Recombinant Cronobacter sakazakii UPF0266 membrane protein ESA_01432 (ESA_01432)

How is UPF0266 membrane protein characterized in experimental settings?

The UPF0266 membrane protein is typically characterized through membrane protein extraction protocols utilizing detergent-based methods. For experimental work, researchers commonly employ Triton X-114 for membrane protein extraction, as seen in studies of C. sakazakii membrane proteins . The extraction process typically involves:

• Cell culture and harvesting (approximately 2g wet weight)

• Suspension in phosphate buffer saline (PBS, pH 7.4) at 4°C

• Sonication in ice bath to disrupt cells

• Phase separation using Triton X-114

• Acetone precipitation to remove excess salts

• Protein dissolution in appropriate lysis buffer (e.g., 5 M urea, 2 M thiourea, 2% SB3-10, 2% CHAPS, 65 mM DTT, 40 mM Tris)

• Quantification using bicinchoninic acid (BCA) assay

For recombinant protein work, the protein is typically stored in Tris-based buffer with 50% glycerol at -20°C, with extended storage recommended at -80°C to maintain stability .

What is known about the biological function of UPF0266 membrane protein in Cronobacter sakazakii?

• Bacterial adhesion to and invasion of human cells

• Biofilm formation

• Flagellar motility

• Virulence mechanisms

Comparative proteomic studies between virulent and attenuated C. sakazakii strains have identified several membrane proteins with differential expression that contribute to virulence differences, though ESA_01432 specifically has not been thoroughly characterized in this context . Future research utilizing gene knockout studies, complementation assays, and protein-protein interaction analyses would greatly enhance our understanding of this protein's specific functions.

What are recommended protocols for recombinant expression of UPF0266 membrane protein?

For successful recombinant expression of UPF0266 membrane protein ESA_01432, the following methodological approach is recommended:

Expression System Selection:

• E. coli-based expression systems are commonly used for recombinant membrane proteins

• BL21(DE3) or C41/C43(DE3) strains are preferred for membrane protein expression

• Consider using pET-based vectors with inducible promoters

Optimization Parameters:

• IPTG concentration: 0.1-1.0 mM (typically start with 0.5 mM)

• Induction temperature: 16-30°C (lower temperatures often improve membrane protein folding)

• Induction time: 4-16 hours

• Media supplements: Consider adding glycerol (0.5-1%) to stabilize membrane proteins

Extraction Protocol:
The extraction should follow specialized membrane protein protocols using appropriate detergents. The specific characteristics of UPF0266 membrane protein (152 amino acids with transmembrane domains) necessitate careful selection of solubilization conditions .

For validation of successful expression, Western blotting using antibodies against the tag incorporated during production is recommended, as the tag type is determined during the production process for this particular protein .

How can researchers effectively analyze UPF0266 membrane protein in comparative proteomic studies?

Two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry has proven effective for comparative analysis of membrane proteins in C. sakazakii. The following methodology is recommended based on established protocols:

Sample Preparation:

• Extract membrane proteins using Triton X-114 phase partitioning

• Precipitate proteins with acetone to remove contaminants

• Resuspend in appropriate lysis buffer (5 M urea, 2 M thiourea, 2% SB3-10, 2% CHAPS, 65 mM DTT, 40 mM Tris)

• Quantify using BCA assay

2-DE Analysis:

• First dimension: Isoelectric focusing using pH 3-10 IPG strips

• Second dimension: SDS-PAGE

• Stain gels with Coomassie blue or silver stain

• Analyze using image analysis software to identify >2-fold changes in protein abundance

Protein Identification:

• Excise spots of interest

• Perform in-gel tryptic digestion

• Analyze peptides using MALDI-TOF/TOF MS

• Search databases (MASCOT) to identify proteins

Validation:
Validate differential expression using RT-PCR for the gene encoding the protein, as demonstrated in studies of other membrane proteins in C. sakazakii .

What approaches can be used to determine the topology and membrane insertion of UPF0266 membrane protein?

Determining the membrane topology of UPF0266 membrane protein requires a multi-faceted approach:

Computational Prediction:

• Use transmembrane prediction algorithms (TMHMM, HMMTOP, Phobius)

• Apply hydropathy analysis to identify hydrophobic regions

• Predict signal peptides using SignalP

Experimental Validation:

• PhoA/LacZ fusion approach: Create fusion proteins at various positions and determine periplasmic/cytoplasmic localization

• Cysteine scanning mutagenesis: Introduce cysteines at various positions and test accessibility to membrane-impermeable reagents

• Protease protection assay: Expose membrane vesicles to proteases to determine exposed regions

• Fluorescence spectroscopy: Utilize environmentally sensitive fluorophores to probe membrane interfaces

Based on the amino acid sequence provided , UPF0266 membrane protein ESA_01432 appears to have multiple hydrophobic regions consistent with transmembrane segments, suggesting a multi-pass membrane protein topology.

What experimental approaches are recommended for studying protein-protein interactions involving UPF0266 membrane protein?

For investigating protein-protein interactions of UPF0266 membrane protein ESA_01432, the following methodological approaches are recommended:

In vitro methods:

• Pull-down assays: Using tagged recombinant UPF0266 protein to identify binding partners

• Co-immunoprecipitation: Using antibodies against UPF0266 to precipitate protein complexes

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry to identify proximal proteins

In vivo methods:

• Bacterial two-hybrid system: Modified for membrane proteins

• FRET/BRET: For detecting interactions in live bacterial cells

• Split-GFP complementation: To visualize interaction locations

Computational approaches:

• Protein-protein interaction prediction: Using algorithms to identify potential interacting partners

• Co-expression network analysis: Identifying genes with similar expression patterns

Given the importance of membrane proteins in C. sakazakii virulence , identifying interaction partners of UPF0266 membrane protein could provide valuable insights into its functional role in bacterial pathogenesis.

What is the relationship between UPF0266 membrane protein expression and growth conditions that promote Cronobacter sakazakii virulence?

The expression of membrane proteins in C. sakazakii can be significantly influenced by environmental conditions that also affect virulence. Research should investigate UPF0266 membrane protein expression under the following conditions:

Temperature effects:
C. sakazakii shows different growth rates at 22°C versus 35°C, with more rapid growth at the higher temperature . Studies should examine UPF0266 membrane protein expression at:

• Room temperature (22°C) - representing environmental conditions

• Body temperature (35-37°C) - representing host conditions

Growth phase influence:
Expression should be monitored across:

• Lag phase

• Exponential growth phase

• Stationary phase

Nutrient conditions:

• Growth in powdered infant formula (PIF) versus laboratory media

• Low iron conditions (mimicking host environments)

• Varying pH levels

Based on studies of C. sakazakii growth dynamics, the bacterium exhibits different generation times depending on temperature (GT of 0.67h at 22°C versus 0.41h at 35°C) . These conditions should be replicated when studying UPF0266 membrane protein expression, as they may regulate virulence factor production.

How can UPF0266 membrane protein research contribute to understanding Cronobacter sakazakii pathogenesis in infants?

C. sakazakii is particularly dangerous for infants under 2 months old who consume contaminated powdered infant formula (PIF), with reported mortality rates of 42-80% for neonatal meningitis and 15-25% for septicemia 3. Research on UPF0266 membrane protein can contribute to pathogenesis understanding through:

• Host-pathogen interaction studies: Investigating whether UPF0266 protein participates in adhesion to or invasion of intestinal epithelial cells or blood-brain barrier penetration

• Immune response analysis: Determining if UPF0266 protein elicits specific immune responses or participates in immune evasion

• Comparative genomics: Analyzing UPF0266 protein sequence conservation across various clinical isolates, particularly those associated with invasive disease like CC4 (clonal complex 4) strains that are associated with neonatal meningitis

• Animal model studies: Using appropriate animal models to assess the contribution of UPF0266 protein to in vivo virulence

Such research could help explain why certain strains of C. sakazakii, particularly ST-4/CC4, have enhanced ability to cause invasive disease in infants .

What methodological approaches are recommended for studying the immunogenicity of UPF0266 membrane protein?

To evaluate the immunogenicity of UPF0266 membrane protein ESA_01432, researchers should consider the following methodological approaches:

In vitro studies:

• Stimulation of immune cells: Expose purified recombinant UPF0266 protein to:

  • Human peripheral blood mononuclear cells (PBMCs)

  • Dendritic cells

  • Macrophage cell lines

• Cytokine profiling: Measure production of:

  • Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α)

  • Anti-inflammatory cytokines (IL-10)

  • Chemokines

• Pattern recognition receptor (PRR) activation: Determine which receptors (TLRs, NLRs) recognize the protein

In vivo studies:

• Immunization studies: Administer purified protein with appropriate adjuvants

• Antibody response characterization: Measure antibody titers, isotypes, and neutralizing capacity

• T-cell response analysis: Evaluate T-cell proliferation and cytokine production in response to protein stimulation

Bioinformatic approaches:

• Epitope prediction: Identify potential B-cell and T-cell epitopes within the protein sequence

• Cross-reactivity analysis: Assess similarity to host proteins to predict autoimmunity risk

This research could potentially identify UPF0266 membrane protein as an antigen for diagnostic or vaccine development purposes.

How does the expression of UPF0266 membrane protein compare between clinical and environmental isolates of Cronobacter sakazakii?

Comparing UPF0266 membrane protein expression between clinical and environmental isolates requires a comprehensive approach:

Sample collection strategy:

• Clinical isolates from:

  • Patients with invasive disease (meningitis, septicemia)

  • Patients with non-invasive disease (urinary tract infections, diarrhea)

• Environmental isolates from:

  • Powdered infant formula

  • Manufacturing environments

  • Domestic environments (feeding bottles, preparation utensils)

Analytical methods:

• Proteomic analysis:

  • 2-DE coupled with mass spectrometry, as successfully used for comparing membrane proteins between virulent and attenuated C. sakazakii strains

  • Label-free quantitative proteomics

• Transcriptomic analysis:

  • RT-PCR to measure gene expression levels

  • RNA-Seq for genome-wide expression analysis

• Genetic analysis:

  • Sequencing of the UPF0266 gene region to identify polymorphisms

  • Promoter analysis to identify regulatory differences

This research would help determine whether UPF0266 membrane protein expression correlates with source of isolation and virulence potential, similar to other membrane proteins like OmpA and OmpX that show differential expression between virulent and attenuated strains .

What structural biology approaches would advance understanding of UPF0266 membrane protein function?

Advancing structural understanding of UPF0266 membrane protein requires specialized approaches for membrane protein structural biology:

X-ray crystallography approach:

• Optimize expression with fusion partners (e.g., T4 lysozyme) to enhance crystallization

• Screen detergents for optimal protein stability

• Utilize lipidic cubic phase (LCP) crystallization methods

• Consider antibody fragment co-crystallization to stabilize protein

Cryo-electron microscopy (cryo-EM):

• Reconstitute protein in nanodiscs or amphipols

• Optimize sample vitrification conditions

• Consider single-particle analysis for structure determination

NMR spectroscopy:

• Express isotope-labeled protein (13C, 15N)

• Optimize detergent micelles or bicelles for solution NMR

• Consider solid-state NMR for membrane-embedded protein

Computational approaches:

• Homology modeling based on structurally characterized proteins in the same family

• Molecular dynamics simulations to predict membrane interactions

• Deep learning-based structure prediction (AlphaFold, RoseTTAFold)

The amino acid sequence of UPF0266 membrane protein (152 residues) suggests it may be amenable to structural studies, potentially revealing functional domains and interaction surfaces.

How might UPF0266 membrane protein research contribute to novel antimicrobial strategies against Cronobacter sakazakii?

If UPF0266 membrane protein is confirmed to play a role in C. sakazakii virulence, it could become a target for novel antimicrobial strategies:

Potential therapeutic approaches:

• Small molecule inhibitors:

  • High-throughput screening of compound libraries

  • Structure-based drug design following structural determination

  • Peptidomimetic inhibitors targeting protein-protein interactions

• Antibody-based approaches:

  • Develop neutralizing antibodies targeting surface-exposed regions

  • Antibody-antibiotic conjugates for targeted delivery

• Vaccine development:

  • Evaluate recombinant UPF0266 protein as a vaccine antigen

  • Design peptide vaccines based on immunogenic epitopes

• Gene silencing strategies:

  • Antisense oligonucleotides targeting mRNA

  • CRISPR interference to suppress gene expression

Testing methodologies:

• In vitro growth inhibition assays

• Cell culture infection models

• Biofilm inhibition assays

• Animal models of C. sakazakii infection

Given the serious nature of C. sakazakii infections in infants, with mortality rates of 42-80% for neonatal meningitis , novel antimicrobial strategies targeting virulence factors like membrane proteins represent an important research direction.