Recombinant Salmonella paratyphi B Probable 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol flippase subunit ArnF (arnF)

What is the basic structure of Salmonella paratyphi B ArnF protein?

The Salmonella paratyphi B ArnF protein is a full-length protein consisting of 125 amino acids. The complete amino acid sequence is: MGVMWGLISVAIASLAQLSLGFAMMRLPSIAHPLAFISGLGAFNAATLALFAGLAGYLVSVFCWQKTLHTLALSKAYALLSLSYVLVWVASMLLPGLQGAFSLKAMLGVLCIMAGVMLIFlpars . Based on related flippase research, this protein likely contains multiple transmembrane helices as part of its structure, which is characteristic of membrane flippases .

What is the predicted function of the ArnF subunit in Salmonella paratyphi B?

The ArnF protein in Salmonella paratyphi B functions as a subunit of the 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol flippase. This protein complex is involved in membrane lipid translocation, specifically flipping 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol from the cytoplasmic side to the periplasmic side of the bacterial membrane . Similar to other flippases studied in different organisms, it likely plays a role in phospholipid asymmetry and membrane composition maintenance . This function may be critical for bacterial membrane integrity and potentially contributes to antimicrobial resistance mechanisms.

What are the synonyms and identifiers for this protein?

The protein is known by several names and identifiers:

• Gene name: arnF

• Synonyms: SPAB_00678, L-Ara4N-phosphoundecaprenol flippase subunit ArnF, Undecaprenyl phosphate-aminoarabinose flippase subunit ArnF

• UniProt ID: A9N5A8

What expression systems are recommended for producing recombinant Salmonella paratyphi B ArnF?

The recommended expression system for recombinant Salmonella paratyphi B ArnF is E. coli . For optimal expression, the protein should be expressed with an N-terminal His-tag, which facilitates later purification steps. When designing expression constructs, it's advisable to include the full-length protein (amino acids 1-125) to maintain complete structural and functional properties . Researchers should consider codon optimization for E. coli if expression levels are suboptimal, as bacterial codon usage may differ between species.

What are the optimal storage conditions for purified recombinant ArnF protein?

The purified recombinant ArnF protein should be stored according to the following protocol:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• For reconstituted protein, add 5-50% glycerol (final concentration) before long-term storage

• The recommended final concentration of glycerol is 50%

What is the recommended protocol for reconstituting lyophilized ArnF protein?

For optimal reconstitution of lyophilized ArnF protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (preferably 50%)

• Prepare small aliquots for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles which can degrade the protein

How can the purity of recombinant ArnF be validated in experimental settings?

The purity of recombinant ArnF protein should be validated using SDS-PAGE analysis. High-quality preparations should show a purity greater than 90% as determined by this method . For more sensitive analysis, researchers may employ additional techniques such as size exclusion chromatography or mass spectrometry. When working with membrane proteins like flippases, it's also important to verify proper folding, which can be assessed through circular dichroism spectroscopy or functional assays that test phospholipid flipping activity.

What methodologies can be used to study ArnF flippase activity in vitro?

To study ArnF flippase activity in vitro, researchers can adapt phospholipid uptake assays similar to those used for other flippases. One approach is to use fluorescently labeled phospholipids such as NBD-labeled phosphatidylserine (NBD-PS) to track internalization . The protocol would involve:

• Prepare liposomes or membrane vesicles containing the recombinant ArnF protein

• Incubate with fluorescently labeled substrate (60 μM NBD-labeled phospholipids)

• Measure internalization at various time points using fluorescence spectroscopy

• Compare with appropriate controls (vesicles lacking ArnF)

• Quantify uptake rates to determine flippase activity

This methodology can be adapted to specifically study the flipping of 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol, the native substrate of ArnF.

How might the structure-function relationship of ArnF be investigated using site-directed mutagenesis?

Investigation of the structure-function relationship of ArnF can be approached through systematic site-directed mutagenesis, focusing on:

• Conserved residues identification: Perform sequence alignment with other bacterial flippases to identify evolutionarily conserved amino acids

• Transmembrane domain analysis: Target residues within the predicted transmembrane regions that may be involved in substrate recognition

• Functional domain mapping: Mutate key residues in cytosolic domains that may participate in ATP binding or hydrolysis

• Activity assessment: Evaluate each mutant using phospholipid uptake assays to quantify changes in flippase activity

This approach is supported by research on related flippases where conserved glycine residues (such as G234 in Arabidopsis ALA3) have been shown to be essential for flippase function . Similar conserved residues in ArnF could be primary targets for mutagenesis studies.

What is the relationship between ArnF function and antimicrobial resistance in Salmonella paratyphi B?

The relationship between ArnF function and antimicrobial resistance in Salmonella paratyphi B likely involves modification of bacterial membrane components. As ArnF participates in the translocation of 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol, it may contribute to lipopolysaccharide (LPS) modifications that reduce the binding affinity of certain antimicrobials, particularly cationic antimicrobial peptides and polymyxins.

Research approaches to investigate this relationship could include:

• Gene knockout studies: Generate arnF deletion mutants and assess changes in antimicrobial susceptibility

• Complementation assays: Restore function in knockout strains to confirm specificity

• Membrane composition analysis: Compare lipid profiles between wild-type and mutant strains

• Antimicrobial susceptibility testing: Use standard methods like agar dilution with EUCAST breakpoints to determine minimum inhibitory concentrations (MICs)

• Whole genome sequencing (WGS): Analyze genomic context and potential co-expression patterns with other resistance genes

How does ArnF compare structurally and functionally with homologous proteins in other bacterial species?

Comparative analysis of ArnF across bacterial species reveals important evolutionary and functional insights:

Functional conservation analysis suggests that despite sequence variations, the core mechanism of phospholipid flipping is preserved across species. The additional amino acids in homologs like the Shewanella sediminis version may contribute to species-specific regulation or substrate specificity. Research approaches to explore these differences could include:

• Complementation studies in heterologous systems

• Chimeric protein construction to identify functional domains

• Structural modeling to predict species-specific substrate binding pockets

• Comparative assessment of flipping activity using standardized in vitro assays

What are effective strategies for optimizing recombinant ArnF expression yield and solubility?

Optimizing expression yield and solubility for recombinant ArnF, a membrane protein, requires special considerations:

• Expression vector selection: Use vectors with tunable promoters to control expression rate

• Growth conditions optimization:

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

  • Evaluate different induction points (OD600 0.4-0.8)

  • Vary inducer concentration (0.1-1.0 mM IPTG for T7-based systems)

• Solubilization strategies:

  • Test different detergents (DDM, LDAO, OG) for membrane protein extraction

  • Consider fusion partners that enhance solubility (MBP, SUMO, Trx)

  • Evaluate co-expression with chaperones (GroEL/ES, DnaK/J)

• Purification optimization:

  • Use immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Include low concentrations of detergent in all buffers

  • Consider size exclusion chromatography as a polishing step

If inclusion bodies form despite optimization, protocols for refolding membrane proteins from inclusion bodies can be implemented, though with typically lower yields of functional protein.

What analytical methods can be used to assess conformational changes in ArnF during substrate binding?

To assess conformational changes in ArnF during substrate binding, researchers can employ several biophysical techniques:

The choice of method depends on available equipment, protein quantity, and the specific research question being addressed.

How does ArnF function potentially relate to Salmonella paratyphi B virulence and infection mechanisms?

The function of ArnF in Salmonella paratyphi B may contribute to virulence through several mechanisms:

• Host immune evasion: By modifying the bacterial outer membrane lipid composition, ArnF activity may help the bacteria evade recognition by host immune surveillance mechanisms.

• Persistence mechanisms: ArnF-mediated membrane modifications may contribute to bacterial survival within host cells or environments, potentially relating to the phenomenon of asymptomatic carriage observed in clinical cases .

• Antimicrobial peptide resistance: Modification of lipopolysaccharide structure through ArnF activity may confer resistance to host-derived antimicrobial peptides, enhancing bacterial survival during infection.

Research approaches could include infection models using wild-type and arnF-deficient strains to compare colonization, persistence, and virulence. Clinical investigations could examine whether strain variations in the arnF gene correlate with disease severity or carrier state duration .

What experimental approaches could be used to evaluate ArnF as a potential therapeutic target?

To evaluate ArnF as a potential therapeutic target, researchers could employ these approaches:

• Target validation:

  • Gene knockout studies to confirm essentiality or significant attenuation of virulence

  • Conditional expression systems to demonstrate that ArnF inhibition is bactericidal or significantly affects pathogenicity

• High-throughput screening:

  • Development of activity-based assays suitable for screening compound libraries

  • Fluorescence-based lipid flipping assays adapted to microplate formats

• Structure-based drug design:

  • Obtaining crystal or cryo-EM structures of ArnF for in silico screening

  • Molecular docking studies to identify potential binding pockets

• Lead compound evaluation:

  • In vitro assessment of identified inhibitors against purified protein

  • Bacterial culture studies to confirm cellular activity

  • Cytotoxicity screening against mammalian cells to assess selectivity

  • Animal infection models to evaluate in vivo efficacy

• Resistance development assessment:

  • Serial passage experiments to determine the frequency of resistance emergence

  • Whole genome sequencing to identify resistance mechanisms

The clinical relevance of such approaches is supported by cases where treatment of asymptomatic carriers has been necessary, particularly in patients with underlying conditions like gallstones, which can harbor Salmonella paratyphi B .

What challenges might researchers encounter when attempting to determine the crystal structure of ArnF, and how can these be addressed?

Determining the crystal structure of ArnF presents several challenges characteristic of membrane proteins:

• Protein stability issues:

  • Challenge: Membrane proteins often denature outside their native lipid environment

  • Solution: Screen multiple detergents and lipid-like additives (e.g., amphipols, nanodiscs) to maintain stability

• Conformational heterogeneity:

  • Challenge: Multiple conformational states can hinder crystal formation

  • Solution: Use of conformation-specific antibodies or nanobodies as crystallization chaperones

• Crystal packing limitations:

  • Challenge: Limited hydrophilic surfaces for crystal contacts

  • Solution: Consider fusion proteins (e.g., T4 lysozyme) to increase hydrophilic surface area

• Phase determination difficulties:

  • Challenge: Lack of molecular replacement models for novel membrane protein structures

  • Solution: Prepare selenomethionine-substituted protein or heavy atom derivatives for experimental phasing

Alternative approaches to classical crystallography include cryo-electron microscopy, which has revolutionized membrane protein structural biology and may be particularly suitable for ArnF structure determination.

How can systems biology approaches incorporate ArnF function into broader understanding of Salmonella pathogenicity?

Systems biology approaches can contextualize ArnF function within the broader framework of Salmonella pathogenicity through:

• Multi-omics integration:

  • Transcriptomics: Identify co-regulated genes under infection-relevant conditions

  • Proteomics: Map protein-protein interactions involving ArnF

  • Metabolomics: Measure changes in lipid profiles dependent on ArnF activity

  • Genomics: Compare arnF sequence variations across clinical isolates with virulence phenotypes

• Network analysis:

  • Construct regulatory networks to identify master regulators of arnF expression

  • Develop metabolic models incorporating membrane lipid biosynthesis and modification pathways

  • Create signaling pathway maps connecting environmental sensing to membrane remodeling

• Mathematical modeling:

  • Develop kinetic models of lipid flipping and its impact on membrane composition

  • Create population-level models of bacterial survival under antimicrobial pressure

• Experimental validation:

  • Generate targeted mutations based on model predictions

  • Perform fitness assessments under various stress conditions

  • Utilize whole genome sequencing to track evolutionary adaptations

These integrative approaches can reveal how ArnF-mediated membrane modifications contribute to bacterial adaptation and virulence in the complex host environment.