Recombinant Listeria monocytogenes serotype 4b Translation initiation factor IF-2 (infB), partial

What is the role of Translation Initiation Factor IF-2 (infB) in Listeria monocytogenes serotype 4b?

Translation Initiation Factor IF-2 (infB) plays a critical role in bacterial protein synthesis by facilitating the binding of initiator tRNA (fMet-tRNA) to the 30S ribosomal subunit during translation initiation. In Listeria monocytogenes serotype 4b, IF-2 functions similarly to other bacterial species, promoting proper formation of the initiation complex and ensuring accurate translation of mRNA . This protein is essential for bacterial survival and virulence expression, as it enables the pathogen to synthesize proteins necessary for host invasion and immune evasion. Serotype 4b strains, particularly the 4bV variant, contain distinctive genomic elements that may influence protein expression patterns, including translation initiation factors .

How does L. monocytogenes serotype 4b differ from other Listeria serotypes?

Listeria monocytogenes serotype 4b is distinguished by its significantly higher association with listeriosis outbreaks compared to other serotypes. Recent research has identified variant strains (4bV) that contain a 6.3 kb segment of DNA normally restricted to lineage II strains while maintaining the serological characteristics of 4b strains . This genetic region contains six genes (lmo0734–lmo0739) that encode proteins with various functions, including a transcriptional regulator, metabolic enzymes, and transport systems . The 4bV strains have been linked to multiple listeriosis outbreaks in 2014-2016 in the USA, highlighting their clinical importance. Unlike other serotypes, serotype 4b (along with 1/2a and 1/2b) is responsible for the majority of human listeriosis cases, making its molecular components, including translation factors, particularly relevant for clinical research .

What methods are commonly used to express recombinant IF-2 from L. monocytogenes serotype 4b?

Recombinant expression of Listeria monocytogenes proteins, including translation factors like IF-2, typically employs Escherichia coli expression systems. Based on established protocols for similar Listeria proteins, the process involves:

• Gene amplification: PCR amplification of the infB gene from L. monocytogenes serotype 4b genomic DNA

• Vector construction: Cloning into expression vectors containing appropriate promoters and affinity tags

• Expression conditions: Optimization of temperature, IPTG concentration, and induction time for maximal protein yield

• Purification: Affinity chromatography using His-tag or other fusion tags, followed by size exclusion chromatography

For internalin proteins from L. monocytogenes, researchers have successfully used pAE vectors for expression in E. coli, followed by protein A-affinity chromatography for purification . Similar approaches can be adapted for the expression of translation initiation factors, with modifications to account for protein-specific characteristics.

How can whole genome sequencing (WGS) be integrated with recombinant protein studies to characterize IF-2 variants in L. monocytogenes 4b strains?

Integrating whole genome sequencing with recombinant protein studies provides a powerful approach to characterizing IF-2 variants in L. monocytogenes serotype 4b strains. A comprehensive methodological framework includes:

• Genomic analysis: Use WGS data to identify polymorphisms in the infB gene across different L. monocytogenes 4b isolates. Apply the CFSAN SNP Pipeline to detect single nucleotide polymorphisms that might affect protein function .

• Variant selection: Prioritize IF-2 variants based on:

  • Proximity to functional domains

  • Conservation across strains

  • Association with virulence or antibiotic resistance

• Recombinant expression: Express identified variants as recombinant proteins using optimized E. coli systems.

• Functional characterization: Compare biochemical properties of wild-type and variant IF-2 proteins through:

  • Ribosome binding assays

  • GTPase activity measurements

  • Thermal stability analyses

• Structural studies: Determine if amino acid substitutions alter protein structure using X-ray crystallography or cryo-EM.

This integrated approach has successfully revealed important insights into other L. monocytogenes proteins and could be particularly valuable for understanding the functional significance of conservation or variation in translation factors across different serotype 4b strains, including the clinically important 4bV variants .

What are the challenges in distinguishing serotype 4b variant (4bV) strains using recombinant protein-based detection methods?

Developing recombinant protein-based detection methods for 4bV strains presents several significant challenges:

• Serological similarity: 4bV strains are serologically identical to 4b strains despite containing a 6.3 kb segment of DNA normally restricted to lineage II strains, making antibody-based discrimination difficult .

• Conserved epitopes: Traditional antibodies might not distinguish between standard 4b and 4bV strains if targeting highly conserved epitopes.

• Technical solutions:

  • Generate antibodies against proteins encoded by the lineage II-specific genes (lmo0734–lmo0739) present in 4bV strains

  • Design sandwich ELISA systems using combinations of antibodies against both common and variant-specific targets

  • Develop phage-display antibody libraries against specific epitopes unique to 4bV strains

• Validation requirements: Any new detection method requires extensive validation against diverse strain collections, including:

Recent successes with phage display-derived monoclonal antibodies against InlA and InlB suggest similar approaches might be applicable for developing detection systems that can distinguish between standard 4b and 4bV strains based on their unique protein profiles .

How can translation efficiency of recombinant IF-2 be optimized for functional studies in heterologous systems?

Optimizing translation efficiency of recombinant L. monocytogenes IF-2 in heterologous systems requires a multifaceted approach:

• Codon optimization: Analyze codon usage bias between L. monocytogenes and the expression host (typically E. coli). Adjust rare codons to match the host's preferences while maintaining critical structural elements.

• Vector selection: Choose expression vectors with:

  • Strong, inducible promoters (T7, tac)

  • Optimal ribosome binding sites

  • Appropriate fusion tags that don't interfere with IF-2 function

• Expression conditions optimization:

  • Test multiple induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Vary inducer concentration (0.1-1.0 mM IPTG)

  • Optimize growth media (rich vs. minimal, supplemented with amino acids)

  • Consider co-expression with chaperones for proper folding

• Protein solubility enhancement:

  • Use solubility-enhancing tags (SUMO, MBP, TRX)

  • Test different cell lysis conditions to minimize aggregation

  • Employ additives (arginine, detergents) in purification buffers

• Activity preservation:

  • Include GTP in buffers to stabilize IF-2

  • Minimize freeze-thaw cycles

  • Determine optimal storage conditions through activity assays

Researchers working with similar recombinant proteins from L. monocytogenes have successfully preserved activity by carefully optimizing expression conditions and purification protocols, as demonstrated with recombinant InlA and InlB proteins .

How does IF-2 from L. monocytogenes serotype 4b differ structurally and functionally from other bacterial pathogens?

Translation initiation factor IF-2 from L. monocytogenes serotype 4b shares the core functional domains with other bacterial pathogens but exhibits several distinguishing features:

• Structural comparison:

  • Contains conserved G-domain (GTPase) and C2 domain for ribosome binding

  • May possess unique linker regions that influence flexibility and interactions with Listeria-specific ribosomal components

  • Predicted to maintain the three-domain architecture typical of bacterial IF-2 proteins

• Functional distinctions:

  • Adapted to function optimally at lower temperatures consistent with Listeria's psychrotrophic nature

  • May exhibit differential GTP hydrolysis rates compared to enteric pathogens

  • Potentially shows altered interactions with fMet-tRNA that reflect Listeria's adaptation to different host environments

• Regulatory mechanisms:

  • The expression and activity of IF-2 in L. monocytogenes likely responds to environmental stresses encountered during infection

  • May be subject to post-translational modifications unique to Listeria's intracellular lifestyle

Research approaches comparing IF-2 across diverse bacterial species could help identify pathogen-specific characteristics that might be exploited for targeted antimicrobial development. Similar comparative approaches have been successfully applied to other Listeria proteins, revealing important insights into their functional adaptation .

What experimental methods can be used to investigate the role of IF-2 in L. monocytogenes stress response and virulence?

Investigating the role of IF-2 in L. monocytogenes stress response and virulence requires a multi-faceted experimental approach:

• Gene expression analysis:

  • qRT-PCR to measure infB expression under various stress conditions (acid, oxidative, heat, cold stresses)

  • RNA-seq to identify co-regulated genes in stress response networks

  • Ribosome profiling to assess translation efficiency during stress adaptation

• Genetic manipulation strategies:

  • Construction of conditional infB mutants (since complete deletion may be lethal)

  • Site-directed mutagenesis of key functional domains

  • CRISPR interference for partial knockdown of expression

• Protein-level analyses:

  • Pull-down assays to identify IF-2 interaction partners during infection

  • Phosphoproteomics to detect stress-induced post-translational modifications

  • In vitro translation assays comparing activity under various stress conditions

• Infection models:

  • Tissue culture invasion and intracellular replication assays with IF-2 mutants

  • Murine infection models to assess virulence in vivo

  • Competition assays between wild-type and IF-2 variant strains

• Structural biology approaches:

  • Cryo-EM studies of IF-2 bound to Listeria ribosomes

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon stress

These methodological approaches build upon successful strategies used to characterize other L. monocytogenes virulence factors, such as the internalins, and can provide insights into how translation factors contribute to pathogenesis .

How can recombinant IF-2 be used to develop novel diagnostic tools for L. monocytogenes serotype 4b detection?

Recombinant IF-2 offers promising opportunities for developing novel diagnostic tools for L. monocytogenes serotype 4b detection, particularly for difficult-to-identify variant strains:

• Antibody development:

  • Generate highly specific monoclonal antibodies against IF-2 epitopes unique to serotype 4b

  • Employ phage display technology similar to that used for InlA/InlB antibodies

  • Develop sandwich ELISA systems with >95% sensitivity and specificity

• Aptamer-based detection:

  • Select RNA or DNA aptamers with high affinity for L. monocytogenes IF-2

  • Incorporate aptamers into lateral flow devices or biosensors

  • Optimize detection limits to reach 10³ CFU/mL or better

• Recombinant phage-based detection:

  • Engineer bacteriophages to specifically target L. monocytogenes serotype 4b

  • Incorporate reporter genes for visual detection upon infection

  • Develop protocols compatible with food safety testing workflows

• Multiplex approaches:

  • Combine IF-2 detection with other serotype-specific markers

  • Develop assays that simultaneously detect multiple virulence factors

  • Create diagnostic panels that differentiate between standard 4b and 4bV strains

These approaches build on successful strategies for developing diagnostic tools for Listeria detection, as evidenced by recent advances in antibody development and phage-based methods .

What strategies can overcome solubility issues when expressing recombinant L. monocytogenes IF-2 in E. coli?

Expressing recombinant L. monocytogenes IF-2 in E. coli often presents solubility challenges that can be addressed through multiple complementary strategies:

• Fusion partners optimization:

  • Test multiple solubility-enhancing tags in parallel:

    • Maltose-binding protein (MBP)

    • Small ubiquitin-like modifier (SUMO)

    • Thioredoxin (TRX)

    • Glutathione S-transferase (GST)

  • Evaluate different tag positions (N-terminal vs. C-terminal)

  • Incorporate flexible linkers between tag and IF-2

• Expression condition modifications:

  • Reduce expression temperature to 16-20°C

  • Decrease inducer concentration (0.1-0.2 mM IPTG)

  • Use specialized E. coli strains (Rosetta, Arctic Express, SHuffle)

  • Test auto-induction media formulations

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Include rare tRNA supplementation

  • Co-express with interaction partners that may stabilize IF-2

• Domain-based approaches:

  • Express individual domains separately

  • Design truncated constructs based on structural predictions

  • Create domain-swapped chimeras with well-expressing bacterial homologs

Similar approaches have proven successful for expressing other challenging Listeria proteins, including surface-associated virulence factors like InlA and InlB .

How can researchers distinguish between pathogenic and non-pathogenic Listeria species when developing IF-2-based detection methods?

Developing IF-2-based detection methods that can reliably distinguish between pathogenic and non-pathogenic Listeria species requires careful consideration of multiple factors:

• Sequence analysis approach:

  • Perform comprehensive alignment of infB sequences across all Listeria species

  • Identify regions with species-specific signatures, particularly in L. monocytogenes

  • Design primers or probes targeting these discriminatory regions

• Epitope mapping strategy:

  • Use phage display to identify peptide epitopes unique to L. monocytogenes IF-2

  • Generate monoclonal antibodies against these specific epitopes

  • Validate antibody specificity against a panel of Listeria species

• Multiplex detection systems:

  • Combine IF-2 detection with established pathogenicity markers (e.g., internalins)

  • Develop assays that simultaneously detect multiple virulence-associated proteins

  • Create diagnostic algorithms that integrate multiple biomarkers for increased specificity

• Validation requirements:

Research has shown that combining targets like IF-2 with species-specific markers like InlA and InlB can achieve 100% sensitivity (CI 29.24–100.0) and specificity (CI 88.78–100.0) in distinguishing pathogenic from non-pathogenic Listeria, providing a model for robust diagnostic development .

What are the critical quality control parameters for validating recombinant L. monocytogenes IF-2 preparations?

Ensuring the quality and consistency of recombinant L. monocytogenes IF-2 preparations requires rigorous quality control measures across multiple parameters:

• Purity assessment:

  • SDS-PAGE analysis (target: >95% purity)

  • Size exclusion chromatography to detect aggregates

  • Mass spectrometry for molecular weight confirmation and contaminant detection

  • Endotoxin testing (<1 EU/mg protein for functional studies)

• Structural integrity verification:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal shift assays to assess stability

  • Limited proteolysis to verify proper folding

  • Dynamic light scattering to evaluate size distribution

• Functional activity testing:

  • GTPase activity assays (comparative analysis with commercial standards)

  • 30S ribosomal subunit binding assays

  • fMet-tRNA binding capacity

  • In vitro translation initiation efficiency

• Storage stability monitoring:

  • Activity retention after freeze-thaw cycles

  • Long-term stability at -80°C, -20°C, and 4°C

  • Buffer optimization for maintained functionality

  • Aggregation assessment over time

• Batch-to-batch consistency:

  • Standardized activity units definition

  • Reference standard comparison for each batch

  • Lot-specific certificate of analysis documentation

These quality control parameters ensure that recombinant IF-2 preparations are suitable for downstream applications in structural biology, functional characterization, and diagnostic development. Similar quality control approaches have been successfully implemented for other recombinant Listeria proteins used in diagnostic and research applications .

How might IF-2 variants in L. monocytogenes 4bV strains contribute to their epidemiological success?

The epidemiological success of L. monocytogenes 4bV strains may be influenced by variations in translation factors like IF-2 through several potential mechanisms:

• Translation efficiency adaptation:

  • IF-2 variants might optimize translation initiation under specific environmental conditions

  • Enhanced translation of stress response proteins could provide survival advantages

  • Altered translation preferences may influence the expression of virulence factors

• Host interaction modulation:

  • Modified translational machinery could affect the production of surface proteins involved in host cell invasion

  • Variations in translation factors might influence the bacterial response to host immune defenses

  • Subtle changes in virulence factor expression timing may enhance pathogenicity

• Stress response coordination:

  • 4bV strains contain a 6.3 kb segment with genes encoding metabolic enzymes and regulators

  • IF-2 variants might specifically enhance translation of these acquired genes

  • Coordinated expression between core genome and acquired elements may provide fitness advantages

• Research approaches to test these hypotheses:

  • Comparative ribosome profiling between standard 4b and 4bV strains

  • Proteomics analysis under various stress conditions

  • Experimental evolution studies under selective pressures

  • Genetic complementation experiments swapping IF-2 variants between strains

The adaptation of translational machinery in 4bV strains represents an intriguing research direction that may explain their involvement in recent listeriosis outbreaks and their apparent emergence as significant pathogens .

What potential exists for developing antimicrobial agents targeting L. monocytogenes IF-2?

Translation initiation factor IF-2 presents a promising target for novel antimicrobial development against L. monocytogenes based on several advantageous characteristics:

• Target validation rationale:

  • IF-2 is essential for bacterial survival

  • The protein has sufficient structural differences from human translation factors

  • Translation initiation represents a bottleneck in protein synthesis

• Drug discovery approaches:

  • High-throughput screening against the GTPase domain

  • Fragment-based drug design targeting IF-2/ribosome interface

  • Structure-based virtual screening for binding pocket inhibitors

  • Repurposing of existing translation inhibitors with optimization for Listeria-specificity

• Potential advantages of IF-2 inhibitors:

  • May be effective against antibiotic-resistant L. monocytogenes strains

  • Could have activity against both replicating and slow-growing intracellular bacteria

  • Might show synergistic effects with existing antibiotics

• Development challenges to address:

  • Ensuring selectivity over human translation factors

  • Achieving sufficient penetration into mammalian cells to reach intracellular bacteria

  • Optimizing pharmacokinetic properties for clinical applications

This approach aligns with the growing need for new antimicrobial strategies against Listeria, particularly given increasing concerns about antibiotic resistance in clinical isolates .

How can structural biology approaches enhance our understanding of L. monocytogenes IF-2 function in translation initiation?

Advanced structural biology approaches offer powerful tools to elucidate the precise mechanisms of L. monocytogenes IF-2 function in translation initiation:

• Cryo-electron microscopy applications:

  • Capture IF-2 in complex with Listeria-specific ribosomes at different functional states

  • Resolve structures at near-atomic resolution (2-3 Å)

  • Visualize conformational changes during GTP hydrolysis and fMet-tRNA positioning

  • Compare structures between standard 4b and variant 4bV strains

• X-ray crystallography strategies:

  • Determine high-resolution structures of individual IF-2 domains

  • Co-crystallize with GTP/GDP and ribosome components

  • Analyze binding sites for potential inhibitor development

  • Map serotype-specific structural variations

• Integrative structural approaches:

  • Combine small-angle X-ray scattering (SAXS) with NMR for full-length protein dynamics

  • Apply hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Use cross-linking mass spectrometry to map interaction interfaces

  • Employ molecular dynamics simulations to predict functional motions

• Functional correlation analyses:

  • Connect structural features to biochemical activities

  • Identify structural determinants of temperature sensitivity

  • Map species-specific structural elements that could influence host adaptation

  • Correlate structural variations with translation efficiency differences

These structural biology approaches would provide unprecedented insights into how L. monocytogenes IF-2 functions at the molecular level, potentially revealing unique adaptations that contribute to this pathogen's virulence and environmental persistence. Similar structural approaches have been successfully applied to other bacterial translation factors, revealing important mechanistic insights into their function .