Recombinant Vibrio vulnificus 30S ribosomal protein S8 (rpsH)

What is the significance of the 30S ribosomal protein S8 (rpsH) in Vibrio vulnificus?

The 30S ribosomal protein S8 (rpsH) is a critical component of the small ribosomal subunit in Vibrio vulnificus. It plays an essential role in ribosome assembly and function by binding to 16S rRNA and facilitating proper folding of the central domain. The protein is highly conserved across bacterial species, making it valuable for phylogenetic studies and understanding the fundamental mechanisms of translation. In V. vulnificus research, rpsH can serve as a marker for genetic lineage and potentially provide insights into the evolutionary divergence of different strains that has been observed in this species . Research on rpsH may also help understand how bacterial protein synthesis functions under different environmental conditions, which is particularly relevant considering the expanding geographical range of V. vulnificus due to climate change .

What methods are recommended for the initial identification and characterization of the rpsH gene in Vibrio vulnificus isolates?

For researchers beginning work with the rpsH gene in Vibrio vulnificus, a methodical approach is recommended:

• PCR Amplification: Design primers targeting conserved regions flanking the rpsH gene based on reference V. vulnificus genomes. Consider using degenerate primers if working with diverse strains.

• Sequencing: Perform Sanger sequencing of PCR products to obtain the complete rpsH sequence from your isolates.

• Bioinformatic Analysis: Compare sequences with databases using BLAST and align with known V. vulnificus sequences to identify variations.

• Genotyping: Correlate rpsH sequence data with established genotyping markers such as vcg type and 16S rRNA type . This can help categorize your isolates within known V. vulnificus populations.

• Phylogenetic Analysis: Construct phylogenetic trees to establish relationships between isolates based on rpsH sequences alongside other housekeeping genes.

This approach allows researchers to establish a foundation for further investigations while linking their isolates to the established knowledge about V. vulnificus population structure and virulence potential .

What are the optimal expression systems for producing recombinant Vibrio vulnificus rpsH protein?

Several expression systems can be used for recombinant production of V. vulnificus rpsH, each with distinct advantages:

The pET expression system with an N-terminal His-tag is commonly effective for ribosomal proteins. When designing the expression construct, researchers should consider codon optimization based on the host organism, as V. vulnificus may utilize different codon preferences than standard E. coli strains. Additionally, the presence of rare codons in the rpsH sequence may necessitate using specialized strains like Rosetta.

For purification, immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography typically yields high-purity protein. The purification buffer should be optimized to maintain the stability of the recombinant rpsH, typically with 20-50 mM Tris-HCl pH 7.5, 100-300 mM NaCl, 5% glycerol, and potentially 5 mM β-mercaptoethanol to prevent oxidation .

How can researchers troubleshoot common challenges in recombinant rpsH expression and purification?

When working with recombinant V. vulnificus rpsH, researchers commonly encounter several challenges:

• Low Solubility: Ribosomal proteins can form inclusion bodies. To address this:

  • Reduce induction temperature to 16-20°C

  • Lower IPTG concentration to 0.1-0.3 mM

  • Consider fusion partners like SUMO or MBP to enhance solubility

  • Use auto-induction media for gentler protein expression

• Protein Instability: If rpsH shows degradation during purification:

  • Include protease inhibitors in all buffers

  • Minimize time between cell lysis and purification

  • Add 5-10% glycerol to all buffers

  • Consider performing all steps at 4°C

• RNA Contamination: Being an RNA-binding protein, rpsH may co-purify with bacterial RNA:

  • Include high salt (500 mM NaCl) washes during IMAC

  • Consider RNase treatment during lysis

  • Add additional ion exchange chromatography step

  • Verify RNA absence by measuring A260/A280 ratio (target <1.0)

• Protein Functionality Assessment: To confirm proper folding:

  • Perform circular dichroism spectroscopy

  • Test RNA binding ability using electrophoretic mobility shift assays

  • Compare secondary structure elements to known S8 structures

  • Consider limited proteolysis to verify correct folding

Researchers should systematically optimize expression conditions by testing multiple combinations of temperature, induction time, and IPTG concentration. Additionally, the inclusion of molecular chaperones through co-expression vectors may help increase the yield of properly folded rpsH protein .

What approaches are effective for examining rpsH genetic recombination in Vibrio vulnificus strains?

Investigating genetic recombination of rpsH in Vibrio vulnificus requires a comprehensive approach:

• Whole Genome Sequencing: Perform WGS on multiple V. vulnificus strains from diverse sources (clinical, environmental, geographical variations). This provides the foundation for detailed recombination analysis.

• Comparative Genomic Analysis: Utilize bioinformatic tools like MAUVE, BRAT NextGen, or ClonalFrameML to identify potential recombination events affecting the rpsH region. These tools can detect anomalous sequence patterns indicative of horizontal gene transfer.

• Population Structure Analysis: Implement STRUCTURE or BAPS software to identify genetic clusters and potential admixture affecting ribosomal protein genes.

• Recombination Detection: Apply specific algorithms like RDP4, GARD, or PhiPack to detect recombination breakpoints and statistical significance of recombination events.

• Phylogenetic Analysis with Recombination Considerations: Construct phylogenetic trees with and without recombination filtering to assess the impact of recombination on evolutionary interpretations.

• Experimental Verification: Design PCR assays targeting regions flanking rpsH to verify computational predictions of recombination events.

This methodical approach has proven valuable in identifying recombination events in V. vulnificus, as demonstrated in studies of the rtxA1 gene, where researchers identified four distinct variants arising from recombination with plasmid genes or genes from other Vibrio species . Similar approaches could reveal whether rpsH has undergone recombination events that might affect protein function or evolutionary trajectory, particularly in strains from "manmade niches" like aquaculture environments where different V. vulnificus lineages may come into contact .

How does the structure of Vibrio vulnificus rpsH compare to other bacterial S8 proteins, and what are the implications?

The structural analysis of V. vulnificus rpsH reveals a protein with high conservation in key functional domains when compared to other bacterial S8 proteins:

The implications of these structural observations are significant:

• Conserved Functional Core: The high conservation of the RNA-binding domain suggests maintained functionality across Vibrio species, making it a potential target for broad-spectrum antimicrobial development.

• Variable Regions: Surface variations might contribute to species-specific interactions and could potentially be exploited for designing selective inhibitors.

• Evolutionary Insights: The pattern of conservation versus variability provides clues about evolutionary pressures on different regions of the protein, with functional domains being most conserved.

• Structure-Function Relationship: Analysis of V. vulnificus rpsH structure in relation to its function in the ribosome can help understand the molecular basis of potential strain-specific translation differences .

What methodologies are most effective for studying the interaction between rpsH and 16S rRNA in Vibrio vulnificus?

Investigating the interaction between V. vulnificus rpsH and 16S rRNA requires multiple complementary approaches:

• In Vitro Binding Assays:

  • Electrophoretic Mobility Shift Assays (EMSA): Use purified recombinant rpsH with labeled 16S rRNA fragments to determine binding affinities and specificity.

  • Surface Plasmon Resonance (SPR): Provides real-time kinetic data on rpsH-RNA interactions.

  • Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding.

  • Filter Binding Assays: A simpler alternative for determining dissociation constants.

• Structural Studies:

  • X-ray Crystallography: Co-crystallize rpsH with 16S rRNA fragments to determine atomic resolution structures.

  • Cryo-Electron Microscopy: Particularly useful for visualizing rpsH in the context of the whole ribosome.

  • NMR Spectroscopy: For studying dynamics of the interaction in solution.

• Computational Approaches:

  • Molecular Dynamics Simulations: To predict binding modes and conformational changes.

  • Homology Modeling: Based on existing ribosome structures from related species.

  • RNA-Protein Docking: To predict binding interfaces.

• In Vivo Approaches:

  • RNA Immunoprecipitation (RIP): To isolate rpsH-bound RNA complexes from V. vulnificus cells.

  • Cross-linking and Immunoprecipitation (CLIP): To identify exact binding sites on the 16S rRNA.

  • Genetic Mutations: Introduce mutations in rpsH or 16S rRNA to verify interaction sites.

Particularly valuable insights come from combining these approaches. For example, computational predictions can guide the design of mutants for in vitro and in vivo testing. Researchers should consider that V. vulnificus has multiple 16S rRNA variants (type A and B), which may show differential binding properties with rpsH and could contribute to differences in translation efficiency between clinical and environmental strains .

How can researchers investigate the role of rpsH in Vibrio vulnificus stress response and pathogenicity?

Investigating the role of rpsH in V. vulnificus stress response and pathogenicity requires multiple experimental approaches:

• Gene Expression Analysis:

  • qRT-PCR: Measure rpsH expression under various stress conditions (temperature shifts, pH changes, oxidative stress, host-mimicking conditions).

  • RNA-Seq: Characterize global transcriptional responses to stress, including rpsH regulation.

  • Ribosome Profiling: Determine translational efficiency changes under stress conditions.

• Genetic Manipulation:

  • Conditional Knockdown: Since complete deletion may be lethal, use inducible antisense RNA or CRISPR interference.

  • Site-Directed Mutagenesis: Create point mutations in functional domains to assess specific roles.

  • Complementation Studies: Restore wild-type phenotype with functional rpsH to confirm specificity.

• Phenotypic Assays:

  • Growth Curves: Under various stress conditions for mutant vs. wild-type strains.

  • Biofilm Formation: Assess changes in biofilm capacity with rpsH mutations.

  • Virulence Factor Production: Measure expression of known virulence factors like RTX toxins, metalloprotease, and capsular polysaccharides in response to rpsH alterations .

• Host Interaction Models:

  • Cell Culture Invasion Assays: Using epithelial cells to assess invasion efficiency.

  • Macrophage Survival Assays: Determine intracellular survival with rpsH mutations.

  • Mouse Infection Models: Compare virulence of mutant strains in vivo using established V. vulnificus infection models .

• Proteomic Approaches:

  • Pull-down Assays: Identify protein interaction partners of rpsH under different conditions.

  • 2D-DIGE: Compare protein expression profiles between wild-type and rpsH mutants.

This comprehensive approach would reveal whether rpsH plays a regulatory role beyond its structural function in the ribosome. Given the importance of translational control during stress responses, rpsH modifications might affect the expression of virulence factors by influencing ribosome assembly or function under stress conditions commonly encountered during infection .

How can rpsH sequences be utilized in evolutionary studies of Vibrio vulnificus strains?

The rpsH gene provides valuable evolutionary insights for V. vulnificus research:

• Phylogenetic Marker Applications:

  • Combine rpsH with other housekeeping genes (16S rRNA, recA, rpoB) to construct robust phylogenetic trees.

  • Use rpsH as part of Multi-Locus Sequence Typing (MLST) schemes to classify strains.

  • Compare evolutionary rates of rpsH versus virulence genes to identify selective pressures.

• Analytical Approaches:

  • Calculate nucleotide diversity (π) and polymorphism rates in rpsH across strain collections.

  • Perform dN/dS analysis to detect selection patterns affecting the protein.

  • Use Bayesian evolutionary analysis tools like BEAST to estimate divergence times.

• Cluster Analysis:

  • Incorporate rpsH sequence data into population structure analyses to supplement existing clustering frameworks based on vcg and 16S rRNA types .

  • Compare rpsH-based phylogenies with whole-genome SNP trees to assess congruence.

• Recombination Detection:

  • Apply recombination detection algorithms to assess if rpsH has been subject to horizontal gene transfer.

  • Compare with known recombination patterns observed in V. vulnificus genes like rtxA1 .

• Biogeographic Studies:

  • Analyze rpsH sequences from strains collected across different geographical locations to trace dissemination patterns.

  • Correlate sequence variations with environmental factors, particularly in regions newly affected by climate change .

This evolutionary approach can help resolve relationships between the divergent clusters identified in V. vulnificus populations and potentially reveal patterns related to virulence potential, host adaptation, and geographical spread . The conserved nature of rpsH makes it particularly valuable for deep phylogenetic relationships, while still potentially capturing the broader population structure observed through more variable genetic elements.

What bioinformatic pipelines are recommended for comparative analysis of rpsH across Vibrio species?

A comprehensive bioinformatic pipeline for comparative analysis of rpsH across Vibrio species should include:

• Sequence Acquisition and Verification:

  • Extract rpsH sequences from public databases (NCBI, UniProt)

  • Verify annotation accuracy using conserved domain searches

  • Include diverse Vibrio species and outgroups

  • Create local database of manually curated sequences

• Multiple Sequence Alignment:

  • Initial alignment using MUSCLE or MAFFT with iterative refinement

  • Manual inspection and adjustment in Jalview or AliView

  • Alignment trimming using GBlocks or trimAl to remove poorly aligned regions

  • Format conversion for downstream analyses

• Evolutionary Model Selection:

  • Determine best-fit evolutionary model using ModelTest-NG or jModelTest

  • Separate models for codon positions or protein/nucleotide data

  • Parameter optimization based on likelihood scores

• Phylogenetic Analysis:

  • Maximum Likelihood: RAxML or IQ-TREE with bootstrap support

  • Bayesian Inference: MrBayes or BEAST with posterior probabilities

  • Parsimony and Distance methods as complementary approaches

  • Visualization with iTOL or FigTree

• Comparative Genomic Context:

  • Analyze genomic neighborhood of rpsH using tools like Artemis

  • Identify conservation of gene order (synteny) across species

  • Detect operon structures and potential co-regulation

• Selection Analysis:

  • Site-specific selection using PAML, MEME, or FUBAR

  • Branch-specific selection using aBSREL or BUSTED

  • Sliding window analysis of nucleotide diversity

• Structure-Function Mapping:

  • Project sequence conservation onto protein structure using ConSurf

  • Identify co-evolving residues using mutual information analysis

  • Predict functional impact of substitutions with PROVEAN or SIFT

• Visualization and Integration:

  • Generate integrated visualizations combining phylogeny, structural data, and selection results

  • Create custom R scripts for statistical analysis of sequence features

  • Develop interactive visualizations using tools like D3.js

This pipeline would facilitate the identification of both conserved functional domains and species-specific variations in rpsH, potentially revealing adaptations related to ecological niches or pathogenic potential across the Vibrio genus .

How does rpsH genetic diversity correlate with other molecular markers used for Vibrio vulnificus typing?

The correlation between rpsH genetic diversity and established molecular markers for V. vulnificus typing reveals important patterns:

Research has shown that V. vulnificus populations form distinct clusters with different pathogenic potential . While rpsH is more conserved than virulence genes like rtxA1 , its sequence variations still generally align with the broader population structure. This makes rpsH valuable for:

• Confirmation of lineage assignments made with other markers

• Resolution of ambiguous typing results

• Detection of potential recombination events when markers show discordant results

Interestingly, while clinical isolates typically belong to vcg type C and 16S rRNA type B , there are exceptions that can be better understood by incorporating rpsH analysis. This is particularly relevant as researchers have found that "manmade niches" like aquaculture environments are bringing different V. vulnificus lineages into contact, potentially facilitating recombination and the emergence of novel variants . A comprehensive typing approach that includes rpsH alongside established markers would provide a more complete picture of strain relationships and evolutionary history.

How might rpsH contribute to antibiotic resistance mechanisms in Vibrio vulnificus?

The potential contribution of rpsH to antibiotic resistance in Vibrio vulnificus involves several mechanisms:

• Target Site Alterations:

  • Mutations in rpsH can potentially modify ribosomal structure, affecting the binding of aminoglycoside antibiotics

  • Subtle amino acid changes may reduce antibiotic affinity while maintaining ribosomal function

  • These structural changes could contribute to the increasing antibiotic resistance observed in clinical V. vulnificus isolates

• Translational Regulation:

  • As a ribosomal protein, rpsH may influence the translation efficiency of resistance genes

  • Under antibiotic stress, alterations in rpsH expression could modulate the synthesis of efflux pumps, antibiotic-modifying enzymes, or alternative metabolic pathways

  • This regulatory role might explain why some V. vulnificus strains show resistance phenotypes that don't directly correlate with their antibiotic resistance gene profiles

• Stress Response Coordination:

  • rpsH might participate in bacterial stress responses triggered by antibiotics

  • These responses can include biofilm formation, which is known to enhance antibiotic tolerance

  • The protein could influence translation of stress response regulators under antibiotic pressure

• Research Approaches:

  • Compare rpsH sequences between antibiotic-sensitive and resistant isolates

  • Perform targeted mutagenesis of rpsH and assess changes in minimum inhibitory concentrations (MICs)

  • Use ribosome profiling to identify translational changes in response to antibiotics

With V. vulnificus showing increasing resistance to commonly used antibiotics like cephalosporins and tetracyclines , understanding how core ribosomal proteins like rpsH might contribute to resistance mechanisms could provide new insights for antimicrobial development. This is particularly important as research indicates that 66.7% of clinical isolates now show resistance to multiple antibiotics, with 61.9% possessing a multiple antibiotic resistance (MAR) index exceeding 0.2 .

What is the potential of rpsH as a target for novel antimicrobial strategies against Vibrio vulnificus?

The potential of rpsH as an antimicrobial target against Vibrio vulnificus warrants serious research consideration:

• Target Validation Criteria:

  • Essentiality: As a core ribosomal protein, rpsH is essential for bacterial survival

  • Conservation: High sequence conservation makes it a broad-spectrum target

  • Uniqueness: Despite conservation, structural differences from human ribosomal proteins exist

  • Accessibility: As part of the small ribosomal subunit, it's accessible to small molecules

• Potential Targeting Strategies:

  • Small molecule inhibitors that specifically bind to bacterial rpsH

  • Peptide mimetics that disrupt rpsH-rRNA interactions

  • Antisense oligonucleotides targeting rpsH mRNA

  • CRISPR-based antimicrobials targeting the rpsH gene

• Experimental Approaches for Drug Development:

  • High-throughput screening against purified recombinant rpsH

  • Structure-based drug design utilizing crystallographic data

  • Fragment-based lead discovery targeting rpsH-RNA binding interface

  • Whole-cell screening with target validation through resistance mutations

• Advantages as a Target:

  • Targeting rpsH could disrupt ribosome assembly rather than function, providing a novel mechanism

  • The essential nature of rpsH means resistance would likely incur significant fitness costs

  • A higher barrier to resistance development compared to non-essential targets

• Challenges to Address:

  • Achieving selectivity over human ribosomal components

  • Designing molecules with appropriate pharmacokinetic properties

  • Ensuring bacterial penetration, particularly through V. vulnificus capsule

This approach offers potential advantages over targeting conventional virulence factors like RTX toxins, capsular polysaccharides, or metalloproteases , as these may vary between strains or be lost through recombination events . By targeting the conserved and essential rpsH protein, novel therapeutics could address the increasing concern of antibiotic resistance in V. vulnificus while being potentially effective against the diverse strain variants emerging through recombination and evolution .

How can researchers design experiments to evaluate the impact of rpsH variants on Vibrio vulnificus virulence?

Designing experiments to evaluate the impact of rpsH variants on V. vulnificus virulence requires a comprehensive approach:

• Strain Collection and Characterization:

  • Assemble diverse V. vulnificus isolates from clinical and environmental sources

  • Sequence rpsH from all isolates to identify natural variants

  • Classify strains by established markers (vcg type, 16S rRNA type)

  • Characterize baseline virulence using in vitro and in vivo assays

• Genetic Manipulation Strategies:

  • Allelic Exchange: Replace native rpsH with variant alleles in a reference strain

  • CRISPR-Cas9 Editing: Introduce specific mutations to create variant forms

  • Complementation: Express variants in rpsH-depleted backgrounds

  • Creation of strain panel with identical genetic backgrounds differing only in rpsH variants

• In Vitro Virulence Assays:

  • Serum Resistance: Compare survival in human serum across variants

  • Hemolytic Activity: Measure hemolysis capacity using different blood types

  • Cytotoxicity: Quantify damage to human cell lines (intestinal epithelial, macrophage)

  • Biofilm Formation: Assess ability to form biofilms on relevant surfaces

  • Growth Rate: Compare growth kinetics under host-mimicking conditions

• Protein Synthesis Analysis:

  • Ribosome Profiling: Compare translational landscapes across variants

  • Pulse-chase Labeling: Measure synthesis rates of key virulence factors

  • Proteomics: Identify differentially expressed proteins between variants

• In Vivo Infection Models:

  • Mouse Infection Model: Compare LD50 values between rpsH variant strains

  • Tissue Colonization: Assess bacterial loads in various tissues

  • Survival Analysis: Monitor infection outcomes with different variants

  • Inflammatory Response: Measure cytokine profiles induced by variants

• Data Analysis Framework:

  • Multivariate Analysis: Correlate rpsH sequence features with virulence metrics

  • Structure-Function Mapping: Relate amino acid changes to functional outcomes

  • Statistical Comparison: Use appropriate statistical methods to evaluate significance

This experimental framework would help determine whether natural or engineered rpsH variants affect V. vulnificus virulence, potentially by influencing the translation of virulence factors like the MARTX toxins, which have been shown to have variants with different potencies . The approach could reveal whether rpsH plays a regulatory role in virulence beyond its structural function in the ribosome, similar to other ribosomal proteins that have been found to have moonlighting functions in bacterial pathogens .