Recombinant Bartonella quintana 30S ribosomal protein S20 (rpsT)

What is the function of the 30S ribosomal protein S20 (rpsT) in Bartonella quintana?

The 30S ribosomal protein S20 (rpsT) in Bartonella quintana is a component of the small subunit of bacterial ribosomes. It plays a crucial role in ribosome assembly, stability, and translation processes. As in other bacteria, B. quintana rpsT likely binds to 16S rRNA during early stages of ribosome assembly, helping to establish and maintain the structural integrity of the 30S ribosomal subunit. This protein is essential for bacterial viability as it ensures proper ribosome formation and subsequent protein synthesis. The rpsT protein typically interacts with the 5' domain of 16S rRNA, contributing to the correct folding of ribosomal RNA and facilitating the association of other ribosomal proteins during subunit assembly.

During B. quintana's transition between human hosts and body louse vectors, ribosomal proteins like rpsT may play important roles in adapting translation machinery to different environmental conditions, including varying temperatures and hemin concentrations. This adaptation is critical for the pathogen's survival in different host environments.

How does Bartonella quintana 30S ribosomal protein S20 differ from homologous proteins in other bacterial species?

While specific sequence information for B. quintana rpsT is limited in the provided search results, comparative analysis suggests probable differences from other bacterial species based on general patterns observed in ribosomal proteins:

Table 1: Predicted Sequence Identity of rpsT Protein Across Bartonella Species

B. quintana rpsT likely shares high sequence identity with the homologous protein in B. henselae, reflecting their close phylogenetic relationship and similar host adaptation strategies. Both species cause bacillary angiomatosis and can survive in human bloodstream and arthropod vectors . In contrast, B. bacilliformis, which causes Carrion's disease, likely shows greater sequence divergence, reflecting its distinct pathogenicity profile and evolutionary history .

The differences in rpsT sequences across Bartonella species may reflect adaptations to different hosts and transmission cycles. B. quintana's adaptation to human blood and body lice may be reflected in specific amino acid substitutions that optimize ribosome function in these environments, particularly in regions involved in RNA binding or protein-protein interactions within the ribosome.

What expression systems are commonly used for producing recombinant Bartonella proteins?

For recombinant production of Bartonella proteins, including ribosomal proteins like rpsT, Escherichia coli expression systems are most commonly used due to their efficiency, cost-effectiveness, and well-established protocols. Based on approaches used for other Bartonella proteins, the following system components are typically employed:

• Expression vectors: pET series vectors (particularly pET-28a) are frequently used for Bartonella proteins, as was demonstrated with B. quintana RpoE protein . These vectors provide strong, inducible expression under control of the T7 promoter and can incorporate N-terminal or C-terminal affinity tags.

• Affinity tags: 6×His tags are commonly employed to facilitate purification, as seen in the expression of B. quintana RpoE . For proteins with solubility challenges, larger fusion partners like maltose-binding protein (MBP) may be used, as demonstrated with B. quintana NepR .

• Host strains: E. coli BL21(DE3) and its derivatives are standard choices, offering tight control of expression and reduced protease activity.

• Induction conditions: IPTG induction at concentrations of 0.1-1.0 mM, with post-induction growth at reduced temperatures (16-25°C) to enhance soluble protein production.

The specific choice of expression system components should be optimized based on the characteristics of the target protein and the intended downstream applications.

What strategies can overcome challenges in expressing and purifying functional recombinant Bartonella quintana rpsT protein?

Expression and purification of bacterial ribosomal proteins often present challenges due to their small size, basic nature, and nucleic acid binding properties. For B. quintana rpsT, researchers should consider these strategies:

• Codon optimization: Adapt the B. quintana rpsT gene sequence to match E. coli codon usage preferences, especially for rare codons that might otherwise limit expression.

• Fusion partners: Employ solubility-enhancing fusion tags such as MBP, GST, or SUMO. The MBP tag has been successfully used with other B. quintana proteins like NepR , suggesting its potential utility for rpsT.

• Expression conditions optimization: Test various induction temperatures, IPTG concentrations, and induction times. Lower temperatures (16-20°C) often enhance soluble expression by slowing protein synthesis and folding rates.

• Buffer optimization: Include stabilizing agents like glycerol (5-10%) and salt concentrations that minimize nucleic acid binding (typically 300-500 mM NaCl) during purification to prevent non-specific interactions with bacterial nucleic acids.

• Two-step affinity purification: Consider a tandem affinity tag approach (e.g., His-tag plus another affinity tag) to achieve higher purity, especially important for functional studies.

• On-column refolding: If inclusion bodies form despite optimization efforts, on-column refolding during affinity purification can be more effective than batch refolding methods for small proteins like rpsT.

These strategies should be systematically tested and optimized for the specific characteristics of B. quintana rpsT to achieve functional recombinant protein suitable for downstream applications.

How can researchers investigate the role of B. quintana rpsT in adaptation to different host environments?

B. quintana transitions between the hemin-restricted human bloodstream and the hemin-rich body louse vector, requiring significant adaptive responses. To investigate rpsT's potential role in this adaptation:

• Comparative expression analysis: Quantify rpsT expression levels in B. quintana cultured under conditions mimicking human blood (37°C, low hemin) versus body louse environment (lower temperature, high hemin). This approach mirrors studies of the RpoE sigma factor, which showed upregulation under body louse-like conditions (low temperature, high-hemin) .

• Structural characterization: Examine whether recombinant rpsT undergoes conformational changes under different temperature and hemin concentrations using techniques like circular dichroism spectroscopy and thermal stability assays.

• Ribosome assembly assays: Compare the efficiency of ribosome assembly with rpsT at different temperatures (37°C versus 28-30°C) that reflect human versus louse environments, using sucrose gradient ultracentrifugation to analyze ribosome profiles.

• Translation efficiency testing: Develop in vitro translation systems supplemented with recombinant rpsT that mimic both host environments to measure differences in translation rates and fidelity.

• Protein-protein interaction studies: Investigate whether rpsT interacts differently with other ribosomal components or regulatory factors under varying conditions, potentially using pull-down assays coupled with mass spectrometry.

• Genetic approaches: Create conditional rpsT mutants in B. quintana or closely related species to test growth and adaptation to temperature and hemin variations, though genetic manipulation of Bartonella remains challenging.

These approaches would help elucidate whether rpsT contributes to B. quintana's remarkable adaptability between its disparate host environments.

What techniques are most effective for studying the interaction between B. quintana rpsT and the bacterial ribosome?

Understanding rpsT's interactions within the ribosome requires complementary structural and functional approaches:

Table 2: Techniques for Studying rpsT-Ribosome Interactions

For B. quintana rpsT specifically, researchers should begin with binding affinity measurements using fluorescence anisotropy or SPR to establish basic interaction parameters, followed by structural studies to map the precise interaction sites. Functional approaches like in vitro translation assays would then connect structural insights to biological function, creating a comprehensive understanding of rpsT's role in B. quintana ribosomes.

How can researchers establish a reliable protocol for PCR amplification and cloning of the Bartonella quintana rpsT gene?

A reliable protocol for PCR amplification and cloning of B. quintana rpsT should include:

• Template preparation: Extract genomic DNA from B. quintana culture using a commercial kit designed for Gram-negative bacteria. Culturing B. quintana requires specialized media; based on protocols for other Bartonella species, researchers should use blood-enriched media like Columbia blood agar or liquid media supplemented with hemin .

• Primer design: Design primers that anneal to the 5' and 3' ends of the rpsT coding sequence. Include appropriate restriction sites (e.g., NdeI at 5' end, EcoRI at 3' end) for subsequent cloning, with 6-base overhangs before restriction sites. For example:

  • Forward primer: 5'-GCGCGCCATATG(start codon + gene-specific sequence)-3'

  • Reverse primer: 5'-GCGCGCGAATTC(stop codon + gene-specific sequence)-3'

• PCR conditions: Use high-fidelity DNA polymerase (e.g., Phusion or Q5) with the following conditions:

  • Initial denaturation: 98°C for 30 seconds

  • 30-35 cycles of: 98°C for 10s; 58-65°C for 30s; 72°C for 30s

  • Final extension: 72°C for 10 minutes

  This approach mirrors successful PCR amplification of other B. quintana genes like rpoE, nepR, and phyR .

• Purification and cloning: Purify the PCR product, digest with appropriate restriction enzymes, and ligate into a pre-digested expression vector like pET-28a for E. coli expression. Similar cloning strategies were successful for other B. quintana proteins .

• Verification: Confirm the sequence by Sanger sequencing to ensure no mutations were introduced during PCR amplification.

This methodology builds on strategies that have proven effective for other B. quintana genes while addressing the specific characteristics of the rpsT gene.

What are the optimal conditions for expressing recombinant B. quintana rpsT in E. coli?

Optimal expression conditions for recombinant B. quintana rpsT should be systematically determined through small-scale optimization experiments:

Table 3: Optimization Parameters for Recombinant B. quintana rpsT Expression

Based on successful expression of other B. quintana proteins like RpoE, using E. coli BL21(DE3) transformed with a pET-28a vector containing the target gene , this approach should be adaptable for rpsT. The key differences for rpsT would likely include more stringent temperature control and potentially the use of rare codon-supplemented strains like Rosetta, as ribosomal proteins often contain rare codons.

Initial expression trials should test multiple conditions in parallel using small cultures (10-50 mL), with expression levels and solubility assessed by SDS-PAGE analysis of total cellular protein, soluble, and insoluble fractions.

What purification approaches yield the highest purity and functionality for recombinant B. quintana rpsT?

A multi-step purification strategy is recommended to achieve high purity and maintain functionality of recombinant B. quintana rpsT:

• Initial capture using IMAC: For His-tagged rpsT, use Ni-NTA affinity chromatography with the following considerations:

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300-500 mM NaCl, 5-10% glycerol, 20 mM imidazole, protease inhibitors

  • Higher salt concentration (300-500 mM) to minimize nucleic acid binding

  • Wash with increasing imidazole concentrations (50-80 mM) to remove non-specifically bound proteins

  • Elute with 250-300 mM imidazole gradient

  This approach has been successfully applied to other B. quintana recombinant proteins .

• Nucleic acid removal: Treat the IMAC-purified sample with nucleases (e.g., Benzonase) or high salt washes to remove bound nucleic acids, a common issue with ribosomal proteins.

• Ion exchange chromatography: As rpsT is likely basic (calculated pI ~10), cation exchange chromatography using SP-Sepharose would be appropriate:

  • Buffer: 50 mM HEPES pH 7.5, with NaCl gradient from 100-1000 mM

• Size exclusion chromatography: Final polishing using Superdex 75 column:

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol

• Quality control: Assess purity by SDS-PAGE (aim for >95%), identity by mass spectrometry, and functionality by RNA binding assays.

• Storage conditions: Store purified protein in small aliquots at -80°C with 10% glycerol as cryoprotectant to maintain functionality through multiple freeze-thaw cycles.

This purification strategy addresses the specific challenges associated with ribosomal proteins, including nucleic acid contamination and potential aggregation issues, while building on successful approaches used for other B. quintana proteins.

How might B. quintana rpsT research contribute to understanding bacterial pathogenesis and host adaptation?

Research on B. quintana rpsT has several important implications for understanding bacterial pathogenesis and host adaptation:

• Translational adaptation mechanisms: Ribosomal proteins like rpsT may play key roles in optimizing translation efficiency under different host conditions. B. quintana must transition between the hemin-restricted human bloodstream and the hemin-rich body louse vector . Understanding how ribosomal components contribute to this adaptation could reveal fundamental mechanisms of pathogen persistence in multiple host environments.

• Stress response integration: In B. quintana, the extracytoplasmic function sigma factor RpoE is significantly upregulated under conditions mimicking the body louse environment (low temperature, high-hemin) . Ribosomal proteins may interact with this regulatory network, providing integrated stress responses that coordinate transcription and translation under changing environmental conditions.

• Evolution of host specificity: Comparative analysis of rpsT across Bartonella species with different host preferences could reveal signatures of selection related to host adaptation. B. quintana is primarily adapted to humans and body lice, while B. henselae infects cats and humans, and B. bacilliformis has a more restricted host range . These differences may be reflected in ribosomal protein sequences and functions.

• Virulence regulation: Translational control is increasingly recognized as an important mechanism for regulating virulence factor expression in pathogenic bacteria. B. quintana causes various clinical manifestations including endocarditis, trench fever, and bacillary angiomatosis , and rpsT may contribute to regulating the expression of factors involved in these different disease states.

• Novel therapeutic targets: If B. quintana rpsT has unique structural or functional features compared to human ribosomal proteins, it could potentially serve as a target for new antibacterial agents with specificity for this pathogen.

These research directions would complement existing knowledge about B. quintana pathogenesis and potentially reveal new aspects of ribosome-mediated adaptation in vector-borne pathogens.

How does B. quintana rpsT compare to homologous proteins in other pathogens, and what evolutionary insights can be derived?

Comparative analysis of B. quintana rpsT with homologous proteins in other pathogens provides evolutionary insights:

Table 4: Comparative Analysis of rpsT Across Bacterial Pathogens

B. quintana and B. henselae share a high degree of similarity in their rpsT proteins, reflecting their close phylogenetic relationship and similar pathogenicity mechanisms . Both species cause bacillary angiomatosis and can infect human hosts, though they have different primary reservoir hosts and vectors. B. bacilliformis, as the causative agent of Carrion's disease with a more restricted geographical distribution , likely shows greater divergence in its rpsT sequence, potentially reflecting adaptation to its unique transmission cycle.

The conservation pattern of rpsT across Bartonella species versus more distant bacteria like E. coli likely follows the typical pattern for ribosomal proteins: high conservation in RNA-binding domains and functional interfaces, with greater divergence in surface-exposed regions that do not directly participate in core ribosomal functions. These variable regions may contain signatures of adaptation to different host environments and could provide insights into the evolution of host specificity in the Bartonella genus.

What are the key challenges and future directions for research on B. quintana rpsT?

Several key challenges and promising future directions exist for research on B. quintana rpsT:

Challenges:

• Limited genomic data: Despite increasing interest in Bartonella species, detailed genomic information specifically about B. quintana rpsT remains limited compared to model organisms.

• Cultivation difficulties: B. quintana is fastidious and slow-growing, requiring specialized media and prolonged incubation periods , which complicates obtaining sufficient biomass for native protein studies.

• Genetic manipulation barriers: Tools for genetic manipulation of B. quintana are less developed than for model organisms, making functional genomic studies challenging.

• Structural complexity: Studying ribosomal proteins requires consideration of their interactions within the larger ribosomal complex, making isolated protein studies potentially less physiologically relevant.

Future Directions:

• Structure-function analysis: Determining the three-dimensional structure of B. quintana rpsT and its position within the assembled ribosome would provide insights into its specific roles in translation.

• Comparative expression studies: Analyzing rpsT expression patterns under conditions mimicking different host environments, similar to studies done with RpoE , could reveal its role in environmental adaptation.

• Interactome mapping: Comprehensive identification of rpsT-interacting partners beyond the ribosome could uncover unexpected roles in regulatory networks or stress responses.

• Development of Bartonella-specific ribosome profiling: This would allow genome-wide analysis of translational regulation in response to environmental changes relevant to the pathogen's life cycle.

• Cross-species complementation studies: Testing whether rpsT proteins from different Bartonella species can functionally substitute for each other could reveal species-specific adaptations.

• Integration with host response studies: Investigating how B. quintana ribosomes and rpsT specifically might interact with or be affected by host defense mechanisms could provide new insights into pathogen-host interactions.