Recombinant Bordetella bronchiseptica 50S ribosomal protein L1 (rplA)

What is Bordetella bronchiseptica 50S ribosomal protein L1 (rplA) and what is its function?

Bordetella bronchiseptica 50S ribosomal protein L1 (rplA) is a 232 amino acid protein belonging to the universal ribosomal protein uL1 family . This protein serves a dual function: first as a ribosomal protein binding to 23S rRNA as part of the ribosomal L1 protuberance, and second as a translational repressor protein that controls the translation of the L11 operon by binding to its mRNA .

The protein functions within Bordetella bronchiseptica, a Gram-negative coccobacilli (0.2-0.7 μm) of the phylum Proteobacteria . This respiratory pathogen is closely related to Bordetella pertussis, the causative agent of whooping cough, but unlike B. pertussis (which only infects humans), B. bronchiseptica infects a broad range of mammalian hosts .

Methodologically, when investigating the function of rplA, researchers should consider both its structural role in ribosome assembly and its regulatory role in translation, requiring experimental designs that can distinguish between these two functions.

Several expression systems have been successfully used for the recombinant production of Bordetella ribosomal proteins. The most common sources include:

• E. coli expression systems - Most frequently used due to ease of manipulation and high yield

• Yeast expression systems - Useful when post-translational modifications are required

• Baculovirus expression systems - Employed for larger-scale production

• Mammalian cell expression systems - Used when specific mammalian folding is necessary

When choosing an expression system, researchers should consider:

• The intended application of the recombinant protein

• Required purity levels (typically >85% by SDS-PAGE is achievable)

• Need for specific tags for purification or detection

• Proper folding requirements

For optimal results with E. coli expression, methodological considerations include:

• Codon optimization for E. coli if needed

• Using appropriate vectors with inducible promoters

• Optimizing culture conditions (temperature, induction time, media composition)

• Including appropriate affinity tags for purification

• Implementing proper storage protocols (typically with glycerol at -20°C/-80°C)

How can researchers investigate rplA's role in the virulence regulation of B. bronchiseptica?

Investigating rplA's potential contribution to virulence regulation in B. bronchiseptica requires sophisticated approaches that connect ribosomal function with virulence gene expression. Recent research on related ribosomal operons provides valuable methodological insights.

Studies have shown that mutations affecting ribosomal protein operons can have profound effects on virulence factor expression in Bordetella species. For example, a G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and caused global dysregulation of gene expression in B. pertussis . This led to downregulation of virulence factors despite not directly affecting the BvgAS virulence regulatory system.

To investigate rplA's potential role, researchers could employ:

• Transcriptome analysis (RNA-Seq) comparing wild-type and rplA-mutant strains under both virulence-activating and virulence-repressing conditions

• Proteome comparison using techniques similar to those that identified 472 differentially expressed proteins between wild-type and ribosomal operon mutant strains

• Assessment of virulence factor production and secretion

• In vivo colonization studies using animal models

A methodological framework based on existing research would include:

• Gene knockout or site-directed mutagenesis of rplA

• qPCR verification of expression changes

• Western blot analysis of key virulence regulators

• Analysis of interactions with virulence regulatory systems like BvgAS, PlrSR, and RisA

What methods can be used to study the impact of rplA mutations on B. bronchiseptica fitness during infection?

Studying the impact of rplA mutations on B. bronchiseptica fitness during infection requires a combination of in vitro and in vivo approaches. Research on related ribosomal proteins provides methodological guidance.

High-throughput transposon sequencing (Tn-seq) has been successfully used to identify bacterial genes contributing to tracheal colonization in B. bronchiseptica . This approach could be applied to investigate how rplA mutations affect bacterial fitness during infection.

A comprehensive methodological approach would include:

• Generation of rplA mutant strains using:

  • Site-directed mutagenesis

  • CRISPR-Cas9 genome editing

  • Allelic exchange techniques

• In vitro fitness assessment:

  • Growth curve analysis under various conditions

  • Biofilm formation assays

  • Motility assays

  • Assessment of c-di-GMP levels, which are linked to virulence in Bordetella

• Co-infection experiments:

  • Competitive index determination between wild-type and mutant strains

  • Analysis of tracheal colonization efficiency

  • Assessment of persistence in the lower respiratory tract

• Transcriptome and proteome analysis:

  • RNA-Seq to identify differentially expressed genes

  • Proteome analysis to detect changes in protein levels

  • Secretome analysis to evaluate effects on protein secretion

The effectiveness of these methods has been demonstrated in studies of other regulators in B. bronchiseptica, such as RpoN (sigma factor 54), which was shown to support bacterial colonization by regulating various bacteriological functions including motility and biofilm formation .

How does the structure-function relationship of rplA in B. bronchiseptica compare with other bacterial species?

The structure-function relationship of rplA in B. bronchiseptica can be compared with other bacterial species through comparative genomics and structural biology approaches. The L1 protein is highly conserved across bacterial species but may exhibit species-specific functional adaptations.

Methodologically, researchers should:

• Perform sequence alignment of rplA from B. bronchiseptica with homologs from:

  • Closely related Bordetella species (B. pertussis, B. parapertussis, B. avium)

  • More distantly related bacteria with well-characterized L1 proteins (e.g., T. thermophilus, S. acidocaldarius)

• Conduct structural comparison through:

  • X-ray crystallography of B. bronchiseptica rplA

  • Homology modeling based on existing L1 structures

  • Analysis of domain organization and RNA-binding surfaces

• Evaluate functional conservation through:

  • RNA-binding assays comparing affinities for conserved RNA targets

  • Complementation studies in heterologous systems

  • Domain-swapping experiments between species

Studies of L1 from other species have revealed important structural insights. For example, the crystal structure of L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus at 2.65 Å resolution has provided critical information about the L1 protuberance of the 50S ribosomal subunit .

The structure-function relationship has practical implications for researchers, as demonstrated by the finding that domain I of L1 is sufficient for specific RNA binding, while domain II provides additional contacts that stabilize the L1-rRNA complex . The differential binding affinity between ribosomal and messenger RNA is the basis for the feedback inhibition mechanism of L1 proteins .

What techniques can be employed to investigate the role of rplA in B. bronchiseptica stress response and adaptation?

Investigating rplA's role in stress response and adaptation requires sophisticated methods to link ribosomal function with bacterial physiology under challenging conditions. Recent studies on the response of Bordetella to environmental signals provide methodological insights.

Bordetella species respond to environmental cues such as temperature, nutrient availability, and CO2 levels, which modulate virulence gene expression . The ribosome plays a central role in translational control during stress adaptation.

A comprehensive methodological approach would include:

• Stress exposure experiments:

  • Growth under various stress conditions (temperature shifts, nutrient limitation, oxidative stress)

  • CO2 response assays, as Bordetella has a CO2-responsive regulon that may intersect with ribosomal function

  • Antibiotic challenge at sub-inhibitory concentrations

• Molecular analysis of stress response:

  • Transcriptome analysis (RNA-Seq) under stress conditions

  • Ribosome profiling to assess translational efficiency

  • Proteome analysis to detect stress-induced changes

  • Analysis of ppGpp levels (alarmone involved in stringent response)

• Genetic approaches:

  • Construction of rplA variants with mutations in key functional regions

  • Complementation studies with heterologous rplA genes

  • Analysis of interactions with stress-response regulators

• In vivo approaches:

  • Animal infection models with exposure to different stressors

  • Assessment of bacterial persistence in different host microenvironments

Two-component regulatory systems like PlrSR, which responds to CO2 and is required for persistence in the lower respiratory tract, represent potential interaction partners for investigating how ribosomal function may integrate with environmental sensing . Similarly, the connection between the BvgAS system and bacterial fitness in different environments provides a framework for studying how ribosomal proteins like rplA may contribute to adaptive responses .

How can rplA be utilized as a potential target for developing novel antimicrobials against Bordetella infections?

Utilizing rplA as a target for antimicrobial development requires understanding both its essential function in protein synthesis and its unique characteristics in Bordetella species. Several methodological approaches can guide this research direction:

• Target validation approaches:

  • Essentiality assessment through conditional knockdown systems

  • Determination of minimum inhibitory concentrations of ribosome-targeting antibiotics

  • Identification of species-specific structural features in B. bronchiseptica rplA

• High-throughput screening methods:

  • RNA-binding assays to identify compounds disrupting rplA-RNA interactions

  • Bacterial growth inhibition assays with compound libraries

  • In silico screening targeting specific pockets in the rplA structure

• Rational drug design approaches:

  • Structure-based design targeting the RNA-binding interface

  • Fragment-based drug discovery focused on domain I of rplA

  • Peptide mimetics designed to compete with natural binding partners

• Evaluation of candidate compounds:

  • Assessment of specificity for bacterial versus mammalian ribosomes

  • Determination of bactericidal versus bacteriostatic effects

  • Evaluation of resistance development frequency

  • In vivo efficacy in animal models of Bordetella infection

The dual function of rplA as both a ribosomal protein and a translational repressor presents unique opportunities for antimicrobial development. Targeting the regulatory function might disrupt the balance of ribosomal protein synthesis, while targeting the structural function could inhibit protein synthesis directly.