Recombinant Klebsiella pneumoniae subsp. pneumoniae Urocanate hydratase (hutU), partial

Advanced Research Questions

• What are the challenges in expressing recombinant K. pneumoniae hutU in different host systems?

Expression of recombinant K. pneumoniae hutU presents several host-specific challenges that require tailored strategies:

When expressing related proteins such as urocanate reductase from Shewanella oneidensis, researchers observed significant reduction in cell growth and partial cell lysis , suggesting careful optimization is necessary when expressing hutU from K. pneumoniae.

The expression system selection should be guided by research requirements, with E. coli typically providing highest yields but potentially requiring extensive optimization to maintain protein solubility and activity.

• How can interdomain interactions in K. pneumoniae hutU be experimentally characterized?

Interdomain interactions in K. pneumoniae hutU can be systematically characterized through multiple complementary approaches:

This multi-method approach can reveal how domain interactions influence:

• NAD+ binding and retention

• Substrate recognition and binding

• Catalytic activity and efficiency

• Conformational dynamics during the catalytic cycle

Similar approaches with K. pneumoniae FimH revealed how interdomain interactions create a conformational equilibrium between low-affinity and high-affinity states , and could provide equivalent insights into hutU function.

• What strategies can optimize the purification of recombinant K. pneumoniae hutU while maintaining enzymatic activity?

Optimizing purification of enzymatically active recombinant K. pneumoniae hutU requires careful consideration of multiple factors:

• Affinity tag selection and placement:

  • N-terminal vs. C-terminal His6-tag positioning based on structural predictions

  • Alternative tags (GST, MBP) for enhanced solubility and affinity purification

  • TEV or PreScission protease cleavage sites for tag removal

  • Dual-tag strategies for sequential purification steps

• Buffer optimization:

  • pH range testing (typically 7.0-8.0) for stability

  • Salt concentration adjustment (100-500 mM NaCl) to prevent aggregation

  • NAD+ supplementation (50-200 μM) to maintain cofactor occupancy

  • Addition of stabilizing agents:

    • Glycerol (10-20%)

    • Reducing agents (1-5 mM DTT or TCEP)

    • Protease inhibitors (PMSF, EDTA-free cocktail)

• Chromatography strategy:

  • Initial capture: IMAC with optimized imidazole gradients (20-250 mM)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography in activity-preserving buffer

  • Activity verification at each stage

• Storage conditions:

  • Concentration optimization (typically 1-5 mg/ml)

  • Stabilizing additives for freezing (10% glycerol)

  • Flash-freezing in liquid nitrogen vs. slow cooling

  • Short-term (4°C) vs. long-term (-80°C) storage evaluation

Throughout purification, monitor NAD+ retention using absorbance at 340 nm and verify enzymatic activity through urocanate conversion assays to ensure the purification process yields functionally relevant protein.

• How does temperature affect hutU expression and activity in K. pneumoniae, and what are the experimental approaches to investigate this relationship?

Temperature effects on hutU expression and activity can be systematically investigated through multiple experimental approaches:

• Expression analysis techniques:

  • qRT-PCR measurement of hutU transcript levels across temperature range (4-37°C)

  • Reporter gene fusions (hutU promoter-lacZ) to quantify temperature-responsive expression

  • Western blot analysis of hutU protein levels at various temperatures

  • Primer extension and 5' RACE to identify temperature-dependent promoter usage

• Enzymatic characterization across temperatures:

  • Determination of temperature-activity profiles (5-50°C)

  • Arrhenius plot analysis to calculate activation energy

  • Thermal stability assessments through activity retention after heat exposure

  • Differential scanning fluorimetry to determine melting temperatures

  • Comparison of kinetic parameters (Km, kcat) at different temperatures

• Structural adaptation assessment:

  • Circular dichroism spectroscopy to monitor secondary structure changes

  • Intrinsic fluorescence measurement of tertiary structure alterations

  • Hydrogen-deuterium exchange rates at varying temperatures

Studies in Pseudomonas syringae demonstrated 10-14 fold higher hutU expression at 4°C compared to 22°C , suggesting temperature-responsive regulation. Similar methodologies applied to K. pneumoniae would reveal whether its hutU exhibits comparable temperature-dependent expression and activity profiles, potentially informing the bacterium's ability to adapt to different environmental niches.

• What are the methodological approaches for using recombinant K. pneumoniae hutU in vaccine development?

Utilizing recombinant K. pneumoniae hutU in vaccine development requires systematic investigation through the following methodological pipeline:

• Antigen preparation strategies:

  • Full-length protein vs. immunogenic fragment identification

  • Expression and purification optimization for GMP-compatible processes

  • Stability and homogeneity assessment under storage conditions

  • Endotoxin removal validation for in vivo testing

• Immunogenicity assessment pipeline:

  • Epitope mapping through peptide arrays or phage display

  • B-cell epitope prediction algorithms validation

  • T-cell epitope identification through MHC-binding assays

  • In vitro dendritic cell activation and antigen presentation studies

• Formulation optimization:

  • Adjuvant screening (alum, oil-in-water, TLR ligands)

  • Delivery platform evaluation (soluble protein, nanoparticles, virus-like particles)

  • Dose-response studies to determine optimal antigen amount

  • Stability studies under various storage conditions

• Preclinical evaluation protocol:

  • Animal model selection based on K. pneumoniae infection susceptibility

  • Immunization schedule optimization (prime-boost regimens)

  • Challenge studies with relevant clinical isolates

  • Correlates of protection identification (antibody titers, cellular responses)

• Safety and efficacy metrics:

  • Toxicity assessment in appropriate animal models

  • Cross-reactivity testing with human proteins

  • Duration of protective immunity evaluation

  • Cross-protection against diverse K. pneumoniae strains

This systematic approach leverages recombinant K. pneumoniae hutU protein's potential as a vaccine antigen candidate against this increasingly antibiotic-resistant pathogen identified by the CDC as a pathogen of urgent concern .

• What experimental designs are most effective for investigating hutU's role in K. pneumoniae virulence and pathogenesis?

To rigorously investigate hutU's potential role in K. pneumoniae virulence and pathogenesis, researchers should implement multifaceted experimental designs:

• Genetic manipulation approaches:

  • Generation of precise hutU deletion mutants using allelic exchange

  • Complementation with wild-type and mutant hutU alleles

  • Construction of conditional expression systems

  • CRISPR interference for controlled hutU downregulation

  • Site-directed mutagenesis of catalytic residues to separate enzymatic from structural roles

• In vitro virulence phenotype assessment:

  • Growth kinetics in standard and histidine-limited media

  • Biofilm formation quantification

  • Stress resistance profiling (oxidative, acid, antimicrobial)

  • Comparative proteomic and transcriptomic analysis of WT vs. hutU mutants

  • Metabolomic profiling to identify pathway alterations

• Host-pathogen interaction models:

  • Adhesion and invasion assays with relevant cell lines

  • Macrophage survival and replication studies

  • Neutrophil killing resistance assessment

  • Cytokine induction profiling

• In vivo infection models:

  • Murine urinary tract infection model (reflecting K. pneumoniae's role in UTIs)

  • Pulmonary infection model

  • Galleria mellonella invertebrate model for high-throughput screening

  • Competition assays between wild-type and hutU mutants in vivo

  • Tissue burden and histopathological analysis

These approaches would establish whether hutU contributes to virulence directly through metabolic adaptation during infection or indirectly through regulatory effects on other virulence factors, potentially identifying new therapeutic targets against this increasingly antibiotic-resistant pathogen .

• How can comparative genomics approaches be applied to study hutU variation across K. pneumoniae clinical isolates?

Comparative genomics approaches to study hutU variation across K. pneumoniae clinical isolates should employ a systematic workflow:

• Sequence acquisition and analysis pipeline:

  • Collection of diverse clinical isolates from different infection sites

  • Whole genome sequencing using short-read (Illumina) and long-read (PacBio, Nanopore) technologies

  • Assembly and annotation with specialized bacterial genome pipelines

  • hutU gene and operon structure identification across isolates

  • Promoter region and regulatory element comparative analysis

• Variation characterization methodologies:

  • Single nucleotide polymorphism (SNP) identification in coding and regulatory regions

  • Insertion/deletion (indel) detection and functional impact prediction

  • Selection pressure analysis using dN/dS ratios

  • Identification of recombination events and horizontal gene transfer

  • Correlating sequence variations with isolation source (UTI, pneumonia, bloodstream)

• Structure-function correlation approaches:

  • Mapping variations onto protein structural models

  • Predicting functional impacts using computational tools

  • Experimental validation of variant effects on enzyme activity

  • Assessment of temperature adaptation signatures in variants

• Clinical correlation analysis:

  • Association studies linking hutU variants to antibiotic resistance profiles

  • Connection between hutU variants and clinical outcomes

  • Analysis of hutU conservation in hypervirulent K. pneumoniae lineages

Similar approaches with other K. pneumoniae virulence factors like FimH have revealed that variants from different patient isolates can shift proteins into different functional states, potentially favoring persistence in specific environments like the urinary tract . Such methodologies could reveal whether hutU undergoes similar adaptive variations with clinical significance.

• What techniques can characterize the interaction between hutU and other components of the histidine utilization pathway in K. pneumoniae?

Characterizing interactions between hutU and other histidine utilization pathway components requires a comprehensive experimental approach:

• Physical interaction analysis methods:

  • Co-immunoprecipitation with tagged hutU protein

  • Bacterial two-hybrid screening for protein-protein interactions

  • Pull-down assays with purified recombinant proteins

  • Surface plasmon resonance for quantitative binding kinetics

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction validation

• Functional cooperation assessment techniques:

  • Metabolic flux analysis using isotope-labeled histidine

  • Enzyme activity assays with combined pathway components

  • Substrate channeling investigation using transient kinetics

  • Metabolite profiling in wild-type vs. pathway mutants

• Regulatory interaction characterization:

  • Chromatin immunoprecipitation to identify regulatory protein binding

  • Electrophoretic mobility shift assays for DNA-protein interactions

  • Reporter fusion assays to monitor coordinated expression

  • Comparative transcriptomics across environmental conditions

• Structural biology approaches:

  • Co-crystallization of hutU with interacting proteins

  • Cryo-EM analysis of multi-enzyme complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Integrative structural modeling combining multiple data sources

These methodological approaches would reveal whether hutU functions independently or as part of a multi-enzyme complex, potentially facilitating substrate channeling for efficient histidine catabolism. Such enzyme-enzyme interactions could represent potential targets for disrupting K. pneumoniae metabolism during infection.

• What are the challenges and solutions in structural studies of K. pneumoniae hutU?

Structural studies of K. pneumoniae hutU face significant challenges that require specialized methodological solutions:

Integrative structural biology approaches combining multiple techniques (X-ray crystallography, cryo-EM, SAXS, HDX-MS, computational modeling) provide the most promising route to understanding the structural basis of hutU function. Recent advances in AlphaFold and related prediction tools can also provide initial structural models to guide experimental design.

• What methodological approaches can optimize the use of recombinant K. pneumoniae hutU in drug discovery against antibiotic-resistant strains?

Optimizing recombinant K. pneumoniae hutU for drug discovery against antibiotic-resistant strains requires a systematic methodology:

• Target validation strategies:

  • Genetic essentiality testing in infection-relevant conditions

  • Chemical validation using known inhibitors of related enzymes

  • Transposon sequencing to determine fitness contributions

  • Metabolic bypass analysis to identify potential resistance mechanisms

• Assay development for screening:

  • Primary high-throughput spectrophotometric assays monitoring urocanate conversion

  • Secondary orthogonal assays measuring NAD+ involvement

  • Counter-screening against human homologs to assess selectivity

  • Cellular assays measuring growth inhibition in hutU-dependent conditions

  • Thermal shift assays for ligand binding detection

• Structure-based drug design approaches:

  • Fragment-based screening using crystallography or NMR

  • Virtual screening against hutU active site and allosteric pockets

  • Molecular dynamics simulations to identify transient binding pockets

  • Rational design targeting the unique NAD+ electrophilic mechanism

• Lead optimization workflow:

  • Medicinal chemistry focused on enhancing potency and selectivity

  • ADME-Tox property optimization

  • Structure-activity relationship development

  • Resistance development assessment through serial passage

• Translational research considerations:

  • In vivo efficacy testing in K. pneumoniae infection models

  • Combination studies with existing antibiotics

  • Activity assessment against clinical isolate panels

  • PK/PD relationship determination for dosing strategies

This methodological framework leverages hutU as a potential novel target against K. pneumoniae, recognized by the CDC as a pathogen of urgent concern due to increasing multidrug resistance , potentially providing alternative therapeutic strategies beyond conventional antibiotics.