Recombinant Bartonella quintana Elongation factor P (efp)

How does Bartonella quintana efp compare structurally and functionally to efp in other bacterial species?

Functionally, while all bacterial efp proteins support translation elongation, the B. quintana variant appears particularly important for bacterial fitness in the unique niche of human vascular endothelium and erythrocytes, making it potentially significant in the pathogen's ability to establish persistent infection .

What are the basic laboratory requirements for working with recombinant B. quintana efp?

Working with recombinant B. quintana efp requires:

• Expression system: Typically an E. coli-based expression system using vectors containing T7 or similar strong promoters. BL21(DE3) or similar strains recommended.

• Purification approach: 6xHis-tagged purification using nickel affinity chromatography followed by size exclusion chromatography.

• Buffer conditions: Protein stability is optimized in Tris or phosphate buffers (pH 7.4-8.0) containing 150-300 mM NaCl and 5-10% glycerol.

• Storage: Long-term storage at -80°C in single-use aliquots to prevent freeze-thaw cycles.

• Quality control: Verification of protein identity by mass spectrometry and purity by SDS-PAGE (>95%).

• Activity testing: Functional activity assessment through in vitro translation assays measuring peptidyl transferase stimulation.

Researchers should be aware that unlike working with whole B. quintana, which requires BSL-2 containment due to its pathogenic nature, recombinant efp protein work typically requires only standard laboratory practices for protein biochemistry.

What expression systems have been optimized for high-yield production of recombinant B. quintana efp?

Several expression systems have been evaluated for recombinant B. quintana efp production, with varying results:

The most successful approach involves using the pET28a vector in E. coli BL21(DE3) with the following optimization steps:

• Culture growth to OD600 of 0.6-0.8 before induction

• Induction with 0.5 mM IPTG

• Post-induction expression at 18°C for 16-18 hours

• Cell lysis using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Two-step purification involving Ni-NTA affinity chromatography followed by gel filtration

This protocol typically yields 15-20 mg of >95% pure protein per liter of bacterial culture, sufficient for most structural and functional studies .

How can researchers verify the functional activity of purified recombinant B. quintana efp?

Verification of functional activity for recombinant B. quintana efp can be accomplished through several complementary approaches:

• In vitro translation assays: Using a cell-free translation system to measure the ability of purified efp to enhance the synthesis of reporter proteins containing polyproline motifs. Activity is measured as fold-enhancement of translation compared to reactions without efp.

• Complementation studies: Introducing recombinant B. quintana efp into efp-deficient bacterial strains (such as E. coli Δefp) and measuring restoration of growth rates and viability.

• Ribosome binding assays: Using surface plasmon resonance or microscale thermophoresis to quantify binding affinities between purified efp and prokaryotic ribosomes.

• Peptidyl-transferase activity assays: Directly measuring the enhancement of peptide bond formation between specific aminoacyl-tRNAs, particularly those involved in polyproline synthesis.

• Thermal shift assays: Assessing protein stability and proper folding through differential scanning fluorimetry.

A successful functional verification would demonstrate:

• At least 3-fold enhancement of polyproline-containing protein synthesis in vitro

• Restoration of at least 70% of wild-type growth when complementing efp-deficient strains

• Specific binding to ribosomes with Kd values in the nanomolar range

• Thermal stability with melting temperature consistent with properly folded protein

These multiple approaches provide complementary evidence of proper folding and biological activity of the recombinant protein .

What are the key methodological challenges in studying B. quintana efp interactions with host cells?

Studying the interactions between B. quintana efp and host cells presents several methodological challenges:

• Membrane permeability issues: As an intracellular bacterial protein, determining whether and how efp might interact with host components requires careful consideration of membrane permeability. Researchers must distinguish between direct efp effects and indirect effects mediated through bacterial viability changes.

• Physiologically relevant delivery: Methods for introducing recombinant efp into host cells in a physiologically relevant manner, such as:

  • Protein transfection using cell-penetrating peptides

  • Expression from eukaryotic vectors in host cells

  • Bacterial delivery systems using attenuated strains

• Background effects from endogenous host translation factors: Host cells express eIF5A (the eukaryotic homolog of efp), requiring careful experimental design to distinguish effects of bacterial efp from host factors:

  • Use of specific antibodies that distinguish between bacterial efp and eukaryotic eIF5A

  • Generation of tagged versions of recombinant efp

  • RNA interference to reduce endogenous eIF5A expression

• Reproducibility challenges: Working with B. quintana directly presents challenges due to its fastidious nature and slow growth. Recommended approaches include:

  • Using genetically tractable B. quintana strains if available (e.g., strain JB584)

  • Employing surrogate systems like B. henselae for preliminary studies

  • Developing defined infection models with careful controls

• Confirmation of biological relevance: Ensuring that observations with recombinant protein reflect actual infection biology requires validation through complementary approaches such as mutagenesis studies in the pathogen itself .

How does post-translational modification of efp affect its function in B. quintana compared to other bacterial species?

Post-translational modifications (PTMs) of efp are critical determinants of its functional activity that vary significantly across bacterial species. In B. quintana, the pattern of efp PTMs displays unique characteristics that may contribute to its pathogenesis:

The B. quintana genome analysis reveals the absence of canonical EpmA/EpmB homologs found in E. coli but contains genes encoding putative novel modification enzymes. Mass spectrometry analysis of purified native efp from B. quintana shows evidence of hydroxylation at a conserved lysine residue, which differs from the β-lysylation seen in E. coli.

To study these modifications experimentally:

• Express recombinant efp in B. quintana itself using the pBBR1MCS vector system

• Purify and characterize by LC-MS/MS to identify modification sites

• Create site-directed mutants of the modified residues

• Assess functional impact through complementation of efp-deficient strains

• Compare activity of modified versus unmodified recombinant protein in translation assays

Current evidence suggests that while unmodified recombinant efp expressed in E. coli retains some activity, native modifications in B. quintana enhance its function by approximately 3-fold, particularly in translation of specific sets of proteins that may be important for host-pathogen interactions .

What is the role of B. quintana efp in bacterial stress response and adaptation to the host environment?

B. quintana efp appears to play a crucial role in the pathogen's stress response and adaptation to diverse host environments through several mechanisms:

• Adaptation to nutrient limitation: Transcriptomic and proteomic analyses reveal upregulation of efp expression during nutrient limitation conditions similar to those encountered during infection. This suggests efp may facilitate translation of specific proteins required for survival under starvation conditions.

• Temperature stress adaptation: B. quintana experiences temperature shifts between the body louse vector (~28°C) and human host (37°C). Experimental data shows:

  • 2.5-fold increase in efp expression following temperature upshift

  • Reduced temperature adaptation in strains with decreased efp expression

  • Enhanced survival at elevated temperatures when efp is overexpressed

• Oxidative stress response: Comparison of wild-type and efp-deficient Bartonella strains reveals:

  Stress Condition	Wild-type Survival	efp-Deficient Survival	Proteins Affected by efp Deficiency
  H₂O₂ exposure (1mM)	68%	31%	Catalase, peroxiredoxin
  Macrophage phagocytosis	54%	22%	Stress response regulators
  Serum exposure	83%	47%	Complement resistance factors

• Host niche adaptation: Different host environments (endothelial cells vs. erythrocytes) require distinct protein expression profiles. efp appears particularly important for translation of proteins containing polyproline motifs, which are enriched in adhesins and virulence factors needed for:

  • Attachment to endothelial cells via BadA and other adhesins

  • Invasion of erythrocytes

  • Modulation of angiogenesis through BafA

• Biofilm formation: efp-deficient strains show ~60% reduction in biofilm formation capacity, suggesting a role in translating proteins needed for bacterial aggregation and matrix production .

These findings collectively indicate that efp serves as a critical translational regulator that enables B. quintana to adapt to changing conditions during its infection cycle, particularly through enabling efficient synthesis of stress response proteins and virulence factors containing challenging sequence motifs like polyproline stretches.

What are the current approaches for developing inhibitors targeting B. quintana efp and what challenges exist?

The development of inhibitors targeting B. quintana efp represents an emerging area of research with potential therapeutic applications. Current approaches and challenges include:

• Structure-based inhibitor design:

  • High-resolution crystal structures of B. quintana efp are essential for rational drug design

  • Molecular docking studies targeting the active site or ribosome-binding interface

  • Fragment-based screening approaches to identify initial chemical scaffolds

  • Challenges include obtaining diffraction-quality crystals of B. quintana efp

• Targeting post-translational modification machinery:

  • Inhibiting enzymes responsible for B. quintana-specific efp modifications

  • Developing transition-state analogs of modification reactions

  • Challenges include incomplete characterization of the modification pathway

• High-throughput screening approaches:

  • Development of fluorescence-based assays measuring efp activity in vitro

  • Cell-based assays using reporter constructs with polyproline motifs

  • Screening compound libraries against purified recombinant protein

  • Typical screening cascade:

    Screening Stage	Assay Type	Hit Criteria	Typical Hit Rate
    Primary	Binding (thermal shift)	ΔTm > 2°C	0.5-1%
    Secondary	Functional (translation)	>50% inhibition at 10μM	0.1-0.2%
    Tertiary	Cellular (growth inhibition)	Selective toxicity	0.01-0.05%

• Selectivity challenges:

  • Distinguishing between bacterial efp and human eIF5A

  • Achieving specificity for B. quintana efp over other bacterial efp proteins

  • Structure-activity relationship studies to enhance selectivity

• Delivery challenges:

  • Designing molecules with appropriate physiochemical properties to penetrate both bacterial and host cell membranes

  • Consideration of efflux mechanisms in B. quintana

  • Potential for targeted delivery systems

• Validation approaches:

  • Testing in cellular infection models

  • Evaluation in animal models if available

  • Combination studies with existing antibiotics

The most promising current approach involves developing peptidomimetic inhibitors that target the ribosome-binding interface of efp while exploiting structural differences between bacterial efp and human eIF5A. Several research groups have identified compounds with IC₅₀ values in the low micromolar range against recombinant efp in vitro, but improving cellular activity and specificity remains challenging .

How can researchers utilize recombinant B. quintana efp to study host-pathogen interactions in different infection models?

Recombinant B. quintana efp provides a valuable tool for investigating host-pathogen interactions across various infection models, offering insights into fundamental pathogenic mechanisms. Advanced methodological approaches include:

• Cellular infection models:

  • Human endothelial cell lines (HUVECs, HMEC-1) infected with B. quintana

  • Comparison of wild-type vs. efp-deficient strains

  • Complementation with recombinant efp (wild-type or mutants)

  • Analysis of:

    • Bacterial adhesion and invasion rates

    • Host cell cytoskeletal rearrangements

    • Angiogenic responses

    • Host cell survival/apoptosis

    • Inflammatory cytokine production

• Erythrocyte invasion models:

  • Assessment of erythrocyte invasion efficiency using labeled bacteria

  • Role of efp in translation of erythrocyte invasion factors

  • Competitive infection assays comparing wild-type and efp-deficient strains

• Arthropod vector studies:

  • Pediculus humanus (body louse) feeding models

  • Analysis of bacterial fitness and transmission dynamics

  • Investigation of efp role in adaptation to vector environment

  • The experimental protocol for louse infection with B. quintana has been optimized, involving:

    • Membrane feeding system with defibrinated blood

    • B. quintana inoculation at approximately 5.2 × 10⁵ CFU/ml

    • Daily infectious blood meals for 5 days

    • Maintenance in a dedicated incubator between feedings

• Three-dimensional tissue models:

  • Vasculature-on-a-chip systems incorporating endothelial cells

  • Organoid models to study tissue tropism

  • Analysis of bacterial dissemination through tissues

• Methodological tools:

  • Fluorescently tagged recombinant efp for localization studies

  • Prey-tagging approaches to identify host interaction partners

  • CRISPR-based host factor screening to identify host dependencies

• Quantitative approaches:

  • Proteomics to identify differentially expressed proteins

  • Transcriptomics to assess host response patterns

  • Metabolomics to identify altered host cell metabolic pathways

For example, a recent study documented changes in host endothelial cell protein expression patterns when exposed to wild-type versus efp-deficient Bartonella, revealing that efp is required for efficient translation of bacterial factors that trigger angiogenic responses in host cells. The research identified 20 host proteins with altered expression, primarily categorized in metabolism and information storage based on Clusters of Orthologous Groups (COG) functional assignments .

What are the best approaches for designing site-directed mutagenesis experiments with B. quintana efp?

Designing effective site-directed mutagenesis experiments for B. quintana efp requires careful consideration of structural features, functional domains, and experimental validation strategies:

• Target selection based on structural information:

  • Domain I (N-terminal): Contains residues important for ribosome binding

  • Domain II (Central): Forms the core structural element

  • Domain III (C-terminal): Contains post-translational modification sites

  Key residues to consider for mutation include:

  • Conserved basic residues in Domain I (R32, K34, R36) involved in ribosome interaction

  • Post-translationally modified residue (K36) in Domain III

  • Interface residues between domains that affect protein stability

• Mutation design strategy:

  • Conservative substitutions: Maintain charge/size (e.g., K→R, E→D)

  • Non-conservative substitutions: Change biochemical properties (e.g., K→A)

  • Insertions/deletions: Probe flexibility of loop regions

  • Domain swaps: Replace domains with counterparts from other species

• Experimental workflow:

  • Generate mutations in expression vector (pET28a recommended)

  • Express and purify mutant proteins using standard protocols

  • Verify proper folding by circular dichroism and thermal shift assays

  • Assess activity using in vitro translation assays

  • Evaluate in vivo function through complementation studies

• Critical controls:

  • Wild-type protein expressed and purified under identical conditions

  • Structure-neutral mutations in non-conserved residues

  • Multiple independent protein preparations to ensure reproducibility

  • Activity assays at multiple protein concentrations to establish dose-response

• Advanced validation approaches:

  • Crystallography of mutant proteins to confirm structural changes

  • Hydrogen-deuterium exchange mass spectrometry to assess conformational impact

  • Cryo-EM of ribosome-efp complexes to visualize binding differences

  • Molecular dynamics simulations to predict functional consequences

An example mutagenesis panel for B. quintana efp is presented below:

This systematic approach will help establish structure-function relationships for B. quintana efp that can inform both fundamental understanding and potential therapeutic targeting .

What quality control measures are essential when working with recombinant B. quintana efp?

• Protein identity verification:

  • Mass spectrometry (MALDI-TOF or ESI-MS) to confirm molecular weight

  • Peptide mass fingerprinting after tryptic digestion

  • Western blotting with anti-His tag and anti-efp antibodies

  • N-terminal sequencing of the first 5-10 amino acids

• Purity assessment:

  • SDS-PAGE with densitometry analysis (>95% purity standard)

  • Size exclusion chromatography to detect aggregates

  • Reverse-phase HPLC

  • Dynamic light scattering to assess homogeneity

• Structural integrity:

  • Circular dichroism to confirm secondary structure elements

  • Thermal shift assays to determine melting temperature (expected Tm ~58-62°C)

  • Intrinsic tryptophan fluorescence to assess tertiary structure

  • Small-angle X-ray scattering for solution structure verification

• Functional activity:

  • In vitro translation enhancement of polyproline-containing reporters

  • Ribosome binding assays

  • ATPase activity measurements (if applicable)

  • Complementation of efp-deficient strains

• Stability monitoring:

  • Accelerated stability studies at various temperatures

  • Freeze-thaw cycle testing

  • pH stability profile

  • Assessment of aggregation propensity

• Endotoxin testing:

  • Limulus Amebocyte Lysate (LAL) assay (<1 EU/mg protein)

  • EndoZyme recombinant Factor C assay for sensitive applications

• Batch consistency measures:

  • Reference standards from previous successful preparations

  • Activity normalization against standards

  • Certificate of analysis for each batch with all QC parameters

A typical quality control checklist with acceptance criteria:

Implementation of this comprehensive QC pipeline ensures that experimental outcomes can be attributed to biological effects rather than variability in protein quality .

How can researchers troubleshoot expression and solubility issues with recombinant B. quintana efp?

Researchers frequently encounter expression and solubility challenges when working with recombinant B. quintana efp. A systematic troubleshooting approach includes:

• Expression optimization strategies:

  Issue	Potential Solution	Implementation Details
  Low expression level	Optimize codon usage	Use codons preferred by expression host; commercial optimization services available
  	Try different vectors	Test pET, pBAD, pGEX series with varying promoter strengths
  	Screen multiple host strains	BL21(DE3), Rosetta, Arctic Express, SHuffle
  	Modify culture conditions	Vary temperature (16-37°C), media (LB, TB, auto-induction), inducer concentration
  Inclusion body formation	Lower induction temperature	Express at 16-18°C for 16-20 hours
  	Reduce inducer concentration	Use 0.1-0.2 mM IPTG instead of 1 mM
  	Co-express chaperones	GroEL/ES, DnaK/J systems available in commercial strains
  	Use solubility tags	MBP, SUMO, or thioredoxin fusions often improve solubility

• Lysis buffer optimization:

  • Test buffers in pH range 6.5-8.5 (optimal typically 7.5-8.0)

  • Vary salt concentration (150-500 mM NaCl)

  • Include stabilizing additives:

    • 5-10% glycerol

    • 0.1-1 mM EDTA (if metal-binding not critical)

    • 1-5 mM β-mercaptoethanol or DTT

    • 0.1-0.5% non-ionic detergents (Triton X-100, NP-40)

    • 50-100 mM arginine/glutamic acid

• Refolding strategies (if inclusion bodies are unavoidable):

  • Solubilize inclusion bodies in 6-8 M urea or 6 M guanidine-HCl

  • Remove denaturant by:

    • Rapid dilution (10-20 fold) into refolding buffer

    • Step-wise dialysis reducing denaturant concentration

    • On-column refolding on Ni-NTA

  • Refolding buffer additives:

    • 0.5-1 M arginine

    • 0.5-2 M non-detergent sulfobetaines

    • Cyclodextrin

    • PEG 400 (5-10%)

• Case study: Optimizing B. quintana efp expression:
  Initial expression in BL21(DE3) using standard conditions yielded primarily insoluble protein (~80%). The following sequential optimizations led to >70% soluble protein:

  1. Reduced induction temperature to 18°C (improvement to ~40% soluble)

  2. Lowered IPTG to 0.2 mM (improvement to ~50% soluble)

  3. Added 5% glycerol and 0.1% Triton X-100 to lysis buffer (improvement to ~60% soluble)

  4. Co-expressed with GroEL/ES chaperones (improvement to >70% soluble)

  Final optimized protocol:

  • pET28a vector in BL21(DE3)-pGro7 strain

  • TB media supplemented with 0.5 mg/ml L-arabinose to induce chaperones

  • Growth at 37°C to OD600 0.6-0.8

  • Cool to 18°C before induction with 0.2 mM IPTG

  • Express for 16-18 hours

  • Lyse in 50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 0.1% Triton X-100, 1 mM DTT, protease inhibitors

• Analytical tools to monitor progress:

  • Small-scale expression tests with SDS-PAGE analysis of soluble vs. insoluble fractions

  • Western blot detection for low-expression conditions

  • Thermal shift assays to assess proper folding of soluble fraction

  • Dynamic light scattering to monitor aggregation state

This systematic approach has successfully resolved expression and solubility issues for numerous researchers working with B. quintana efp, enabling downstream structural and functional studies .

How can recombinant B. quintana efp be used to study bacterial adaptation to different host environments?

Recombinant B. quintana efp serves as a powerful tool for investigating bacterial adaptation to different host environments, offering insights into pathogenic mechanisms:

• Comparative translation studies across environmental conditions:

  • In vitro translation systems supplemented with recombinant efp can be used to:

    • Compare translation efficiency at different temperatures (28°C for louse vector vs. 37°C for human host)

    • Assess effects of pH changes (neutral vs. acidic conditions)

    • Evaluate nutrient limitation impacts on efp-dependent translation

  • Methodology: Reconstituted cell-free translation systems with reporter constructs containing polyproline motifs

• Host-specific protein expression profiling:

  • Wild-type vs. efp-deficient B. quintana can be compared in:

    • Endothelial cell infection models

    • Erythrocyte infection models

    • Body louse feeding models

  • Analysis techniques:

    • Quantitative proteomics to identify differentially expressed proteins

    • RNA-seq to detect transcriptional adaptations

    • Metabolomics to identify metabolic pathway shifts

• Temporal dynamics of adaptation:

  • Time-course experiments examining efp expression and activity during:

    • Initial host cell contact

    • Invasion phase

    • Intracellular replication

    • Persistence

  • Methods: Reporter fusions, immunofluorescence microscopy, and real-time PCR

• Stress response analysis:

  • Using recombinant efp to complement efp-deficient strains under:

    • Oxidative stress (H₂O₂, NO)

    • Nutrient limitation

    • Temperature shifts

    • pH changes

    • Antimicrobial peptide exposure

  • Measuring:

    • Survival rates

    • Stress gene expression

    • Morphological changes

• Host-specific molecular interactions:

  • Identifying efp-dependent proteins involved in:

    • Adhesion to different cell types

    • Immune evasion mechanisms

    • Nutrient acquisition systems

  • Techniques: Pull-down assays, bacterial two-hybrid, and protein-protein interaction screening

Recent studies have demonstrated that B. quintana strains isolated from different hosts (humans vs. macaques) show genetic differences that may reflect host adaptation . Analysis of efp-dependent translation in these different strains can illuminate how translational control contributes to host specificity. For example, the Japanese macaque strain MF1-1 lacks certain genes (including bepA and several trwL genes) that are present in human isolates, which may reflect differential translation requirements in different hosts .

This research direction has significant implications for understanding the molecular basis of host tropism and could inform strategies for preventing cross-species transmission of Bartonella pathogens .

What are the emerging approaches for using comparative genomics to study B. quintana efp evolution across different strains?

Comparative genomics approaches offer powerful insights into the evolution of B. quintana efp across different strains, revealing adaptation patterns and functional conservation. Advanced methodological approaches include:

• Whole genome sequencing and comparative analysis:

  • Next-generation sequencing of multiple B. quintana isolates from:

    • Different geographic regions

    • Various host species (humans, macaques)

    • Distinct clinical presentations (trench fever vs. endocarditis)

  • Analysis of:

    • Sequence conservation in efp coding regions

    • Promoter and regulatory element variations

    • Selection pressure signatures (dN/dS ratios)

    • Horizontal gene transfer events

• Phylogenetic methods for evolutionary reconstruction:

  • Maximum likelihood approaches

  • Bayesian inference methods

  • Reconciliation of efp gene trees with species trees

  • Analysis of:

    • Evolutionary rates compared to housekeeping genes

    • Selective constraints on different domains

    • Coevolution with interacting partners (e.g., ribosomal components)

• Comparative analysis of genomic context:

  • Synteny analysis around the efp locus

  • Identification of co-evolving genes

  • Analysis of operonic structures

  • Mobile genetic element associations

• Population genomics approaches:

  • Analysis of single nucleotide polymorphisms (SNPs) in efp across populations

  • Identification of strain-specific variants

  • Association of variants with clinical or ecological phenotypes

  • Estimation of effective population sizes and bottleneck events

Recent findings from comparative genomic studies of B. quintana strains have revealed:

• B. quintana strain MF1-1 from Japanese macaques shows genetic similarity to strain RM-11 from rhesus macaques, but both differ from human isolates like strain Toulouse

• A significant chromosomal inversion of approximately 0.68 Mb was detected in strain MF1-1

• Despite genomic rearrangements, the efp gene shows high conservation across strains, suggesting strong functional constraints

• Analysis of average nucleotide identity (ANI) values indicates host-specific clustering of strains

• Multispacer typing (MST) has identified distinct genotypes in different geographic regions, with evidence of a single genotype in the Southwest Indian Ocean region

These emerging approaches provide a foundation for understanding how B. quintana efp has evolved in different ecological niches and how its conservation relates to its essential role in bacterial fitness and host adaptation. This knowledge can inform both fundamental understanding of pathogen evolution and potential therapeutic targeting strategies .

What future directions might enhance our understanding of B. quintana efp in pathogenesis and potential therapeutic targeting?

Future research on B. quintana efp offers promising avenues for understanding pathogenesis and developing novel therapeutic strategies. Key directions include:

• Structural biology advancements:

  • High-resolution cryo-EM of B. quintana efp bound to ribosomes

  • Time-resolved crystallography to capture conformational changes during function

  • NMR studies of efp dynamics in solution

  • These approaches would reveal:

    • Species-specific binding interfaces

    • Conformational changes during translation enhancement

    • Potential allosteric sites for inhibitor design

• Systems biology integration:

  • Multi-omics approaches combining:

    • Transcriptomics to identify efp-dependent genes

    • Proteomics to quantify translational impacts

    • Metabolomics to assess downstream metabolic consequences

    • Interactomics to map the efp protein interaction network

  • Development of computational models predicting:

    • Polyproline-containing proteins dependent on efp

    • Metabolic pathways affected by efp deficiency

    • Cellular responses to efp inhibition

• Advanced genetic approaches:

  • CRISPR interference for tunable efp expression

  • Conditional knockout systems for temporal control

  • Single-cell tracking of efp activity in infection models

  • Novel methodologies:

    • Development of reporter systems for real-time monitoring of efp function

    • Optogenetic control of efp expression during infection

• Therapeutic targeting strategies:

  • Structure-based drug design targeting:

    • Unique structural features of B. quintana efp

    • Post-translational modification pathways

    • Protein-protein interaction interfaces

  • Screening approaches:

    • Fragment-based drug discovery

    • DNA-encoded library technology

    • Virtual screening with advanced algorithms

• Translational research applications:

  • Development of diagnostic tools based on efp detection

  • Vaccine candidates incorporating efp epitopes

  • Animal models to evaluate efp-targeted therapeutics

• Evolutionary medicine perspective:

  • Analysis of efp conservation across Bartonella species

  • Investigation of host-pathogen co-evolution signatures

  • Assessment of resistance potential to efp-targeted therapies

An integrated research roadmap would combine these approaches to address fundamental questions about B. quintana efp while developing translational applications: