Recombinant Listeria monocytogenes serotype 4b tRNA modification GTPase MnmE (mnmE)

What is the role of MnmE in L. monocytogenes serotype 4b pathogenicity?

MnmE, a conserved tRNA-modifying GTPase, plays a critical role in the accuracy and efficiency of protein synthesis in bacteria. Similar to its function in other bacterial species, MnmE in L. monocytogenes serotype 4b likely forms an α2β2 heterotetrameric complex with GidA to control the addition of a carboxymethylaminomethyl (cmnm) group at position five of the wobble uridine of tRNAs that read codons ending with adenine or guanine . This modification ensures translational fidelity, which is particularly important for virulence-associated proteins.

The significance of MnmE in pathogenicity can be appreciated by examining similar tRNA-modifying enzymes in related species. For instance, in Streptococcus suis serotype 2, deletion of the mnmE gene resulted in attenuated virulence and altered metabolism . In the context of L. monocytogenes serotype 4b—a strain strongly associated with human listeriosis outbreaks —MnmE likely contributes to the regulation of virulence factors that enable its invasive capacity and intercellular spread.

How does L. monocytogenes serotype 4b differ from other serotypes, and why is this relevant to MnmE research?

L. monocytogenes serotype 4b exhibits the strongest epidemiological association with human listeriosis among all serotypes and is overrepresented in clinical isolates compared to food isolates . This serotype belongs primarily to genetic lineage I and contains specific genomic features that contribute to its virulence:

These distinctive characteristics make MnmE research in serotype 4b particularly relevant, as tRNA modifications may differentially affect the expression of serotype-specific virulence factors. When designing experiments with recombinant MnmE, researchers should consider these serotype-specific genomic features that may interact with or be affected by tRNA modifications .

What are the recommended methods for constructing a recombinant MnmE expression system in L. monocytogenes serotype 4b?

Construction of a recombinant MnmE expression system in L. monocytogenes serotype 4b requires careful consideration of vector design, transformation protocols, and expression optimization. Based on established methodologies for similar recombinant proteins in Listeria, we recommend the following approach:

Vector Selection and Design:

• For stable integration, use site-specific integration vectors like pSET4s that allow for homologous recombination into the L. monocytogenes genome .

• For controlled expression, incorporate a constitutive or inducible promoter system that functions efficiently in L. monocytogenes.

• Include appropriate selection markers (e.g., erythromycin resistance cassette) for screening recombinant strains .

Cloning Procedure:

• Amplify the mnmE gene and its flanking regions using specific primers designed based on available genome sequences of L. monocytogenes serotype 4b strains .

• Create upstream and downstream homologous arms for integration (typically 500-1000 bp each).

• Clone these fragments into the chosen vector, ensuring proper orientation.

• Transform the construct into E. coli for verification and plasmid amplification.

• Electroporate the verified construct into L. monocytogenes serotype 4b.

• Select transformants on appropriate antibiotic-containing media.

• Confirm recombinants by PCR amplification and sequencing of the mnmE gene and adjacent regions .

Expression Verification:

• Quantify mnmE expression using RT-qPCR or Western blotting with appropriate antibodies.

• Assess MnmE protein functionality through complementation studies in mnmE-deficient strains.

How can I create and validate an mnmE deletion mutant in L. monocytogenes serotype 4b?

Creating an mnmE deletion mutant is essential for understanding its function through phenotypic analysis. Based on similar gene deletion studies in bacterial systems, the following methodological approach is recommended:

Deletion Mutant Construction:

• Design primers to amplify approximately 1000 bp upstream and downstream of the mnmE gene.

• Clone these fragments into a temperature-sensitive shuttle vector like pSET4s, flanking an antibiotic resistance marker (e.g., erm^r from pAT18) .

• Electroporate the resulting plasmid into L. monocytogenes serotype 4b.

• Employ a two-step homologous recombination process:

  • Grow transformants at permissive temperature (30°C) with antibiotic selection.

  • Shift to non-permissive temperature (42°C) to select for chromosomal integration.

  • Return to permissive temperature without plasmid selection to allow second recombination.

  • Screen colonies for antibiotic resistance indicating integration of the marker in place of mnmE.

Validation of the Deletion Mutant:

• PCR verification using:

  • Primers spanning the deletion junction to confirm the absence of mnmE.

  • Primers targeting adjacent genes to ensure they remain intact.

  • Primers specific to the antibiotic resistance cassette to confirm its presence .

• RT-PCR to verify the absence of mnmE transcripts.

• Phenotypic analysis to compare the mutant with wild-type strain (growth curves, stress resistance, virulence in infection models).

Complementation Studies:

• Clone the wild-type mnmE gene with its native promoter and terminator into a different vector (e.g., pSET2).

• Transform this construct into the ΔmnmE strain.

• Verify complementation by PCR, expression analysis, and restoration of wild-type phenotypes .

This comprehensive approach ensures a reliable genetic system for studying MnmE function in L. monocytogenes serotype 4b.

What phenotypic changes should I expect in an mnmE deletion mutant of L. monocytogenes serotype 4b?

Based on studies of tRNA modification enzymes in related bacterial species, an mnmE deletion mutant in L. monocytogenes serotype 4b would likely exhibit multiple phenotypic alterations:

Growth and Basic Physiology:

• Reduced growth rate, particularly in nutrient-limited conditions

• Altered colony morphology and potentially smaller cell size

• Increased sensitivity to temperature fluctuations

Stress Response:

• Heightened sensitivity to oxidative stress due to impaired translation of stress response proteins

• Decreased survival under osmotic stress conditions (relevant for food preservation environments)

• Potentially altered resistance to antimicrobials, particularly those targeting protein synthesis

Virulence-Related Phenotypes:

• Attenuated virulence in infection models

• Altered expression of surface proteins critical for host cell invasion

• Decreased intracellular survival and cell-to-cell spread

• Reduced hemolytic and phospholipase activities

• Impaired ability to escape from phagosomes

Metabolic Changes:

• Altered arginine metabolism (as observed in other species with mnmE mutations)

• Changes in amino acid utilization patterns

• Potential dysregulation of carbon source utilization

These phenotypic changes should be systematically quantified through comparative analyses between the wild-type, ΔmnmE mutant, and complemented strains to establish causal relationships between the absence of MnmE and observed phenotypes.

How do I assess the impact of MnmE on tRNA modification patterns in L. monocytogenes serotype 4b?

Evaluating tRNA modification patterns requires a combination of analytical techniques focused on identifying specific modifications at the molecular level:

tRNA Isolation and Purification:

• Extract total RNA using TRIzol or hot phenol methods.

• Enrich for tRNA fraction using size exclusion chromatography or commercial kits.

• Purify specific tRNA species using oligonucleotide-directed affinity capture if targeting particular tRNAs affected by MnmE.

Modification Analysis Techniques:

• Liquid Chromatography-Mass Spectrometry (LC-MS):

  • Digest purified tRNAs with nuclease P1 and phosphatase.

  • Analyze resulting nucleosides by LC-MS to identify and quantify modified nucleosides.

  • Compare modification profiles between wild-type and ΔmnmE strains.

• Primer Extension Analysis:

  • Design primers that anneal downstream of the expected modification site.

  • Perform reverse transcription, which may be impeded by certain modifications.

  • Analyze extension products by high-resolution gel electrophoresis.

• High-Resolution Northern Blotting:

  • Separate tRNAs by acid-urea PAGE.

  • Probe for specific tRNAs to detect mobility shifts resulting from modification absence.

• Next-Generation Sequencing Approaches:

  • Employ techniques like tRNA-seq with modification-sensitive reverse transcriptases.

  • Analyze modification-dependent misincorporation patterns.

The absence of MnmE should specifically affect the cmnm^5U34 modification in the wobble position of tRNAs reading codons ending in A or G. Quantifying these changes across different growth conditions provides insights into how MnmE activity influences translational fidelity in L. monocytogenes serotype 4b.

How does MnmE influence the processing and presentation of L. monocytogenes serotype 4b antigens in host cells?

The influence of MnmE on antigen processing and presentation involves complex interactions between bacterial protein synthesis and host immune recognition systems:

Effect on Bacterial Protein Synthesis:
MnmE-mediated tRNA modifications affect translational efficiency and fidelity, potentially altering the expression levels of immunogenic proteins. In L. monocytogenes serotype 4b, this could impact the synthesis of key antigens like IspC (a ~77 kDa surface-associated autolysin) and other virulence factors recognized by host immune cells.

Impact on MHC Class I Presentation:
L. monocytogenes has a unique ability to escape from phagosomes and access the host cell cytosol, allowing secreted bacterial proteins to enter the MHC class I antigen processing pathway . Studies have shown that recombinant L. monocytogenes proteins undergo distinct processing compared to endogenously synthesized proteins:

• Proteins secreted by L. monocytogenes generate surface pMHC (peptide-MHC) complexes at rates independent of protein half-life .

• The efficiency of pMHC generation from Listeria-derived proteins is approximately 19-fold higher than from the same proteins expressed by other vectors .

An mnmE mutation could alter this processing efficiency by:

• Changing the synthesis rate or conformational properties of secreted antigens

• Affecting the secretion systems responsible for delivering proteins to the host cytosol

• Modifying the amino acid composition of antigenic peptides due to translational errors

Experimental Approach to Assess Impact:
To investigate this relationship, researchers should:

• Generate isogenic wild-type, ΔmnmE, and complemented strains expressing a model antigen (e.g., ovalbumin).

• Infect antigen-presenting cells and measure surface presentation of specific epitopes using antibodies or T-cell activation assays.

• Compare the kinetics and efficiency of epitope presentation between strains.

• Analyze the fidelity of antigen synthesis through mass spectrometry to detect potential amino acid misincorporations.

Can recombinant L. monocytogenes serotype 4b with modified MnmE be used as an improved vaccine vector?

L. monocytogenes has shown promise as a vaccine vector due to its ability to elicit robust CD8+ T cell responses against heterologous antigens . Modifying MnmE function could potentially enhance these vaccine properties:

Rationale for MnmE Modification in Vaccine Design:

• Attenuated Virulence: Partial inhibition of MnmE function could reduce virulence while maintaining immunogenicity, creating a safer vaccine vector.

• Enhanced Antigen Presentation: Specific modifications to MnmE might optimize the translation of vaccine antigens to improve processing and presentation.

• Metabolic Balance: Carefully tuned MnmE activity could create a metabolic state that prolongs bacterial persistence in antigen-presenting cells without causing disease.

Potential Approaches:

• Site-Directed Mutagenesis: Introduce specific mutations in the GTPase domain of MnmE to partially reduce its activity.

• Regulated Expression: Place mnmE under the control of inducible or tissue-specific promoters for contextual regulation.

• Chimeric MnmE: Create fusion proteins combining MnmE with domains that enhance antigen processing or immune stimulation.

Evaluation Framework:
To assess the efficacy of MnmE-modified vaccine vectors, researchers should:

Potential Advantages Over Current Vectors:

• More precise attenuation compared to deletion of essential virulence genes

• Balanced metabolic state that optimizes immunogenicity

• Potential for enhanced cross-presentation of heterologous antigens

This approach would require careful validation to ensure that MnmE modifications do not introduce unexpected effects on vaccine antigen expression or bacterial fitness in vivo .

How does MnmE function differ between L. monocytogenes serotype 4b and other pathogenic serotypes?

Understanding the serotype-specific functions of MnmE requires comparative analysis across different L. monocytogenes serotypes, with particular attention to translational dynamics and virulence expression:

• Protein-protein interactions with GidA and other tRNA modification enzymes

• Regulatory elements controlling mnmE expression

• GTPase activity and nucleotide binding affinity

Serotype-Specific Translational Profiles:
MnmE may differentially impact the translation of serotype-specific genes, particularly those with biased codon usage patterns. In serotype 4b, this could affect:

• Expression of serotype 4b-specific cell wall teichoic acid genes

• Translation of IspC autolysin, which is highly conserved in serotype 4b isolates

• Expression of genes within clonal complex-specific genomic regions

Methodological Approach for Comparative Studies:

• Generate mnmE deletion mutants in representative strains of multiple serotypes.

• Perform systematic phenotypic comparisons under identical conditions.

• Conduct ribosome profiling to identify serotype-specific translational impacts.

• Perform trans-complementation experiments, expressing MnmE from one serotype in the ΔmnmE background of another serotype.

• Measure virulence in standardized infection models to detect serotype-dependent effects.

This comparative approach would illuminate how MnmE function has potentially evolved to optimize the pathogenic potential of serotype 4b strains, which are disproportionately associated with human disease .

What is the relationship between MnmE and stress adaptation mechanisms in L. monocytogenes serotype 4b?

The relationship between tRNA modification and stress adaptation represents a frontier in understanding bacterial persistence in hostile environments. For L. monocytogenes serotype 4b, which exhibits enhanced survival in food processing environments and host tissues, this relationship is particularly significant:

Stress-Responsive tRNA Modification:
Evidence from other bacterial systems suggests that tRNA modification patterns change in response to environmental stressors, potentially serving as a regulatory mechanism to optimize translation during stress . In L. monocytogenes serotype 4b, MnmE activity might be modulated during:

• Osmotic stress encountered in food preservation

• Oxidative stress within phagocytes

• Acid stress in food or the gastrointestinal tract

• Cold stress during refrigeration

Regulatory Networks:
MnmE activity may intersect with established stress response regulators in L. monocytogenes, including:

• Alternative sigma factors (σB, σH)

• Cold shock proteins (Csps)

• Two-component systems responding to environmental signals

Experimental Framework for Investigation:

• Transcriptional Analysis:

  • Quantify mnmE expression under various stress conditions.

  • Assess expression in regulatory mutants (ΔsigB, Δcsp) to identify control mechanisms.

• Translational Fidelity Assessment:

  • Develop reporter systems to measure mistranslation rates during stress.

  • Compare these rates between wild-type and ΔmnmE strains.

• tRNA Modification Profiling:

  • Quantify changes in tRNA modification patterns across stress conditions.

  • Correlate modifications with MnmE activity and expression.

• Proteome Analysis:

  • Perform quantitative proteomics to identify proteins differentially expressed in ΔmnmE mutants during stress.

  • Focus on proteins with codon usage likely affected by MnmE-dependent modifications.

Understanding this relationship could reveal how serotype 4b strains maintain translational integrity during environmental transitions, potentially explaining their enhanced epidemiological success in causing human disease .

What are common challenges in working with recombinant MnmE in L. monocytogenes serotype 4b and how can they be addressed?

Researchers working with recombinant MnmE in L. monocytogenes serotype 4b encounter several technical challenges that require specific troubleshooting approaches:

Expression Level Optimization:

• Challenge: Achieving appropriate expression levels that avoid toxicity while maintaining functional activity.

• Solution: Test multiple promoter systems with different strengths (constitutive vs. inducible). For inducible systems, titrate inducer concentrations to find the optimal expression window.

Protein Solubility and Folding:

• Challenge: Ensuring proper folding of recombinant MnmE, which contains complex domains including a GTPase domain.

• Solution: Consider fusion tags that enhance solubility (e.g., thioredoxin, SUMO), optimize growth temperature (lower temperatures often improve folding), or co-express with GidA partner protein to promote complex formation.

Genetic Stability:

• Challenge: Maintaining stable integration of recombinant constructs in the L. monocytogenes genome.

• Solution: Use site-specific integration at genomic loci known to be stable. Verify construct stability through multiple passages by PCR and sequencing. Consider antibiotic selection maintenance in laboratory conditions.

Functionality Assessment:

• Challenge: Confirming that recombinant MnmE is enzymatically active.

• Solution: Develop assays to measure GTPase activity of purified protein. Alternatively, perform complementation tests in ΔmnmE strains looking for restoration of growth and tRNA modification patterns.

Addressing Polar Effects in Gene Deletions:

• Challenge: Ensuring that phenotypes of ΔmnmE mutants are due to the absence of MnmE rather than effects on adjacent genes.

• Solution: Design deletion constructs that precisely remove the coding sequence without affecting regulatory elements of neighboring genes. Verify expression of flanking genes by RT-PCR. Use complementation to confirm phenotype specificity.

Construct Design Considerations:
When designing recombinant MnmE constructs, researchers should pay particular attention to:

• Preserving all functional domains, especially the G-domain (GTPase), and dimerization interfaces

• Maintaining proper spacing if adding tags or fusion partners

• Including appropriate ribosome binding sites optimized for L. monocytogenes

• Considering codon optimization if expression is problematic

How can I optimize the purification of recombinant MnmE from L. monocytogenes serotype 4b for structural and biochemical studies?

Purification of recombinant MnmE from L. monocytogenes serotype 4b requires a methodical approach to obtain pure, active protein suitable for downstream applications:

Expression System Design:

• Affinity Tag Selection: For optimal purification, incorporate a tag such as His6, FLAG, or Strep-tag at either the N- or C-terminus. C-terminal tags are often preferable as they ensure only full-length proteins are purified.

• Protease Cleavage Site: Include a TEV or PreScission protease recognition sequence between the tag and MnmE to allow tag removal.

• Expression Control: Use an inducible promoter system to control expression timing and level.

Optimized Purification Protocol:

• Cell Lysis:

  • Suspend bacterial cells in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM DTT, and protease inhibitors.

  • Lyse cells by sonication or high-pressure homogenization.

  • Add 5-10 μg/ml DNase I and 5 mM MgCl2 to reduce viscosity.

• Initial Affinity Purification:

  • For His-tagged MnmE, apply clarified lysate to Ni-NTA resin.

  • Wash extensively with buffer containing 20-40 mM imidazole.

  • Elute with 250-300 mM imidazole.

• Secondary Purification:

  • Apply affinity-purified protein to size exclusion chromatography (Superdex 200).

  • Include 0.2 mM GTP or GDP in buffers to stabilize the protein.

  • Assess purity by SDS-PAGE and protein identity by mass spectrometry or Western blotting.

• Activity Preservation:

  • Add 5 mM MgCl2 to all buffers to maintain GTPase domain structure.

  • Include 1 mM DTT or 0.5 mM TCEP to prevent oxidation of cysteine residues.

  • Store purified protein at -80°C with 10% glycerol as a cryoprotectant.

Quality Control Metrics:

• Purity: >95% as assessed by SDS-PAGE and mass spectrometry

• Activity: GTPase activity using colorimetric assays (MESG assay)

• Structural Integrity: Circular dichroism to confirm proper folding

• Oligomeric State: Size exclusion chromatography multi-angle light scattering (SEC-MALS) to verify correct quaternary structure

This purification approach yields high-quality MnmE suitable for crystallization, enzyme kinetics, binding studies, and other biochemical applications essential for understanding the molecular mechanisms of this important tRNA modification enzyme in L. monocytogenes serotype 4b.