Recombinant Listeria monocytogenes serotype 4b 30S ribosomal protein S5 (rpsE)

What is the function of 30S ribosomal protein S5 (rpsE) in Listeria monocytogenes?

The 30S ribosomal protein S5 (rpsE) is a critical component of the small ribosomal subunit in Listeria monocytogenes. While specific research on rpsE in L. monocytogenes is limited in the provided literature, we can draw parallels with other ribosomal proteins such as S21 (rpsU). Similar to other ribosomal proteins, rpsE likely plays essential roles in translation initiation, ribosome assembly, and mRNA decoding. Mutations in ribosomal proteins like rpsU have been shown to affect stress resistance and growth rate in L. monocytogenes, suggesting that rpsE may have similar importance in cellular physiology . Research methodologies for determining rpsE function often include comparative genomics, structural biology approaches, and phenotypic analysis of mutant strains.

How do mutations in ribosomal proteins affect L. monocytogenes stress response?

Mutations in ribosomal proteins can significantly alter L. monocytogenes stress response mechanisms. For example, a point mutation in the rpsU gene (rpsU G50C) encoding ribosomal protein S21 leads to increased multi-stress resistance against acid, heat, and other stressors, along with a reduced maximum specific growth rate . This mutation appears to activate the alternative sigma factor SigB, which regulates the general stress response in L. monocytogenes, through a pathway independent of the classical stressosome and RsbV/RsbW partner switching model .

The increased stress resistance phenotype in rpsU mutants occurs even in strains lacking RsbR1 (a component of the stressosome) and RsbV (the anti-sigma factor antagonist), suggesting an alternative mechanism for SigB activation . By analogy, mutations in rpsE might similarly affect stress response pathways, potentially through alterations in translation efficiency or through specific regulatory interactions.

What methods are recommended for generating recombinant L. monocytogenes strains expressing modified rpsE?

For generating recombinant L. monocytogenes strains with modified rpsE, several established methodologies can be adapted from existing protocols for other ribosomal proteins:

• Site-specific integration vectors: Shuttle integration vectors such as pPL1, which utilizes listeriophage U153 integrase and attachment site within the comK gene, can be used for chromosomal insertion of modified rpsE sequences . These vectors contain useful polylinkers, can be directly conjugated from E. coli into L. monocytogenes, and form stable, single-copy integrants at a frequency of ~10^-4 per donor cell .

• Genome integration approaches: For precise genomic modifications, techniques used for other genes can be adapted, such as those used to introduce the hly and actA genes at comK-attBB in deletion strains .

• Expression systems: Both single-copy genome integration and multicopy episomal expression systems can be considered, similar to the approaches used for E7 protein expression in vaccine vector development . The choice between these methods should be based on the research objectives and the potential impact of expression levels on cellular physiology.

How do ribosomal protein mutations affect virulence and pathogenesis in L. monocytogenes?

Mutations in ribosomal proteins can have profound effects on L. monocytogenes virulence and pathogenesis, though the mechanisms may be complex and multifaceted. In recombinant L. monocytogenes strains used as vaccine vectors, virulence can be significantly decreased compared to wild-type strains . The virulence effects may depend on:

• Altered translation efficiency: Changes in ribosomal proteins like rpsE might affect the translation of virulence factors.

• Stress response alterations: As seen with rpsU mutations, changes in stress resistance can impact survival during host infection .

• Growth rate effects: Ribosomal protein mutations often lead to reduced maximum specific growth rates, which may alter the dynamics of infection .

For studying these effects, methodologies should include:

• In vitro infection models using relevant cell lines

• Animal models to assess colonization and virulence

• Transcriptomic and proteomic analyses to identify differential expression of virulence factors

• Immunological assays to evaluate host response

What are the implications of ribosomal protein modifications on translation fidelity and antibiotic resistance?

Modifications to ribosomal proteins like rpsE may have significant implications for translation fidelity and antibiotic resistance in L. monocytogenes. Based on studies of other ribosomal proteins, researchers should consider:

• Translation accuracy: Changes in rpsE structure might alter codon-anticodon recognition or tRNA binding, affecting translation fidelity.

• Antibiotic resistance: Many antibiotics target the ribosome, and alterations in ribosomal proteins can confer resistance to specific antimicrobials.

• Compensatory mechanisms: Cells may develop compensatory mechanisms to maintain translation efficiency despite ribosomal protein modifications.

Methodological approaches should include:

• Minimum inhibitory concentration (MIC) assays against a panel of antibiotics

• Translation fidelity reporter systems

• Ribosome profiling to assess translation dynamics

• Structural studies of modified ribosomes

How can proteomics approaches be optimized to study rpsE interactions within the ribosome complex?

Proteomics approaches can provide valuable insights into rpsE interactions within the ribosome complex of L. monocytogenes. Based on methods used for studying other ribosomal proteins, researchers should consider:

In a recent study of L. monocytogenes ribosomal proteins, proteomics analysis revealed that mutations in the rpsU gene led to changes in the abundance ratios of ribosomal proteins, including the ratio between the anti-sigma factor RsbW and SigB . Similar approaches could be applied to study how modifications to rpsE affect ribosome composition and function.

What genetic techniques are most effective for studying the effect of point mutations in rpsE?

For studying point mutations in rpsE, several genetic techniques have proven effective in ribosomal protein research:

• Site-directed mutagenesis: Precise introduction of specific mutations to test structure-function hypotheses.

• Allelic exchange: Replacement of the native rpsE gene with mutated versions to study phenotypic effects.

• CRISPR-Cas9 genome editing: For efficient introduction of point mutations in the native genetic context.

• Complementation studies: Introduction of wild-type or mutant genes into deletion strains to confirm phenotype association.

When studying the effects of rpsU G50C mutation, researchers combined phenotypic and proteomic approaches to investigate stress resistance, growth rate, and SigB activation . Similar methodologies could be adapted for rpsE studies, with careful genetic controls to account for potential second-site mutations or compensatory changes.

How can recombinant L. monocytogenes strains with modified rpsE be utilized in vaccine development?

Recombinant L. monocytogenes strains with modified ribosomal proteins could potentially be utilized in vaccine development, drawing on principles established with other recombinant L. monocytogenes vaccine vectors:

• Antigen delivery platforms: L. monocytogenes naturally targets antigen-presenting cells and delivers antigens to both MHC class I and II pathways, making it an effective vaccine vector .

• Attenuated strains: Modifications to ribosomal proteins might serve as attenuation strategies, reducing virulence while maintaining immunogenicity.

• Fusion protein strategies: Similar to the Lm-LLO-E7 approach, where E7 is fused to a non-hemolytic listeriolysin O (LLO), rpsE could potentially be used as part of fusion proteins to enhance antigen processing .

Research has shown that different recombinant L. monocytogenes constructs can induce markedly different immune responses. For example, Lm-LLO-E7 induces regression of E7-expressing tumors in mice, while Lm-E7 does not, despite both strains eliciting measurable CTL responses . This highlights the importance of vector design in determining immunological outcomes.

What controls should be included when studying recombinant L. monocytogenes expressing modified rpsE?

When studying recombinant L. monocytogenes expressing modified rpsE, appropriate controls are essential for result interpretation:

• Wild-type strain: The parental strain without any modifications should be included as a baseline control.

• Empty vector control: For plasmid-based expression, a strain containing the same vector without the rpsE insert.

• Complemented strains: Where possible, complementation with wild-type rpsE can confirm phenotype specificity.

• Multiple independent transformants: To control for potential integration site effects or secondary mutations.

How can the impact of rpsE modifications on ribosome assembly be assessed?

To assess the impact of rpsE modifications on ribosome assembly in L. monocytogenes:

• Sucrose gradient ultracentrifugation: To separate and quantify ribosomal subunits, complete ribosomes, and polysomes.

• Cryo-electron microscopy: For structural analysis of ribosome assembly intermediates and completed ribosomes.

• In vitro ribosome reconstitution assays: To assess the incorporation efficiency of modified rpsE.

• Pulse-chase experiments: To monitor the kinetics of ribosome assembly with radiolabeled proteins.

Previous research on the rpsU G50C mutation suggests that the amino acid substitution from arginine to proline potentially results in loss of functionality and/or exclusion of the mutant protein from the 30S ribosome . Similar approaches could determine whether specific rpsE modifications affect incorporation into the ribosome or alter ribosome assembly kinetics.