Recombinant Bartonella henselae 50S ribosomal protein L1 (rplA)

What is Bartonella henselae and why is it significant for research?

Bartonella henselae is a Gram-negative, rod-shaped bacterium that primarily infects red blood cells and endothelial cells. It is the causative agent of cat-scratch disease (bartonellosis) and is transmitted to humans through scratches, bites, or flea vectors associated with cats. B. henselae is a facultative intracellular microbe that belongs to the genus Bartonella, one of the most common bacterial types globally. In the United States alone, approximately 20,000 cases of B. henselae infection are diagnosed annually, with most patients under 15 years of age. The bacterium's ability to invade human blood cells makes it a significant subject for research, particularly regarding host-pathogen interactions and intracellular survival mechanisms .

What is the 50S ribosomal protein L1 (rplA) and what is its function in B. henselae?

The 50S ribosomal protein L1, encoded by the rplA gene, is an essential component of the bacterial ribosome's large subunit. In B. henselae, as in other bacteria, this protein plays crucial roles in ribosome assembly, stability, and protein synthesis. Specifically, L1 helps maintain the structural integrity of the 50S subunit and participates in interactions with ribosomal RNA. While not specifically mentioned in available data on B. henselae, ribosomal proteins like L1 are generally highly conserved across bacterial species due to their fundamental role in protein biosynthesis. Understanding these proteins can provide insights into bacterial evolution, antibiotic targets, and pathogenicity mechanisms.

How does B. henselae growth differ from other bacterial species, and what implications does this have for recombinant protein research?

B. henselae is notably fastidious in its growth requirements compared to many other bacterial species. Research shows that blood supplementation significantly enhances B. henselae growth, which has direct implications for recombinant protein research. When cultured in media such as BAPGM (Bartonella Alpha Proteobacteria Growth Medium) supplemented with sheep blood, B. henselae exhibits enhanced growth over time. Specifically, after about 12 days at 36°C with 5% CO₂ and 100% humidity, B. henselae growth is strongly enhanced by sheep blood supplementation .

For recombinant protein research, these fastidious growth conditions create challenges for achieving sufficient bacterial biomass for protein extraction. Researchers must optimize culture conditions to maximize growth while maintaining protein expression. The data indicates that BAPGM with sheep blood supplementation generates significantly higher DNA amplification concentrations compared to other media, suggesting this may be the optimal approach for recombinant protein production .

What expression systems are most effective for recombinant B. henselae rplA production?

While the search results don't specifically address rplA expression systems, we can extrapolate from related research on B. henselae recombinant proteins. For the recombinant Pap31 protein of B. henselae, researchers successfully used the pET200D/TOPO expression system in Escherichia coli BL21 (DE3) clones. This system allowed for correct insertion of the gene in the proper reading frame and orientation, resulting in successful protein expression. Sequence verification confirmed 100% homology with the target B. henselae protein .

For recombinant rplA production, a similar approach using E. coli expression systems would likely be effective, given their established use with other B. henselae proteins. The pET expression systems are particularly valuable as they provide tight control of protein expression, high yields, and compatibility with various purification methods. When designing such systems for rplA, researchers should ensure codon optimization for E. coli and include appropriate fusion tags to facilitate downstream purification.

What purification strategies yield the highest purity and functional activity for recombinant B. henselae rplA?

Based on successful approaches with other B. henselae recombinant proteins, a multi-step purification strategy is recommended for rplA. For the Pap31 protein, researchers achieved high purity using nickel affinity chromatography for His-tagged recombinant proteins. SDS-PAGE and Western blot analysis confirmed the purity of the final product .

For rplA purification, a recommended protocol would include:

• Immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins if a His-tag is incorporated

• Ion exchange chromatography as a secondary purification step to remove remaining contaminants

• Size exclusion chromatography for final polishing and buffer exchange

• Quality control by SDS-PAGE, Western blotting, and mass spectrometry

Functional activity assessment would require ribosomal assembly assays or RNA binding studies specific to the L1 protein's known functions.

How can researchers verify the structural integrity and proper folding of recombinant B. henselae rplA?

Verification of structural integrity and proper folding of recombinant rplA requires multiple complementary approaches:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure content and compare with predicted models based on homologous proteins.

• Thermal Shift Assays: To evaluate protein stability and proper folding by monitoring the protein's melting temperature.

• Limited Proteolysis: To probe the accessibility of cleavage sites, which differs between properly folded and misfolded proteins.

• Functional Assays: Testing for RNA binding capability and interactions with other ribosomal components.

• Mass Spectrometry: Native MS can provide information about the intact protein and any post-translational modifications.

For ribosomal proteins like L1, proper folding is often dependent on interactions with ribosomal RNA, so RNA binding assays are particularly informative for functional verification.

How can recombinant B. henselae rplA be used to study ribosomal assembly and function in intracellular pathogens?

Recombinant B. henselae rplA provides a valuable tool for investigating ribosomal assembly and function in this important intracellular pathogen. Since B. henselae invades and replicates within erythrocytes and endothelial cells, understanding its ribosomal function is critical for comprehending its pathogenicity .

Research applications include:

• In vitro reconstitution studies: Using purified recombinant rplA along with other ribosomal components to study assembly kinetics and structural requirements.

• Protein-RNA interaction analysis: Identifying specific interactions between rplA and ribosomal RNA using techniques like RNA electrophoretic mobility shift assays (EMSA) or RNA footprinting.

• Cryo-EM structural studies: Incorporating recombinant rplA into ribosomal subunits for structural determination, potentially revealing unique features of B. henselae ribosomes.

• Comparative studies: Examining differences between B. henselae rplA and homologous proteins from other bacteria to identify species-specific features that might relate to its intracellular lifestyle.

• Translation fidelity assays: Using reconstituted ribosomes containing recombinant rplA to assess how B. henselae ribosomal proteins influence translation accuracy and efficiency.

What are the challenges and solutions for studying potential post-translational modifications of B. henselae rplA?

Studying post-translational modifications (PTMs) of bacterial ribosomal proteins presents several challenges, particularly for fastidious organisms like B. henselae:

Challenges:

• Low abundance of naturally modified protein

• Potential loss of labile modifications during purification

• Distinguishing bacterial PTMs from those introduced by expression hosts

• Limited knowledge of B. henselae-specific modification enzymes

Solutions:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics: Enzymatic digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact protein by high-resolution MS

  • Targeted methods for known modification types (e.g., phosphorylation, methylation)

• Modification-specific enrichment:

  • Immobilized metal affinity chromatography for phosphopeptides

  • Antibody-based enrichment for specific modifications

• Native protein isolation:

  • Direct isolation from B. henselae cultures using optimized growth conditions in BAPGM with sheep blood supplementation

  • Gentle extraction procedures to preserve labile modifications

• Comparative analysis:

  • Parallel analysis of recombinant and native proteins to identify host-introduced modifications

  • Comparison with homologous proteins from related Bartonella species

How does genetic diversity in B. henselae strains affect rplA structure and function?

Research indicates that B. henselae exhibits limited genetic diversity among human isolates compared to feline isolates. Studies have identified two distinct alleles of both gltA and 16S ribosomal DNA in all isolates, with high congruity between 16S and gltA inheritance among human pathogens .

While specific data on rplA diversity is not presented in the search results, the patterns observed in other genes suggest:

• Potential conservation: As a critical ribosomal protein, rplA likely shows high conservation across strains, particularly among human isolates which appear to represent a limited subset of B. henselae strains.

• Possible strain-specific variations: Minor differences in rplA sequence might exist between the predominant types (e.g., 16S type I vs. type II strains).

• Impact on function: Given the essential nature of ribosomal proteins, any variations in rplA sequence would likely maintain functional integrity, with potentially subtle effects on efficiency or regulation.

For comprehensive analysis of rplA diversity, researchers should consider:

Can recombinant B. henselae rplA be utilized as a diagnostic target for bartonellosis detection?

While ribosomal proteins represent potential diagnostic targets due to their conserved nature and essential function, their utility for B. henselae specifically requires careful evaluation. The search results don't directly address rplA as a diagnostic target, but provide insights from studies of another B. henselae protein, Pap31, which has been evaluated for diagnostics.

For rplA to be evaluated as a diagnostic target, researchers would need to:

• Develop highly purified recombinant rplA preparations

• Test against well-characterized serum panels from confirmed cases and controls

• Determine optimal cutoff values for ELISA or other serological assays

• Compare performance with existing diagnostic methods

• Evaluate cross-reactivity with other bacterial species

The modest performance of Pap31 suggests careful validation would be required before considering rplA for diagnostic applications.

What methodologies are most effective for detecting immune responses to B. henselae rplA in clinical samples?

Based on approaches used with other B. henselae proteins, several methodologies could be applied to detect immune responses to rplA:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Direct coating of recombinant rplA on plates

  • Indirect detection using labeled secondary antibodies

  • Determination of appropriate cutoff values using ROC curve analysis

• Western Blot Analysis:

  • SDS-PAGE separation followed by transfer and immunodetection

  • Useful for confirming ELISA results and identifying cross-reactive proteins

• Peptide Microarrays:

  • Screening of multiple linear epitopes from rplA simultaneously

  • Identification of immunodominant regions for improved diagnostics

• Flow Cytometry:

  • Detection of anti-rplA antibodies using fluorescently labeled recombinant protein

  • Useful for multiplex detection of antibodies against several B. henselae proteins

For RNA-based detection, similar approaches to those used for other Bartonella targets could be employed, such as a "pan-Bartonella PCR detection" method, which is non-invasive and uses blood or biopsies to diagnose B. henselae infection .

How might structure-function studies of rplA contribute to novel antimicrobial development against B. henselae?

Ribosomal proteins, including L1, are potential targets for antimicrobial development due to their essential role in protein synthesis. Structure-function studies of B. henselae rplA could contribute to drug development in several ways:

• Identification of unique structural features:

  • Comparative analysis with human ribosomal proteins to identify bacterial-specific regions

  • Mapping of functional domains involved in RNA binding and ribosomal assembly

• Binding site characterization:

  • Identification of druggable pockets using computational approaches

  • Experimental validation using biophysical techniques like NMR or X-ray crystallography

• Fragment-based drug discovery:

  • Screening of fragment libraries against purified rplA

  • Development of high-affinity compounds that interfere with ribosomal function

• Peptide inhibitor design:

  • Design of peptides that mimic natural binding partners of rplA

  • Optimization for stability, cellular uptake, and target specificity

What are the optimal conditions for expressing isotopically labeled B. henselae rplA for NMR structural studies?

For NMR structural studies requiring isotopically labeled recombinant rplA, specialized expression and purification approaches are necessary:

Expression System:

• E. coli BL21(DE3) or its derivatives (similar to systems used for other B. henselae proteins )

• Minimal media composition containing:

  • M9 salts supplemented with vitamins and trace elements

  • ¹⁵N-ammonium chloride as the sole nitrogen source (for ¹⁵N labeling)

  • ¹³C-glucose as the sole carbon source (for ¹³C labeling)

  • Deuterated water (for deuteration if required)

Expression Protocol:

• Grow initial culture in rich media to high density

• Wash cells and transfer to minimal media with isotopes

• Allow adaptation period (2-3 hours)

• Induce with IPTG at lower temperatures (16-20°C)

• Extended expression times (16-24 hours) to compensate for slower growth in minimal media

Purification Considerations:

• Maintain reducing conditions to prevent disulfide formation

• Use deuterated buffers for final NMR sample preparation if deuteration was employed

• Carefully monitor and adjust buffer conditions (pH, salt concentration) for optimal NMR spectra

Quality Control:

• SDS-PAGE to confirm purity

• Mass spectrometry to verify isotope incorporation levels

• 1D ¹H-¹⁵N HSQC to assess protein folding before proceeding to complex 3D experiments

How can researchers optimize co-expression systems for B. henselae rplA and its ribosomal binding partners?

Co-expression of rplA with its binding partners (other ribosomal proteins or ribosomal RNA) requires careful design and optimization:

Vector Selection:

• Compatible dual-plasmid systems with different origins of replication

• Polycistronic expression vectors for multiple protein components

• Specialized vectors for in vivo synthesis of ribosomal RNA if required

Expression Strategies:

• Sequential induction: Different inducible promoters (T7/lac, arabinose, tetracycline) controlling different components

• Stoichiometric control: Varying promoter strengths to achieve appropriate ratios of components

• Fusion protein approaches: Co-expression with chaperones or solubility enhancers

Validation Methods:

• Co-purification assays to confirm complex formation

• Size exclusion chromatography to analyze complex integrity

• Pull-down assays to verify specific interactions

• Functional reconstitution assays to test activity of the assembled complex

Optimization Table for Co-expression Systems:

What analytical techniques best characterize the interactions between recombinant B. henselae rplA and its RNA targets?

Multiple complementary techniques are recommended to comprehensively characterize rplA-RNA interactions:

Biophysical Methods:

• Surface Plasmon Resonance (SPR):

  • Quantitative binding kinetics (kon, koff)

  • Determination of equilibrium dissociation constants (KD)

  • Real-time monitoring of binding events

• Isothermal Titration Calorimetry (ITC):

  • Thermodynamic parameters (ΔH, ΔS, ΔG)

  • Stoichiometry determination

  • No labeling or immobilization required

• Microscale Thermophoresis (MST):

  • Low sample consumption

  • Solution-based measurements

  • Wide affinity range detection

Structural Methods:

• Nuclear Magnetic Resonance (NMR):

  • Mapping of interaction interfaces at atomic resolution

  • Dynamics of binding events

  • Structure determination of complexes

• X-ray Crystallography:

  • High-resolution structures of rplA-RNA complexes

  • Detailed view of atomic interactions

  • Identification of water-mediated contacts

• Cryo-Electron Microscopy:

  • Visualization of larger assemblies

  • Less demanding on sample crystallization

  • Structures in more native-like environments

Biochemical Methods:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Qualitative binding assessment

  • Competition studies with mutant RNAs

  • Cooperative binding analysis

• RNA Footprinting:

  • Identification of protected RNA regions

  • Mapping of binding sites

  • Structural changes upon binding

• Cross-linking coupled with Mass Spectrometry:

  • Identification of specific contact points

  • Zero-length or defined-length crosslinkers

  • MS/MS analysis of crosslinked peptide-RNA adducts