human homologs.

Delivery challenges:

• B. quintana's intracellular lifecycle poses drug delivery challenges that would need to be addressed.

• Compounds may need to penetrate both host cell membranes and bacterial membranes to reach their target.

The development of GlyA inhibitors represents a novel approach to treating B. quintana infections that could complement or replace current antibiotic therapies.

How does genetic diversity among clinical isolates of B. quintana impact the structure and function of GlyA?

The genetic diversity among clinical isolates of B. quintana and its impact on GlyA structure and function can be analyzed through several approaches:

Evolutionary context and diversity assessment:

• While B. quintana shows relatively low genetic diversity compared to other bacteria, the BaGTA gene transfer agent facilitates recombination that could contribute to glyA sequence variation .

• Comparison of glyA sequences across clinical isolates could reveal:

  • Conserved regions essential for enzymatic function

  • Variable regions potentially under selection pressure

  • Evidence of recombination events

Sequence variation impacts:

• Even minor sequence variations could impact GlyA function through:

  • Altered substrate binding kinetics

  • Changes in PLP cofactor binding affinity

  • Modified protein stability or oligomerization

  • Differences in regulatory properties

  • Variations in potential moonlighting functions

Analytical approaches to characterize diversity:

• Comparative sequence analysis: Similar to approaches used for the citrate synthase gene (gltA) where sequence similarities between different Bartonella species were 83.8-93.5%, while intraspecies similarity exceeded 99.8% .

• Structural modeling: Homology modeling of GlyA variants to predict functional consequences of polymorphisms.

• Recombinant expression studies: Expressing GlyA variants from different clinical isolates to compare:

  • Enzymatic activities

  • Substrate preferences

  • Inhibitor susceptibilities

  • Protein stability parameters

• Clinical correlation analysis: Associating GlyA sequence variants with:

  • Disease manifestations

  • Treatment responses

  • Host adaptation markers

  • Geographical distribution patterns

Methodological approach for diversity studies:

• PCR amplification of the glyA gene from clinical isolates

• Next-generation sequencing to identify variants across the B. quintana population

• Recombinant expression of representative variants for functional characterization

• Structural analysis through X-ray crystallography or cryo-electron microscopy

Understanding this genetic diversity would provide insights into B. quintana evolution and could inform the development of diagnostics and therapeutics targeting GlyA.