Recombinant Vibrio vulnificus 30S ribosomal protein S9 (rpsI)

How is rpsI expression regulated during Vibrio vulnificus infection?

During Vibrio vulnificus infection, rpsI expression undergoes complex regulation as part of the bacterium's adaptive response to host environments. When V. vulnificus transitions from environmental conditions to host tissues and bloodstream, significant shifts in ribosomal protein expression occur. Research indicates that upon entering human serum, the bacterium must rapidly adapt its translational machinery to overcome innate immune defenses and accommodate new metabolic constraints .

The regulation of rpsI appears to be coordinated with other essential genes involved in protein synthesis. During infection progression, especially as the pathogen invades the bloodstream causing sepsis, expression of ribosomal proteins including rpsI may be modulated to optimize bacterial survival and proliferation in different host niches. Transposon insertion sequencing (TIS) studies have shown that disruption of essential ribosomal components significantly impacts the pathogen's ability to survive in human serum, suggesting tight regulation of these components during infection .

What are the optimal expression systems for producing recombinant V. vulnificus rpsI?

Multiple expression systems can be utilized for recombinant V. vulnificus rpsI production, each with distinct advantages for different research applications. Based on available data, the following expression systems have been successfully employed:

For most structural and biochemical studies, the E. coli expression system (CSB-EP807775VFI) provides sufficient yield and quality . For applications requiring specific modifications or biotinylation, the E. coli system with Avi-tag biotinylation (CSB-EP807775VFI-B) offers an effective alternative. This system utilizes E. coli biotin ligase (BirA) to covalently attach biotin to the 15 amino acid AviTag peptide with high specificity .

The choice of expression system should align with specific research goals, considering factors such as required yield, protein purity, post-translational modifications, and downstream applications.

What purification strategies are most effective for recombinant V. vulnificus rpsI?

Purification of recombinant V. vulnificus rpsI requires strategic approaches to achieve high purity while maintaining protein functionality. Based on established protocols, a multi-step purification strategy is recommended:

• Initial Capture: Affinity chromatography using the appropriate tag (His-tag, AviTag, etc.) provides the most efficient initial purification step, achieving >85% purity as verified by SDS-PAGE .

• Secondary Purification: Ion exchange chromatography utilizing the protein's isoelectric point helps remove remaining contaminants.

• Polishing: Size exclusion chromatography (gel filtration) separates any aggregates or degradation products.

• Quality Assessment: Analysis by SDS-PAGE, Western blotting, and mass spectrometry to confirm purity, identity, and integrity.

For reconstitution of lyophilized protein, it is recommended to briefly centrifuge the vial prior to opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, addition of 5-50% glycerol (final concentration) followed by aliquoting and storage at -20°C/-80°C is advised . This approach prevents repeated freeze-thaw cycles that can compromise protein integrity.

How can transposon insertion sequencing (TIS) be applied to study rpsI function?

Transposon insertion sequencing (TIS) represents a powerful approach for investigating rpsI function in the context of V. vulnificus pathogenesis. This methodology enables genome-wide identification of genes essential for survival under specific conditions. For studying rpsI, the following TIS-based experimental strategy is recommended:

• Library Construction: Generate a high-density transposon mutant library in V. vulnificus (similar to the approach used with strain YJ016) .

• Selection Conditions: Expose the library to relevant conditions such as human serum, which imposes both immune defense and metabolic constraints on the pathogen .

• Comparative Analysis: Compare the relative abundance of insertion mutants before and after exposure to experimental conditions.

• Essential Gene Identification: Identify genes underrepresented for insertions, which indicates their essential nature under the tested conditions.

• Validation: Construct targeted mutants to validate findings from the TIS screen.

When studying rpsI through TIS, researchers should be aware that as an essential ribosomal component, this gene may be inherently underrepresented in transposon libraries. Therefore, conditional depletion approaches may be necessary to study its function. Comparative TIS screens, as demonstrated in previous V. vulnificus research, provide valuable insights into how ribosomal proteins like rpsI contribute to bacterial survival under specific physiological conditions .

What role does rpsI play in V. vulnificus antibiotic resistance mechanisms?

As a component of the 30S ribosomal subunit, rpsI plays a significant role in V. vulnificus antibiotic resistance, particularly against aminoglycosides and tetracyclines that target protein synthesis. Research indicates that subtle variations in ribosomal proteins can confer differential antibiotic susceptibility profiles across bacterial strains.

For researchers investigating rpsI's role in antibiotic resistance, the following methodological approaches are recommended:

• Structural Analysis: Examine the interaction between rpsI and antibiotics through crystallography or cryo-EM studies to identify binding sites.

• Site-Directed Mutagenesis: Introduce specific mutations in the rpsI gene to alter amino acids at potential antibiotic interaction sites.

• Minimum Inhibitory Concentration (MIC) Assays: Compare antibiotic susceptibility between wild-type and mutant strains to quantify resistance changes.

• Ribosome Profiling: Apply this technique to assess how rpsI mutations impact ribosome function during translation in the presence of sub-inhibitory antibiotic concentrations.

How does rpsI contribute to V. vulnificus virulence and survival in human serum?

The 30S ribosomal protein S9 (rpsI) contributes to V. vulnificus virulence and serum survival through both direct and indirect mechanisms. Research using transposon insertion sequencing (TIS) has revealed that ribosomal proteins play crucial roles in enabling bacterial adaptation to the metabolic constraints and immune defenses present in human serum .

For investigating rpsI's specific contributions to serum survival and virulence, the following experimental approaches are recommended:

• Comparative Proteomics: Analyze proteome changes in wild-type versus rpsI-depleted strains during serum exposure to identify downstream effectors.

• Serum Bactericidal Assays: Quantify survival rates of V. vulnificus strains with modified rpsI expression in normal and heat-inactivated human serum.

• Translational Efficiency Analysis: Measure the impact of serum exposure on translation rates of specific virulence factors in wild-type versus rpsI-modified strains.

• In vivo Virulence Studies: Assess pathogenicity of rpsI-modified strains in appropriate animal models to correlate in vitro findings with in vivo outcomes.

Research has demonstrated that V. vulnificus relies on its Na⁺ transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive and proliferate within the metabolic constraints of serum . The ribosomal machinery, including rpsI, likely plays a critical role in facilitating the rapid translation of proteins necessary for these adaptive responses during infection.

What bioinformatic approaches are most effective for analyzing rpsI conservation across Vibrio species?

Comprehensive bioinformatic analysis of rpsI conservation requires a multi-faceted approach to capture both sequence and structural conservation across Vibrio species. For researchers pursuing such analyses, the following methodological pipeline is recommended:

• Sequence Retrieval and Alignment:

  • Extract rpsI sequences from complete Vibrio genomes using BLAST or ortholog databases

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Calculate conservation scores and identify invariant residues

• Phylogenetic Analysis:

  • Construct maximum likelihood or Bayesian phylogenetic trees

  • Apply appropriate evolutionary models (e.g., JTT, WAG)

  • Evaluate node support through bootstrap or posterior probability analysis

• Structural Conservation Assessment:

  • Map conservation data onto 3D structures using homology modeling

  • Identify structurally conserved domains across species

  • Analyze conservation of RNA-binding interfaces

• Coevolution Analysis:

  • Implement methods such as Direct Coupling Analysis (DCA) or Mutual Information

  • Identify coevolving residues within rpsI and between rpsI and interacting partners

  • Correlate coevolution patterns with functional domains

This comprehensive approach enables researchers to distinguish between conserved regions essential for basic ribosomal function and variable regions that may contribute to species-specific adaptations. When analyzing conservation data, it's important to consider both absolute sequence conservation and physicochemical property conservation, as the latter may maintain functional equivalence despite sequence divergence.

How should experiments be designed to study the interaction between rpsI and host immune factors?

Studying interactions between V. vulnificus rpsI and host immune factors requires careful experimental design to capture both direct interactions and downstream effects. The following methodological framework is recommended:

• Protein-Protein Interaction Assays:

  • Co-immunoprecipitation using purified recombinant rpsI and host immune components

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Biolayer interferometry for real-time interaction analysis

  • Crosslinking mass spectrometry to map interaction interfaces

• Cellular Response Assays:

  • Treatment of immune cells with purified rpsI protein (with appropriate controls)

  • Measurement of cytokine production using ELISA or flow cytometry

  • Analysis of immune cell activation markers

  • Assessment of phagocytosis efficiency in the presence of rpsI

• In vivo Models:

  • Comparison of immune responses to wild-type versus rpsI-modified V. vulnificus strains

  • Evaluation of bacterial clearance rates in immunocompetent versus immunocompromised models

  • Histopathological analysis of infected tissues

When conducting these experiments, it's essential to verify that purified rpsI maintains its native conformation using circular dichroism or other structural validation methods. Additionally, appropriate controls should include other ribosomal proteins to distinguish rpsI-specific effects from general bacterial protein responses. The biotinylated version of the protein (CSB-EP807775VFI-B) may be particularly valuable for interaction studies, as the biotin tag provides a consistent orientation for binding assays without interfering with protein structure .

What factors should be considered when interpreting conflicting data on rpsI function?

When confronted with conflicting data regarding rpsI function in V. vulnificus, researchers should implement a systematic approach to resolve discrepancies and develop a coherent understanding. Consider the following methodological framework:

• Experimental Context Analysis:

  • Compare strain backgrounds (clinical vs. environmental isolates)

  • Assess differences in experimental conditions (temperature, media, growth phase)

  • Evaluate protein expression levels and modifications

  • Consider the impact of different tags or expression systems on protein function

• Technical Validation:

  • Repeat experiments with standardized protocols across different laboratories

  • Implement multiple complementary techniques to address the same question

  • Validate antibodies and reagents for specificity

  • Apply appropriate statistical methods for each data type

• Biological Complexity Considerations:

  • Assess potential redundancy in ribosomal function

  • Investigate compensatory mechanisms that may mask phenotypes

  • Consider context-dependent functions that vary across infection stages

  • Evaluate potential moonlighting functions beyond canonical ribosomal roles

• Integrated Data Analysis:

  • Apply meta-analysis techniques to quantitatively synthesize results across studies

  • Develop computational models that accommodate apparent contradictions

  • Utilize Bayesian approaches to weight evidence based on methodological strength

This systematic approach allows researchers to distinguish between genuine biological complexity and technical artifacts. When preparing publications, transparent reporting of all experimental conditions and limitations is essential for advancing collective understanding of rpsI function in V. vulnificus pathogenesis.

How can functional studies distinguish between rpsI's role in normal ribosome function versus potential pathogenesis-specific roles?

Differentiating between rpsI's canonical ribosomal functions and potential pathogenesis-specific roles requires sophisticated experimental approaches that can isolate these potentially overlapping functions. The following methodological strategy is recommended:

By systematically comparing these parameters, researchers can distinguish between general effects on bacterial physiology due to compromised translation and specific impacts on virulence mechanisms. This approach acknowledges that ribosomal proteins like rpsI may have evolved additional functions in pathogenic bacteria that extend beyond their conserved roles in protein synthesis.

What emerging technologies could advance our understanding of rpsI function in V. vulnificus?

Several cutting-edge technologies hold promise for deepening our understanding of rpsI function in V. vulnificus pathogenesis. Researchers should consider incorporating these approaches in future studies:

• Cryo-Electron Microscopy (Cryo-EM):

  • Obtain high-resolution structures of V. vulnificus ribosomes under various conditions

  • Visualize conformational changes in rpsI during different stages of translation

  • Compare ribosome structures from bacteria grown in standard media versus serum

• CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa):

  • Achieve precise, tunable control of rpsI expression levels

  • Implement time-resolved repression or activation during infection progression

  • Create libraries targeting different regions of the rpsI operon to study regulatory elements

• Time-Resolved Proteomics:

  • Apply pulse-SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to track proteome dynamics

  • Measure protein synthesis rates during host-pathogen interactions

  • Identify condition-specific translation programs dependent on rpsI

• Single-Cell Approaches:

  • Utilize single-cell RNA sequencing to capture heterogeneity in bacterial populations

  • Apply single-cell ribosome profiling to assess translational responses

  • Implement microfluidics-based approaches to track individual bacterial cells during infection

These technologies, when integrated with established methods such as transposon insertion sequencing , can provide unprecedented insights into the multifaceted roles of rpsI in V. vulnificus. By combining structural, functional, and dynamic information across multiple scales, researchers can develop comprehensive models of how this essential ribosomal protein contributes to both basic cellular functions and pathogenesis-specific processes.

How might rpsI be targeted for antimicrobial development against Vibrio vulnificus?

Exploiting rpsI as a target for novel antimicrobials against V. vulnificus requires strategic approaches that leverage its essential role while achieving sufficient selectivity. The following methodological framework is recommended for researchers pursuing this direction:

• Structure-Based Drug Design:

  • Utilize high-resolution structural data to identify unique binding pockets in V. vulnificus rpsI

  • Apply computational screening to identify compounds that specifically interact with these sites

  • Optimize lead compounds for improved binding affinity and selectivity

  • Validate binding through techniques such as isothermal titration calorimetry or thermal shift assays

• Peptide-Based Inhibitors:

  • Design synthetic peptides that mimic natural binding partners but introduce inhibitory modifications

  • Screen peptide libraries for sequences that disrupt rpsI interactions

  • Optimize for stability, cell penetration, and resistance to proteolytic degradation

  • Evaluate both antimicrobial activity and impact on virulence factor expression

• Antisense Approaches:

  • Develop modified oligonucleotides targeting rpsI mRNA

  • Optimize for bacterial cell penetration using peptide conjugates or nanoparticle delivery

  • Assess impact on bacterial survival in both standard media and infection-relevant conditions

  • Evaluate potential for resistance development through long-term exposure studies

• Adjuvant Development:

  • Identify compounds that sensitize V. vulnificus to existing antibiotics by partially inhibiting rpsI function

  • Determine synergistic combinations that allow dose reduction of current antibiotics

  • Assess impact on virulence factor expression and production

  • Evaluate potential for accelerating the clearance of infections in animal models

When pursuing these approaches, researchers should implement thorough cytotoxicity testing to ensure selectivity for bacterial over human cells, given the existence of human ribosomal proteins with some structural similarities. Additionally, pharmacokinetic and pharmacodynamic studies will be essential to determine whether candidate compounds can achieve sufficient concentrations at infection sites to be therapeutically viable.

What are the most promising unresolved questions regarding V. vulnificus rpsI?

Despite significant advances in understanding ribosomal protein function, several critical questions regarding V. vulnificus rpsI remain unresolved and represent promising areas for future research:

• Structural Adaptations: How has the structure of rpsI in V. vulnificus evolved specific adaptations that contribute to this pathogen's virulence, particularly its ability to survive in human serum? Comparative structural analyses across non-pathogenic and pathogenic Vibrio species could reveal pathogenesis-specific features.

• Regulatory Networks: What transcriptional and post-transcriptional regulatory mechanisms control rpsI expression during different stages of infection? Understanding these regulatory networks could reveal potential intervention points for disrupting pathogenesis.

• Host-Pathogen Interactions: Does rpsI interact directly with host factors, potentially moonlighting beyond its canonical ribosomal role? Systematic interactome studies under infection-mimicking conditions could reveal unexpected interactions.

• Translational Specialization: Does V. vulnificus modify its translational machinery, including rpsI, to preferentially translate virulence-associated transcripts during infection? Ribosome profiling under various conditions could provide insights into potential translational reprogramming.

• Inter-Strain Variability: How do sequence variations in rpsI across different V. vulnificus strains correlate with virulence potential and host adaptation? Comprehensive comparative genomics and functional validation studies could reveal strain-specific adaptations.

Addressing these questions requires integration of structural biology, molecular genetics, systems biology, and infection models. The resulting insights could significantly advance our understanding of not only V. vulnificus pathogenesis but also the broader role of ribosomal specialization in bacterial adaptation and virulence.

What standardized protocols should researchers adopt when working with recombinant V. vulnificus rpsI?

To enhance reproducibility and facilitate comparison across studies, researchers working with recombinant V. vulnificus rpsI should consider adopting the following standardized protocols:

• Expression and Purification:

  • Use defined expression systems with consistent tags (e.g., His-tag or Avi-tag systems)

  • Document codon optimization strategies for heterologous expression

  • Implement standardized purification protocols with defined buffer compositions

  • Verify protein folding using circular dichroism or thermal shift assays

  • Assess purity by SDS-PAGE and mass spectrometry (>85% purity standard)

• Storage and Handling:

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% for long-term storage

  • Store aliquoted protein at -20°C/-80°C to prevent freeze-thaw cycles

  • Centrifuge vials briefly before opening to bring contents to the bottom

• Functional Validation:

  • Assess RNA binding capacity using electrophoretic mobility shift assays

  • Verify incorporation into ribosomal subunits using sucrose gradient centrifugation

  • Confirm translation activity using cell-free translation systems

• Reporting Standards:

  • Document all modifications to the native sequence, including tags and linkers

  • Report protein concentration methods and values

  • Provide detailed buffer compositions for all experimental conditions

  • Specify lot-to-lot variation assessment methods