Recombinant Klebsiella pneumoniae subsp. pneumoniae Translation initiation factor IF-2 (infB), partial

What is the functional significance of the IF-2 protein in K. pneumoniae translation initiation?

Translation initiation factor IF-2 in K. pneumoniae serves several essential functions in protein synthesis initiation:

• Facilitates binding of formylmethionyl-tRNA (fMet-tRNA) to the 30S ribosomal subunit

• Promotes formation of the pre-initiation complex

• Assists in joining of the 50S ribosomal subunit to form the 70S initiation complex

• Functions as a GTPase, providing energy through GTP hydrolysis

In K. pneumoniae, IF-2 shares significant homology with other Enterobacteriaceae members, particularly E. coli, though strain-specific variations may influence translation efficiency under different environmental conditions . The protein contains multiple domains including the G-domain responsible for GTP binding and hydrolysis, and domains involved in interactions with the ribosome and initiator tRNA.

How should researchers design experimental approaches to study IF-2 function in different K. pneumoniae pathotypes?

When studying IF-2 across different K. pneumoniae pathotypes (classical vs. hypervirulent strains), researchers should implement:

• Comparative sequence analysis of infB genes from well-characterized clinical isolates

• Expression profiling of IF-2 under infection-relevant conditions using qRT-PCR and Western blotting

• Ribosome profiling to identify translation patterns specific to each pathotype

• Mutational analysis targeting key functional residues in different strain backgrounds

Experimental designs should account for the genetic distinctness of classical (cKp) and hypervirulent (hvKp) strains, which inhabit non-overlapping geographical regions and interact differently with host immune systems . A factorial experimental design (e.g., 2×2 or 3×3) can help evaluate multiple variables simultaneously, such as strain type, growth conditions, and stress responses . This approach allows assessment of how pathotype-specific differences in IF-2 might contribute to virulence traits.

What bioinformatic tools are most appropriate for analyzing IF-2 sequence conservation across K. pneumoniae isolates?

For comprehensive IF-2 sequence analysis across K. pneumoniae isolates, researchers should employ:

When analyzing sequence data, researchers should pay particular attention to:

• Sequence variations between different sequence types (STs) and clonal complexes (CCs)

• Comparison of strains with different antibiotic resistance profiles

• Differences between isolates from various infection sites

• Potential correlations between IF-2 sequence and virulence phenotypes

The growing genetic diversity observed in clinical K. pneumoniae isolates makes such comparative analysis particularly valuable for understanding potential adaptation mechanisms related to translation machinery.

What are the optimal expression systems for producing recombinant K. pneumoniae IF-2?

The choice of expression system for recombinant K. pneumoniae IF-2 significantly impacts yield and protein quality:

For optimal results with E. coli expression systems:

• Use pET vectors with T7 promoter for high-level expression

• Include an N-terminal His-tag for purification, with a TEV protease cleavage site

• Optimize codon usage for rare codons in the IF-2 sequence

• Test expression at multiple temperatures (16°C, 25°C, 37°C) and IPTG concentrations (0.1-1.0 mM)

• Monitor growth curves during expression, as overexpression of translation factors can affect host cell growth

The genetic similarity between E. coli and K. pneumoniae makes E. coli an effective host for expression of K. pneumoniae proteins, though careful optimization is required for large translation factors like IF-2.

What purification strategies yield the most active recombinant IF-2 protein?

Purification of active recombinant IF-2 requires careful consideration of buffer conditions and chromatographic techniques:

• Cell lysis considerations:

  • Use gentle lysis methods (e.g., lysozyme treatment followed by sonication)

  • Include protease inhibitors to prevent degradation

  • Maintain reducing conditions with DTT or β-mercaptoethanol (1-5 mM)

• Optimized buffer composition:

  • Tris-HCl or HEPES buffer (pH 7.5-8.0)

  • Moderate salt concentration (150-300 mM NaCl)

  • Glycerol (10-15%) for stability

  • GTP or non-hydrolyzable analogs (0.1-1 mM) to stabilize native conformation

• Multi-step chromatography approach:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for initial capture

  • Ion exchange chromatography to remove nucleic acid contaminants

  • Size exclusion chromatography for final polishing and buffer exchange

• Critical quality controls:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Dynamic light scattering to assess aggregation state

  • GTPase activity assays to confirm functional integrity

  • Mass spectrometry to verify intact mass and post-translational modifications

Throughout purification, it's crucial to maintain GTP in the buffers as nucleotide binding significantly affects IF-2 stability and conformation. For structural studies, consider using stable GTP analogs like GDPNP to capture specific conformational states.

How can researchers distinguish between native and recombinant IF-2 activity in functional assays?

Distinguishing between native and recombinant IF-2 activity requires carefully designed controls and specific assay conditions:

• GTPase activity assays:

  • Compare intrinsic GTPase rates using [γ-32P]GTP or malachite green phosphate detection

  • Measure ribosome-stimulated GTPase activity with purified ribosomes

  • Establish kinetic parameters (Km, kcat) for both native and recombinant proteins

  • Include negative controls with GTPase-deficient IF-2 variants (e.g., mutations in the G-domain)

• Ribosome binding studies:

  • Filter binding assays with labeled ribosomes or labeled IF-2

  • Surface plasmon resonance to determine binding kinetics

  • Sucrose gradient centrifugation to assess 30S initiation complex formation

  • Competition assays between native and recombinant IF-2

• In vitro translation systems:

  • Reconstituted translation systems depleted of endogenous IF-2

  • Complementation with either native or recombinant IF-2

  • Quantify translation of reporter constructs (luciferase, GFP)

  • Dose-response curves to determine relative activities

• Differential labeling strategies:

  • Isotope labeling (15N, 13C) of recombinant protein for NMR studies

  • Fluorescent tagging at non-essential sites for microscopy and FRET assays

  • Mass spectrometry differentiation through incorporation of heavy isotopes

For all functional comparisons, it's critical to ensure equivalent active concentrations of proteins, which can be determined through active site titration assays using fluorescent GTP analogs or by measuring stoichiometric binding to ribosomes.

How should researchers design experiments to study infB gene regulation in K. pneumoniae?

To effectively study infB gene regulation in K. pneumoniae, researchers should implement:

• Promoter mapping and analysis:

  • 5' RACE to identify transcription start sites

  • Reporter gene fusions (lacZ, gfp) to quantify promoter activity

  • Deletion and mutation analysis of promoter elements

  • ChIP-seq to identify transcription factor binding sites

• Transcriptional regulation studies:

  • RNA-seq under various growth and stress conditions

  • qRT-PCR to validate expression changes

  • Northern blotting to identify operon structure and potential processing

  • Analysis of stringent response elements and their role in regulation

• Post-transcriptional regulation investigation:

  • Ribosome profiling to measure translation efficiency

  • RNA structure probing of 5' UTR (SHAPE, DMS-seq)

  • Identification of potential small RNAs affecting infB expression

  • Assessment of mRNA stability under stress conditions

• Integrative approaches:

  • Correlation of infB expression with virulence traits

  • Comparison between different K. pneumoniae pathotypes

  • Examination of infB regulation in antibiotic-resistant isolates

  • Global regulatory network mapping using systems biology approaches

The genetic context of infB in K. pneumoniae likely influences its expression patterns during infection and stress responses. Understanding these regulatory mechanisms could provide insights into how translation machinery adapts during pathogenesis and antibiotic exposure.

What are the methodological considerations for studying sequence variations in infB genes from clinical K. pneumoniae isolates?

Investigating infB sequence variations in clinical K. pneumoniae isolates requires rigorous methodology:

• Sample collection and processing:

  • Diverse sampling from different infection sites, geographical regions, and patient populations

  • Serial isolates from persistent infections to track evolutionary changes

  • Proper documentation of antibiotic treatment history and clinical outcomes

  • Consideration of within-patient genetic diversity as observed in clinical studies

• Sequencing approach selection:

  • Sanger sequencing for targeted infB gene analysis

  • Whole genome sequencing for contextual genomic information

  • Deep sequencing to identify minor variants within populations

  • Long-read sequencing to resolve complex structural variations

• Bioinformatic analysis pipeline:

  • Quality filtering and trimming of sequence data

  • Alignment against reference infB sequences

  • Variant calling with appropriate confidence thresholds

  • Annotation of functional impacts of amino acid substitutions

• Validation and functional assessment:

  • PCR verification of key variants

  • Recombinant expression of variant IF-2 proteins

  • Functional comparison of variant proteins

  • Complementation studies in appropriate genetic backgrounds

Researchers should pay particular attention to sequence variations between different clonal complexes and sequence types, especially comparing ST258 (the predominant clone in many regions) with emerging non-CC258 sequence types that show increasing prevalence in clinical settings .

What experimental approaches can determine the impact of mobile genetic elements on infB gene function in K. pneumoniae?

To investigate how mobile genetic elements affect infB gene function in K. pneumoniae, researchers should employ:

• Genomic context analysis:

  • Whole genome sequencing to identify insertion sequences, transposons, or genomic islands near infB

  • Comparative genomics across multiple strains to identify variable regions

  • Analysis of sequence anomalies indicating horizontal gene transfer

  • Examination of specialized transduction events involving infB

• Functional genomics approaches:

  • Transcriptome analysis to detect altered expression due to mobile element insertion

  • Transposon mutagenesis to identify regulatory elements

  • CRISPR-Cas9 editing to remove or modify mobile elements

  • Reporter systems to monitor effects on gene expression

• Molecular characterization of recombination events:

  • PCR mapping of genomic islands and insertion sites

  • Long-read sequencing to resolve complex structural arrangements

  • Analysis of transposon platforms similar to Tn4401b and Tn6454 observed in K. pneumoniae

  • Characterization of plasmid-mediated transfer of infB variants

• Evolutionary analysis:

  • Phylogenetic reconstruction of infB evolution across K. pneumoniae lineages

  • Molecular clock analysis to date acquisition events

  • Selective pressure analysis (dN/dS ratios) to identify adaptive evolution

  • Correlation with antibiotic resistance acquisition timelines

Recent studies have shown that K. pneumoniae readily exchanges DNA with other members of the human microbiome and acquires mobile genetic elements carrying resistance and virulence genes . Understanding how these processes might affect translation factors like IF-2 could reveal mechanisms of adaptation during infection.

How does IF-2 function contribute to K. pneumoniae virulence during infection?

The potential contributions of IF-2 to K. pneumoniae virulence involve several mechanisms:

• Stress adaptation during infection:

  • Modulation of translation initiation efficiency under host-imposed stresses

  • Selective translation of virulence factors and stress response proteins

  • Maintenance of protein synthesis under nutrient limitation

  • Adaptation to temperature shifts between environment and host

• Pathotype-specific translation regulation:

  • Differential expression or activity between classical and hypervirulent strains

  • Potential role in translating hypervirulence-associated transcripts

  • Contribution to growth rate differences between pathotypes

  • Possible involvement in capsule production regulation

• Immune evasion mechanisms:

  • Role in translating proteins involved in complement resistance

  • Support for rapid adaptation to macrophage and neutrophil encounters

  • Contribution to translation during phagosome residence

  • Potential role in biofilm formation through selective translation

• Antibiotic stress responses:

  • Altered translation initiation under antibiotic exposure

  • Role in expressing resistance determinants

  • Recovery of translation after antibiotic-induced stress

  • Potential modifications affecting ribosome-targeting antibiotics

K. pneumoniae pathogenesis depends heavily on interactions between the bacterium and host immune defenses, including complement, macrophages, neutrophils, and monocytes . Translation machinery components like IF-2 likely play crucial roles in adapting to these host defenses and supporting the expression of virulence factors during infection.

What experimental models are most appropriate for studying IF-2 function during K. pneumoniae infection?

Selecting appropriate experimental models for studying IF-2 during infection requires consideration of various factors:

• In vitro cellular models:

  • Human lung epithelial cell lines for pneumonia models

  • Human bladder epithelial cells for urinary tract infection studies

  • Macrophage cell lines (THP-1, RAW264.7) for phagocytosis studies

  • Primary neutrophils for investigating bacterial survival

• Ex vivo tissue models:

  • Precision-cut lung slices maintaining 3D architecture

  • Urinary tract epithelium explants

  • Human intestinal organoids for colonization studies

  • Whole blood assays for sepsis models

• Animal infection models:

  • Mouse pneumonia models via intranasal infection

  • Urinary tract infection models via transurethral instillation

  • Liver abscess models for hypervirulent strains

  • Galleria mellonella (wax moth) for high-throughput screening

• Specialized approaches for studying translation:

  • Ribosome profiling from infected tissues

  • Fluorescent reporters for translation monitoring in vivo

  • Selective capture of translated mRNAs (TRAP-seq)

  • Isotope labeling to track newly synthesized proteins

Each model has advantages for specific aspects of K. pneumoniae pathogenesis. For studying respiratory infections, models should reflect the interaction with respiratory epithelia, where K. pneumoniae fimbrial types play important roles in adherence . For hypervirulent strains, liver abscess models may be most relevant, while classical strains might be better studied in urinary tract or lung models.

How can researchers investigate potential interactions between host immune factors and bacterial IF-2 during K. pneumoniae infection?

To investigate host immune factor interactions with K. pneumoniae IF-2 during infection, researchers should employ:

• Protein-protein interaction studies:

  • Co-immunoprecipitation of IF-2 from infected cells or tissues

  • Yeast two-hybrid screening against host factor libraries

  • Proximity labeling approaches (BioID, APEX) in infection models

  • Surface plasmon resonance with purified host factors and IF-2

• Localization studies:

  • Immunofluorescence microscopy of IF-2 during infection

  • Live-cell imaging with fluorescently tagged IF-2

  • Electron microscopy with immunogold labeling

  • Subcellular fractionation of infected cells

• Functional interaction assays:

  • Transfection of host cells with IF-2 to identify cellular responses

  • Screening for host factors affecting recombinant IF-2 activity

  • Competition assays with host translation machinery

  • Effect of host antimicrobial peptides on IF-2 function

• Immunological approaches:

  • Analysis of antibody responses to IF-2 during infection

  • T cell epitope mapping of IF-2

  • Cytokine responses to purified IF-2

  • Inflammasome activation studies

These approaches could reveal whether IF-2 is recognized by host pattern recognition receptors or whether host defense mechanisms specifically target bacterial translation machinery. K. pneumoniae interacts with various components of the innate immune system , and understanding how translation factors participate in these interactions could provide new insights into pathogenesis.

What are the methodological challenges in studying IF-2 conformational changes during translation initiation?

Investigating IF-2 conformational dynamics presents significant technical challenges:

• Structural biology approaches and limitations:

  • X-ray crystallography challenges: Capturing discrete conformational states requires stabilization with non-hydrolyzable GTP analogs and crystallization chaperones

  • Cryo-EM considerations: Large size and flexibility make particle classification complex; preferably studied in complex with ribosomes

  • NMR spectroscopy limitations: Full-length IF-2 exceeds size limits; domain-based approach necessary

• Real-time conformational monitoring techniques:

  • Single-molecule FRET to track domain movements during GTP hydrolysis

  • Time-resolved small-angle X-ray scattering for solution-phase conformational transitions

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Optical tweezers or atomic force microscopy for force-extension measurements

• Computational approaches:

  • Molecular dynamics simulations of conformational transitions

  • Enhanced sampling methods to overcome energy barriers

  • Coarse-grained modeling of large-scale conformational changes

  • Integration of experimental constraints with simulation data

• Experimental design considerations:

  • Time-resolved measurements synchronized with GTP hydrolysis

  • Stabilization of intermediate states with modified nucleotides

  • Strategic placement of fluorescent or spin labels at domain interfaces

  • Parallel analysis of wild-type and mutant proteins with altered conformational dynamics

These methodological challenges are significant but addressing them would provide valuable insights into how K. pneumoniae IF-2 functions mechanistically and potentially reveal species-specific features that could be exploited for targeted interventions.

How can multi-omics approaches be integrated to understand IF-2's role in K. pneumoniae adaptation during infection?

Integrating multi-omics approaches for studying IF-2 in K. pneumoniae adaptation requires systematic methodology:

• Comprehensive data collection strategy:

  • Genomics: WGS of isolates from various infection stages

  • Transcriptomics: RNA-seq under infection-relevant conditions

  • Proteomics: Global protein expression and post-translational modifications

  • Translatome: Ribosome profiling to capture translation dynamics

  • Metabolomics: Metabolic changes associated with translation regulation

• Integration frameworks:

  • Correlation networks linking genomic variants to expression changes

  • Pathway enrichment across multiple data types

  • Machine learning approaches to identify patterns across datasets

  • Mathematical modeling of translation regulation in response to stress

• Experimental validation pipeline:

  • Targeted mutagenesis of identified regulatory elements

  • Complementation studies with variant IF-2 proteins

  • Reporter systems monitoring translation of key transcripts

  • Time-course studies during infection progression

• Clinical correlation approaches:

  • Analysis of within-patient diversity as observed in clinical studies

  • Correlation with antibiotic resistance profiles

  • Association with treatment outcomes and infection persistence

  • Comparison across different infection sites

A key consideration is analyzing the data in the context of K. pneumoniae diversity, as studies have shown significant genetic diversity within patients, including multiple unrelated clones with different sequence types and resistance profiles . This complexity requires carefully designed sampling strategies and sophisticated computational approaches for data integration.

What experimental designs are most appropriate for evaluating IF-2 as a potential drug target against multidrug-resistant K. pneumoniae?

Evaluating IF-2 as a drug target against multidrug-resistant K. pneumoniae requires systematic experimental approaches:

• Target validation strategy:

  • Conditional knockdown systems to demonstrate essentiality

  • Complementation studies with resistant mutations to identify mechanism

  • Comparative analysis across diverse clinical isolates

  • Assessment of impact on virulence in animal models

• High-throughput screening approaches:

  • GTPase activity assays adapted to microplate format

  • Fragment-based screening with differential scanning fluorimetry

  • Structure-based virtual screening against binding pockets

  • Phenotypic screens with reporter strains

• Compound evaluation framework:

  • Determination of minimum inhibitory concentrations (MICs)

  • Time-kill kinetics against different K. pneumoniae strains

  • Resistance development assessment through serial passage

  • Cytotoxicity testing in mammalian cell lines

• Advanced drug development considerations:

  • Structure-activity relationship studies for lead optimization

  • In vitro ADME profiling (absorption, distribution, metabolism, excretion)

  • Animal pharmacokinetics and efficacy studies

  • Combination testing with existing antibiotics

• Testing against diverse strains:

  • Evaluation across major lineages, including ST258 and emerging sequence types

  • Assessment against hypervirulent and classical strains

  • Testing against isolates with different resistance mechanisms

  • Efficacy against strains harboring different mobile genetic elements

This experimental framework should be designed as a 2×3×4 factorial approach , where multiple variables (strain type, compound concentration, exposure time) can be systematically evaluated to develop a comprehensive understanding of compound efficacy against the diversity of K. pneumoniae strains encountered in clinical settings.