Recombinant Bartonella quintana Glutamate--tRNA ligase 2 (gltX2)

What is the biological role of Glutamate--tRNA ligase 2 in Bartonella quintana?

Glutamate--tRNA ligase 2 (gltX2) in B. quintana is an essential enzyme responsible for catalyzing the attachment of glutamate to its cognate tRNA (tRNA^Glu), a critical step in protein biosynthesis. Unlike many bacteria that possess a single gltX gene, B. quintana contains two distinct glutamyl-tRNA synthetase genes (gltX1 and gltX2), suggesting specialized functions. The gltX2 enzyme likely plays a crucial role in B. quintana's adaptation to different environments during its infectious cycle, particularly when transitioning between the hemin-restricted human bloodstream (37°C) and the hemin-rich body louse vector environment (28°C) . This adaptation is essential as B. quintana must survive in these dramatically different niches to maintain its transmission cycle.

What are the optimal conditions for expressing recombinant B. quintana gltX2?

For optimal expression of recombinant B. quintana gltX2, researchers should consider a strategy similar to that used for other B. quintana proteins :

• Vector selection: pET-28a(+) vector system with an N-terminal 6×His tag has been successfully used for B. quintana proteins and would likely be appropriate for gltX2 .

• Expression system: E. coli BL21(DE3) is recommended due to its reduced protease activity and compatibility with T7 promoter-based expression.

• Culture conditions:

  • Initial growth at 37°C to OD600 of 0.6-0.8

  • Induction with 0.5-1.0 mM IPTG

  • Post-induction temperature reduction to 18-25°C for 16-20 hours to enhance protein solubility

• Buffer optimization:

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

  • Addition of 0.1-0.5% Triton X-100 can improve solubility

• Purification protocol:

  • Immobilized metal affinity chromatography using Ni-NTA resin

  • Size exclusion chromatography for higher purity

This methodology is based on successful expression strategies for other B. quintana proteins and standard approaches for aminoacyl-tRNA synthetases from related organisms.

How does hemin concentration affect gltX2 activity and expression in B. quintana?

B. quintana has a unique ability to survive exposure to hemin concentrations that are typically bactericidal (>1 mM) . This adaptation is critical for survival in the hemin-rich body louse environment compared to the hemin-restricted human bloodstream. To investigate how hemin concentration affects gltX2:

• Expression analysis: Culture B. quintana under varying hemin concentrations (0.1 mM to 2.0 mM) and quantify gltX2 expression using RT-qPCR . Compare this with expression patterns of known hemin-responsive genes like rpoE, which shows upregulation under high hemin conditions.

• Enzyme activity assays: Measure the aminoacylation activity of purified recombinant gltX2 in the presence of different hemin concentrations using:

  • ATP-PPi exchange assay

  • tRNA aminoacylation assay with [³H]-glutamate

  • Analysis of reaction kinetics (Km and Vmax) to determine if hemin acts as an activator or inhibitor

• Structural analysis: Investigate potential hemin-binding sites in gltX2 through:

  • UV-visible spectroscopy to detect hemin-protein interactions

  • Site-directed mutagenesis of potential hemin-binding residues

  • Crystallography of gltX2 with and without hemin

This comprehensive approach would elucidate whether gltX2 plays a role in B. quintana's remarkable tolerance to high hemin concentrations, which is a key adaptation for survival in the body louse vector .

What is the relationship between gltX2 function and the stress response mechanisms in B. quintana?

B. quintana employs various stress response mechanisms to survive environmental transitions. The extracytoplasmic function (ECF) sigma factor RpoE has been identified as important in mediating B. quintana's tolerance to high hemin concentrations and temperature changes . To investigate potential relationships between gltX2 and stress response pathways:

• Transcriptional regulation analysis:

  • Perform chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against RpoE to determine if gltX2 is part of the RpoE regulon

  • Analyze the gltX2 promoter region for ECF15 sigma factor binding motifs

  • Construct reporter gene fusions to measure gltX2 promoter activity in response to various stressors

• Protein-protein interaction studies:

  • Use bacterial two-hybrid assays to detect interactions between gltX2 and stress response proteins

  • Perform co-immunoprecipitation experiments followed by mass spectrometry to identify protein complexes

  • Investigate if NepR (anti-sigma factor) or PhyR (response regulator) influence gltX2 expression or activity

• Mutant phenotype analysis:

  • Create ΔgltX2 mutant strains (if viable) or conditional knockdowns

  • Compare stress tolerance profiles with ΔrpoE and ΔrpoE ΔnepR mutants

  • Assess survival under conditions mimicking host-vector transition

This research would provide insight into whether gltX2 functions beyond protein synthesis as part of B. quintana's adaptive stress response network.

How can researchers effectively design experiments to assess gltX2 function during B. quintana infection cycles?

Designing experiments to assess gltX2 function during B. quintana's complex infection cycle requires approaches that span in vitro, in vivo, and ex vivo systems:

• In vitro host-vector models:

  • Develop a temperature-shift model (37°C to 28°C) with varying hemin concentrations to mimic transition between host and vector

  • Monitor gltX2 expression and protein levels during transitions

  • Use conditional expression systems to modulate gltX2 levels at different stages

• Ex vivo infection models:

  • Establish body louse gut epithelial cell cultures and human erythrocyte/endothelial cell co-cultures

  • Assess gltX2 expression in bacteria attached to or internalized within different cell types

  • Compare wild-type bacteria with gltX2 mutants for attachment, invasion, and persistence

• In vivo approaches:

  • Develop body louse infection models to study gltX2 expression and bacterial fitness in the vector

  • Consider humanized mouse models for studying mammalian infection aspects

  • Use competition assays between wild-type and gltX2 mutants to assess fitness costs

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data to build a systems-level understanding of gltX2 function

  • Use stable isotope labeling to track glutamate incorporation during different infection stages

This experimental framework would provide comprehensive insights into gltX2 function throughout the B. quintana lifecycle and potentially identify novel therapeutic targets.

What are the best methods for purifying active recombinant B. quintana gltX2?

Purifying active recombinant B. quintana gltX2 requires specific techniques to maintain enzyme functionality:

• Expression optimization:

  • Test multiple fusion tags beyond 6×His, including MBP (maltose binding protein) which has been successful for other B. quintana proteins

  • Use specialized E. coli strains like Rosetta or Arctic Express to address potential codon bias or folding issues

  • Co-express with bacterial chaperones (GroEL/GroES) if solubility is problematic

• Purification protocol:

  Step	Method	Buffer Composition	Notes
  Lysis	Sonication or French press	50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol	Add 0.1% Triton X-100 for membrane dissociation
  IMAC	Ni-NTA affinity	Above + 20-250 mM imidazole gradient	Low imidazole wash (20 mM) followed by elution
  Ion exchange	Q-Sepharose	50 mM Tris-HCl pH 7.5, 50-500 mM NaCl gradient	Removes nucleic acid contaminants
  Size exclusion	Superdex 200	20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT	Ensures homogeneity

• Activity preservation:

  • Include 5-10% glycerol in all buffers to maintain stability

  • Add zinc or magnesium ions (1-5 mM) as cofactors

  • Store purified protein at -80°C in single-use aliquots with 20% glycerol

• Quality assessment:

  • Circular dichroism to confirm proper folding

  • Dynamic light scattering to verify monodispersity

  • Aminoacylation activity assays to confirm function

This comprehensive purification strategy accounts for the challenges often encountered with B. quintana proteins and should yield active recombinant gltX2 suitable for structural and functional studies.

How can researchers develop specific inhibitors targeting B. quintana gltX2?

Developing specific inhibitors for B. quintana gltX2 involves a structured drug discovery approach focusing on selectivity over human glutamyl-tRNA synthetase:

• Structural analysis and target validation:

  • Solve the crystal structure of B. quintana gltX2

  • Perform structural alignment with human glutamyl-tRNA synthetase

  • Identify unique binding pockets or conformational states

  • Validate gltX2 as an essential target using conditional knockdown systems

• Inhibitor discovery strategies:

  • Structure-based virtual screening against identified binding pockets

  • Fragment-based screening using thermal shift assays

  • Repurposing known aminoacyl-tRNA synthetase inhibitors

  • High-throughput biochemical assays using the ATP-PPi exchange reaction

• Compound optimization workflow:

  Stage	Methods	Endpoints	Success Criteria
  Primary screening	Biochemical assays	IC50 for enzyme inhibition	<10 μM potency
  Selectivity	Parallel testing against human enzyme	Selectivity index	>100-fold selectivity
  Cellular activity	B. quintana growth inhibition	MIC determination	MIC <5 μg/ml
  Mechanism validation	Enzyme kinetics, binding studies	Ki, binding mode	Confirmed mechanism
  Lead optimization	Medicinal chemistry, ADME studies	Improved properties	Suitable for in vivo testing

• In vitro and ex vivo efficacy assessment:

  • Test in B. quintana cultures at both 28°C and 37°C conditions

  • Evaluate efficacy in cell infection models

  • Assess activity against intracellular bacteria

  • Determine effect on bacterial persistence and stress tolerance

This systematic approach leverages structural and functional information about gltX2 to develop selective inhibitors that could serve as research tools or potential therapeutic leads for B. quintana infections.

What techniques are most effective for investigating the potential role of gltX2 in B. quintana pathogenesis?

Investigating gltX2's role in B. quintana pathogenesis requires integrating molecular and cellular approaches:

• Genetic manipulation strategies:

  • Conditional gene expression systems using tetracycline-inducible promoters

  • CRISPR interference (CRISPRi) for partial knockdown if complete deletion is lethal

  • Site-directed mutagenesis of catalytic residues to create enzymatically inactive variants

  • Complementation with wild-type or mutant alleles to verify phenotypes

• Host-pathogen interaction models:

  • Endothelial cell infection models to study vascular manifestations seen in patients

  • Erythrocyte colonization assays to evaluate gltX2's role in bloodstream persistence

  • Ex vivo body louse feeding models to assess vector colonization

• Virulence assessment methodologies:

  • Comparative transcriptomics of wild-type vs. gltX2-modified strains

  • Secretome analysis to identify pathogenicity factors dependent on gltX2

  • Immune response profiling to detect gltX2-dependent inflammation patterns

  • Patient sample analysis correlating gltX2 expression with disease severity

• Clinical relevance evaluation:

  • For patients with confirmed B. quintana infection and varied clinical presentations (trench fever, endocarditis, bacillary angiomatosis) , analyze:

  Clinical Presentation	Samples to Analyze	Parameters to Measure	Expected Findings
  Bacteremia	Blood	gltX2 expression levels	Potential correlation with persistence
  Endocarditis	Valve tissue	gltX2 protein localization	Association with vegetation formation
  Neurological symptoms	CSF	Antibody response to gltX2	Potential diagnostic marker

This comprehensive approach would provide insights into whether gltX2 functions as a direct virulence factor or if its role is primarily metabolic but indirectly impacts pathogenicity through bacterial fitness and persistence .

How does B. quintana gltX2 compare to orthologous enzymes in other Bartonella species?

Comparative analysis of gltX2 across Bartonella species provides evolutionary insights and reveals potential species-specific adaptations:

• Phylogenetic analysis approach:

  • Construct multiple sequence alignments of gltX2 from B. quintana, B. henselae, B. vinsonii, and other Bartonella species

  • Calculate sequence conservation and identify species-specific variations

  • Map variations onto structural models to predict functional consequences

  • Trace evolutionary history of gltX duplication events in the Bartonella lineage

• Functional comparison methodology:

  • Express and purify recombinant gltX2 from multiple Bartonella species

  • Compare enzymatic parameters (kcat, Km) for glutamate and tRNA substrates

  • Assess temperature optima and stability profiles in relation to host body temperatures

  • Evaluate response to environmental stressors relevant to each species' lifecycle

• Host-specificity correlation:

  • Analyze gltX2 sequence variations in relation to host range (human-specific B. quintana vs. zoonotic species)

  • Examine if gltX2 properties correlate with vector specificity (body louse vs. flea or tick)

  • Investigate potential co-evolution with host tRNA populations

This comparative approach would determine whether differences in gltX2 contribute to the distinct host preferences, transmission vectors, and disease manifestations observed across Bartonella species.

What potential non-canonical functions might gltX2 serve in B. quintana beyond protein synthesis?

Recent research on aminoacyl-tRNA synthetases has revealed numerous functions beyond their classical role in protein synthesis. To investigate potential non-canonical functions of B. quintana gltX2:

• Protein interaction network analysis:

  • Perform pull-down assays with tagged gltX2 followed by mass spectrometry

  • Use bacterial two-hybrid screens to identify interaction partners

  • Map the interaction network and identify connections to signaling pathways

• Regulatory RNA studies:

  • Investigate whether gltX2 binds non-cognate RNAs using RNA immunoprecipitation

  • Perform CLIP-seq (cross-linking immunoprecipitation-sequencing) to identify all RNA targets

  • Assess potential moonlighting functions in regulating gene expression

• Alternative substrate screening:

  • Test gltX2 activity with non-canonical amino acids or amino acid analogs

  • Investigate potential incorporation of glutamine through mischarging

  • Examine relationships to glutamate-dependent stress response pathways

• Host-pathogen interface functions:

  • Assess if gltX2 or fragments are secreted during infection

  • Test for potential immunomodulatory effects on host cells

  • Investigate if host glutamyl-tRNA synthetase function is affected during infection

This research direction would expand our understanding of gltX2 beyond its canonical role and potentially reveal new therapeutic targets if moonlighting functions contribute to pathogenesis.

How might advances in structural biology techniques enhance our understanding of B. quintana gltX2?

Advanced structural biology approaches offer unprecedented opportunities to understand gltX2 function and dynamics:

• Cryo-electron microscopy applications:

  • Achieve high-resolution structures of gltX2 in different functional states

  • Visualize gltX2-tRNA complexes to understand recognition mechanisms

  • Capture conformational changes during catalysis

  • Examine potential oligomeric states or interaction with other cellular components

• Integrative structural biology approach:

  • Combine X-ray crystallography, NMR, and SAXS data

  • Use hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

  • Apply molecular dynamics simulations to predict functional motions

  • Employ cross-linking mass spectrometry to validate protein interaction networks

• In situ structural studies:

  • Use cryo-electron tomography to visualize gltX2 localization within bacterial cells

  • Apply proximity labeling techniques to map the spatial organization

  • Implement super-resolution microscopy to track gltX2 during different growth conditions

These advanced structural approaches would provide unprecedented insights into how gltX2 functions in the context of B. quintana's adaptation to its unique lifecycle between human hosts and louse vectors , potentially revealing structural features that could be exploited for targeted drug development.

What bioinformatic approaches are most valuable for analyzing gltX2 in the context of B. quintana metabolism?

Comprehensive bioinformatic analysis of gltX2 in the broader metabolic context of B. quintana requires integrative approaches:

• Metabolic network reconstruction:

  • Map gltX2 in relation to glutamate metabolism pathways

  • Identify potential metabolic bottlenecks where gltX2 function is critical

  • Use flux balance analysis to predict the impact of altered gltX2 activity

  • Model metabolic shifts during host-vector transitions

• Comparative genomics methodology:

  • Analyze the genomic context of gltX2 across Bartonella species

  • Identify conserved operon structures or regulatory elements

  • Perform synteny analysis to detect genomic rearrangements affecting gltX2

  • Compare with related alphaproteobacteria to identify unique features

• Regulatory network inference:

  • Predict transcription factor binding sites in the gltX2 promoter region

  • Construct gene regulatory networks incorporating gltX2

  • Integrate with stress response pathways, particularly RpoE-mediated regulation

  • Model the impact of environmental signals on gltX2 expression

• Multi-omics data integration framework:

  Data Type	Analysis Approach	Integration Method	Expected Insights
  Genomics	SNP/variation analysis	Mapping to structure	Potential adaptive mutations
  Transcriptomics	Differential expression	Co-expression networks	Condition-specific regulation
  Proteomics	Protein abundance	Correlation analysis	Post-transcriptional control
  Metabolomics	Glutamate/glutamine flux	Pathway analysis	Metabolic consequences

This systems biology approach would position gltX2 within the broader context of B. quintana metabolism and adaptation to different host environments.

What are the main technical challenges in studying B. quintana gltX2 and how can researchers overcome them?

Researchers face several technical challenges when studying B. quintana gltX2:

• Cultivation difficulties:

  • Challenge: B. quintana is fastidious, requiring specialized media and extended incubation periods (12-14 days for primary isolation)

  • Solution: Use liquid BAPGM (Bartonella alpha-Proteobacteria growth medium) enrichment cultures prior to plating on solid media , optimize hemin concentration based on experimental goals , and implement co-cultivation with eukaryotic cells for enhanced recovery

• Genetic manipulation limitations:

  • Challenge: Low transformation efficiency and limited genetic tools for B. quintana

  • Solution: Adapt techniques from related alphaproteobacteria, optimize electroporation conditions specific to B. quintana, and develop shuttle vectors with appropriate selection markers

• Protein expression and purification hurdles:

  • Challenge: Obtaining sufficient quantities of soluble, active recombinant gltX2

  • Solution: Use specialized expression systems (like IMPACT or cell-free systems), explore fusion partners beyond standard His-tags , and optimize buffer conditions to maintain stability

• In vivo model constraints:

  • Challenge: Limited animal models that recapitulate B. quintana's natural infection cycle

  • Solution: Develop humanized mouse models, establish body louse colonies for vector studies, and create ex vivo systems mimicking human vascular endothelium

• Functional redundancy complications:

  • Challenge: Potential functional overlap between gltX1 and gltX2 complicating phenotypic analysis

  • Solution: Develop conditional expression systems, use CRISPRi for partial knockdowns, and employ isoform-specific inhibitors

By addressing these technical challenges with innovative approaches, researchers can overcome the difficulties inherent to studying this fastidious pathogen and its aminoacyl-tRNA synthetases.

How might research on B. quintana gltX2 contribute to understanding other vector-borne pathogens?

Research on B. quintana gltX2 has broader implications for understanding vector-borne pathogen biology:

• Comparative adaptation mechanisms:

  • Insights from how gltX2 functions in the host-vector transition of B. quintana can inform studies of adaptation in other vector-borne pathogens like Rickettsia, Borrelia, and Anaplasma

  • The temperature-responsive regulation mechanisms may represent conserved strategies across arthropod-transmitted bacteria

• Vector colonization strategies:

  • Understanding how translation machinery components like gltX2 adapt to the vector environment can reveal common mechanisms for vector colonization

  • These insights could lead to novel strategies for blocking transmission at the vector stage

• Co-infection dynamics:

  • As B. quintana infections are sometimes found alongside other vector-borne pathogens , research on gltX2 could reveal how metabolic adaptations influence co-infection dynamics

  • This may explain why certain vector-borne diseases show overlapping epidemiology

• Translational significance:

  • Aminoacyl-tRNA synthetases represent promising broad-spectrum antibiotic targets

  • Insights from B. quintana gltX2 could inform drug development strategies applicable to multiple vector-borne pathogens

  • Understanding vector-specific adaptations could lead to transmission-blocking interventions

This research field thus serves as a model for studying fundamental aspects of vector-pathogen-host interactions applicable across multiple disease systems.