Recombinant Salmonella paratyphi A UPF0060 membrane protein ynfA (ynfA)

What is the UPF0060 membrane protein YnfA in Salmonella paratyphi A?

YnfA (designated as Q5PHH0 in UniProt) is a membrane protein belonging to the UPF0060 family, present in Salmonella paratyphi A. This protein consists of 108 amino acids and functions as an integral membrane protein. The full amino acid sequence is: MLKTTLLFFVTALCEIIGCFLPWLWIKRGASVWWLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT . The protein's hydrophobic profile suggests it contains multiple transmembrane domains, consistent with its role as a membrane protein. While its precise biological function remains under investigation, membrane proteins typically serve critical roles in cellular processes including transport, signaling, and maintaining cellular integrity.

What expression systems are most effective for recombinant YnfA production?

E. coli expression systems are predominantly used for recombinant YnfA production due to their efficiency, cost-effectiveness, and scalability. For the recombinant full-length YnfA protein, E. coli provides suitable yields when the protein is fused to an N-terminal His-tag . Researchers should consider the following methodology:

• Clone the ynfA gene (1-108aa) into a prokaryotic expression vector with an N-terminal His-tag

• Transform into a compatible E. coli strain (BL21(DE3) or similar)

• Induce expression with IPTG at optimal conditions (typically 0.5-1mM IPTG at 16-25°C)

• Harvest cells and extract membrane fractions using detergent solubilization

• Purify using Ni-NTA affinity chromatography

For membrane proteins like YnfA, expression optimization may require testing multiple E. coli strains (C41, C43) specifically engineered for membrane protein expression.

What purification and storage protocols maximize YnfA stability?

Maintaining structural integrity during purification and storage is crucial for membrane proteins like YnfA. The recommended protocol includes:

Purification:

• Solubilize membrane fractions using mild detergents (DDM, LDAO or OG)

• Perform affinity chromatography using Ni-NTA resin

• Consider size exclusion chromatography as a polishing step

• Concentrate to >90% purity as confirmed by SDS-PAGE

Storage:

• Store lyophilized protein at -20°C/-80°C

• After reconstitution, prepare working aliquots and store at 4°C for up to one week

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol (final concentration 5-50%, optimal 50%) for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles that diminish protein stability

For working solutions, researchers should use Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which has been shown to enhance stability of YnfA .

How can membrane mimetics be optimized for functional studies of YnfA protein?

Membrane protein research requires careful selection of mimetic environments to maintain native structure and function. For YnfA functional studies, consider this methodological approach:

Step 1: Detergent Screening
Test multiple detergent classes in a systematic manner:

• Maltosides (DDM, DM)

• Glucosides (OG)

• Phosphocholines (FC-12, FC-14)

• Neopentyl glycols (LMNG)

Step 2: Advanced Mimetic Exploration
If detergents prove suboptimal, explore:

• Nanodiscs (MSP1D1 or MSP1E3D1) with E. coli total lipids

• Amphipols (A8-35)

• Lipid cubic phase formulations

• Polymer-based systems (SMALPs)

Step 3: Stability Assessment
Following reconstitution into the selected mimetic, evaluate protein stability using:

• Size-exclusion chromatography

• Thermal shift assays

• Mass photometry to assess oligomerization state and sample purity

Mass photometry offers particular advantages for membrane proteins like YnfA, as it can directly characterize oligomerization states and complex formations with minimal sample consumption (1-5 μL at 10-100 nM) . This technique allows researchers to rapidly evaluate multiple membrane mimetic conditions to optimize stability.

What experimental approaches can determine the potential role of YnfA in S. paratyphi A pathogenesis?

Understanding YnfA's role in pathogenesis requires multi-faceted experimental strategies:

Genetic Approaches:

• Create ynfA deletion mutants in S. paratyphi A

• Perform complementation studies with wild-type and mutated ynfA

• Analyze virulence in cell culture and animal models

Functional Assays:

• Membrane permeability studies with fluorescent dyes

• Ion flux measurements in reconstituted liposomes

• Protein-protein interaction studies with key virulence factors

In Vivo Analysis:

• Mouse infection models comparing wild-type and ΔynfA strains

• Tissue distribution assays focusing on heart and kidney colonization (tissues particularly relevant for S. paratyphi A infection)

• Competitive index assays with wild-type and mutant strains

Data Analysis Framework:

When evaluating YnfA's role in pathogenesis, it's important to consider its potential interaction with the human immune system during the infection process. S. paratyphi A's ability to invade intestinal epithelial cells, especially M cells overlying Peyer's patches, and subsequently disseminate to systemic tissues may involve membrane proteins like YnfA.

How might structural studies of YnfA inform vaccine development against S. paratyphi A?

Current vaccines against typhoid fever lack adequate cross-protection against S. paratyphi A infections , highlighting the need for paratyphoid A-specific vaccine candidates. Structural studies of membrane proteins like YnfA could contribute significantly:

Structural Determination Approach:

• Express and purify YnfA in sufficient quantities (>5mg)

• Screen crystallization conditions using lipidic cubic phase methods

• Attempt cryo-EM studies if crystallization proves challenging

• Apply computational approaches (AlphaFold2) to predict structure

Epitope Mapping Workflow:

• Identify surface-exposed regions of YnfA

• Synthesize peptides corresponding to predicted epitopes

• Test immunogenicity of peptides in animal models

• Evaluate cross-reactivity with other Salmonella serovars

Integration with Vaccine Development:

• Assess whether YnfA can elicit protective immunity

• Determine if YnfA-derived epitopes can be incorporated into subunit vaccines

• Evaluate YnfA as a carrier protein for S. paratyphi A polysaccharides

The increasing incidence of S. paratyphi A infections globally and the limited cross-protection offered by existing typhoid vaccines underscore the urgency for paratyphoid A-specific vaccines . Membrane proteins like YnfA, if sufficiently exposed on the bacterial surface, could represent promising vaccine candidates.

What role might YnfA play in antibiotic resistance mechanisms of S. paratyphi A?

With S. paratyphi A developing resistance to multiple antibiotics including chloramphenicol, nalidixic acid, ampicillin, and sulfamethoxazole , investigating membrane proteins like YnfA in this context is crucial:

Experimental Framework:

• Compare YnfA expression levels in resistant versus susceptible strains

• Overexpress or delete ynfA and assess changes in antibiotic susceptibility

• Perform binding studies between YnfA and various antibiotics

• Investigate YnfA's potential interaction with known efflux systems

Antibiotic Resistance Profile Assessment:
Clinical isolates of S. paratyphi A have demonstrated resistance to multiple antibiotics:

• Cefuroxime (>16 mg/L)

• Nalidixic acid (≥32 mg/L)

• Ciprofloxacin (>1 mg/L)

• Tigecycline (>1 mg/L)

Alternative Therapeutic Approaches:
If YnfA proves to be involved in resistance mechanisms, researchers could explore:

• YnfA inhibitors as adjuvants to existing antibiotics

• Bacteriophage therapy targeting S. paratyphi A (like phage Sal11TP)

• Combined approaches using both phage therapy and conventional antibiotics

Recent studies with the bacteriophage Sal11TP demonstrated significant reduction in bacterial loads in heart tissues (1.49 ± 0.09 log cfu/g) compared to conventional antibiotic treatment with gentamycin (2.29 ± 0.25 log cfu/g) . This suggests that alternative therapeutic approaches may be effective against antibiotic-resistant S. paratyphi A.

How can mass photometry advance YnfA interaction studies?

Mass photometry offers unique advantages for investigating membrane proteins like YnfA:

Methodological Applications:

• Characterize YnfA oligomerization states with minimal sample (1-5 μL at 10-100 nM)

• Assess detergent or membrane mimetic efficiency through homogeneity analysis

• Quantify binding of YnfA to potential interaction partners

• Monitor the assembly of multiprotein complexes involving YnfA

Protocol Overview:

• Prepare YnfA samples at 10-100 nM concentration

• Apply sample to glass coverslip and allow settling

• Record interferometric scattering for 30-60 seconds

• Analyze single-molecule mass distribution using specialized software

Advantages Over Traditional Methods:

• No labeling required (label-free detection)

• Minimal sample consumption

• Native solution conditions

• Real-time monitoring of complex formation

• Ability to distinguish between different oligomeric species

Mass photometry can overcome challenges associated with traditional membrane protein characterization techniques like SEC-MALS, AUC, or native PAGE, which often require larger sample amounts and may not maintain the native membrane environment .

What are the critical controls for functional assays involving recombinant YnfA?

Rigorous controls are essential for membrane protein functional studies:

Expression and Purification Controls:

• Empty vector control (expressing tag only)

• Negative control membrane protein (unrelated to YnfA)

• Positive control membrane protein (known function/behavior)

Structural Integrity Controls:

• Circular dichroism to confirm secondary structure

• Thermal stability assays at experimental conditions

• Size exclusion profiles before and after functional assays

Functional Assay Controls:

• Detergent-only controls to assess background signals

• Heat-denatured YnfA to confirm specificity

• Site-directed mutants of key residues

• Concentration-dependent responses to establish dose-relationships

For interaction studies, always include:

• GST-tag or His-tag alone controls

• Scrambled peptide controls

• Competitive inhibition controls

When interpreting results, researchers should compare YnfA from S. paratyphi A with homologs from other species to distinguish conserved versus serovar-specific functions.

How can researchers resolve contradictory findings about YnfA function?

Contradictory findings in membrane protein research often stem from methodological differences. A systematic approach to reconcile conflicting results includes:

Step 1: Environmental Parameter Comparison
Analyze differences in:

• Detergent/mimetic systems used

• Buffer compositions (pH, salt concentration)

• Temperature conditions

• Protein constructs (full-length vs. truncated)

Step 2: Independent Validation

• Replicate key experiments using multiple techniques

• Employ orthogonal approaches to measure the same parameter

• Use both in vitro and in vivo systems when possible

Step 3: Integration Framework
Create a hypothesis that can accommodate seemingly contradictory findings by considering:

• Context-dependent protein behavior

• Allosteric regulation mechanisms

• Conformational heterogeneity

Decision Matrix for Resolving Contradictions:

When studying YnfA's role in S. paratyphi A, researchers should be particularly attentive to contradictory findings regarding virulence, as clinical observations have shown conflicting results about whether S. paratyphi A causes milder or equally severe disease compared to S. Typhi .

How might YnfA contribute to innovative therapeutic approaches against S. paratyphi A?

With increasing antibiotic resistance, exploring YnfA's therapeutic potential is valuable:

YnfA as a Drug Target:

• Develop high-throughput screening assays for YnfA inhibitors

• Design structure-based inhibitors if structural data becomes available

• Explore antibody-based targeting of surface-exposed YnfA epitopes

YnfA in Vaccine Development:

• Evaluate YnfA as a component of subunit vaccines

• Assess YnfA-derived peptides as immunogens

• Investigate YnfA as a carrier protein for conjugate vaccines

Combined Therapeutic Approaches:

• Study synergistic effects between YnfA inhibitors and existing antibiotics

• Investigate YnfA's role in bacteriophage infection, particularly with phage Sal11TP

• Develop YnfA-targeted drug delivery systems

Given that current typhoid vaccines lack adequate cross-protection against S. paratyphi A , targeting YnfA could offer complementary approaches to conventional vaccines and antibiotics.

What computational approaches can predict YnfA interactions with host proteins?

Computational methods offer powerful tools to predict how YnfA might interact with host systems:

Sequence-Based Predictions:

• Identify short linear motifs that might interact with host proteins

• Predict post-translational modification sites that could modulate host interactions

• Compare with homologs of known function using multiple sequence alignments

Structure-Based Predictions:

• Apply homology modeling or AlphaFold2 to generate YnfA structural models

• Perform molecular docking with candidate host proteins

• Run molecular dynamics simulations to assess interaction stability

Systems Biology Approaches:

• Network analysis to predict YnfA's position in host-pathogen interaction networks

• Integrate transcriptomic data to identify co-regulated genes

• Apply machine learning to identify patterns associated with YnfA-expressing pathogens

Computational Workflow:

• Generate YnfA structural model

• Identify surface-exposed regions

• Predict binding sites using docking algorithms

• Validate top predictions with targeted binding assays

These computational predictions can guide experimental design and help prioritize which host interactions to investigate first in experimental systems.

What are the key challenges and opportunities in YnfA research?

Research on YnfA presents several distinct challenges and opportunities:

Technical Challenges:

• Maintaining native structure in recombinant expression systems

• Achieving sufficient yield for structural studies

• Developing functional assays for proteins of unknown function

• Distinguishing YnfA-specific effects from general membrane perturbations

Knowledge Gaps:

• Precise biological function remains uncharacterized

• Relationship to virulence not fully established

• Potential as a therapeutic target needs validation

• Role in the broader context of S. paratyphi A biology unclear

Research Opportunities:

• Contribute to novel vaccine development against S. paratyphi A

• Explore as a target for alternative therapeutics like bacteriophage Sal11TP

• Investigate as a component of antibiotic resistance mechanisms

• Advance membrane protein methodology through applied research