Recombinant Yersinia pestis bv. Antiqua UPF0266 membrane protein YPN_2368 (YPN_2368)

What is Yersinia pestis bv. Antiqua UPF0266 membrane protein YPN_2368?

YPN_2368 (UniProt ID: Q1CH34) is a membrane protein belonging to the UPF0266 family, found in Yersinia pestis biovar Antiqua, a causative agent of plague. This protein consists of 153 amino acids and is predicted to be an integral membrane protein with multiple transmembrane domains. The "UPF" designation (Uncharacterized Protein Family) indicates that while the protein has been identified and sequenced, its precise biological function remains incompletely characterized. YPN_2368 is also referred to as YP516_2666 in some databases and literature . The protein is part of the membrane proteome of Y. pestis, which plays critical roles in bacterial survival, virulence, and interaction with host cells during infection processes.

How should recombinant YPN_2368 be stored and handled in laboratory settings?

For optimal stability and experimental reproducibility, recombinant YPN_2368 protein should be stored according to these guidelines:

• Upon receipt, briefly centrifuge the vial to bring contents to the bottom before opening.

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

• Add glycerol to a final concentration of 5-50% (typically 50% is recommended) and aliquot for long-term storage .

• Store aliquots at -20°C or preferably -80°C for extended stability.

• Avoid repeated freeze-thaw cycles as they significantly degrade membrane proteins.

• For working stocks, store aliquots at 4°C for up to one week .

The protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during lyophilization and reconstitution . When handling the protein, maintain sterile conditions and use low-binding microcentrifuge tubes to prevent protein adherence to container walls, which is common with hydrophobic membrane proteins.

What expression systems are commonly used for producing recombinant YPN_2368?

Recombinant YPN_2368 is typically produced using prokaryotic expression systems, with E. coli being the most commonly employed host . The standard expression protocol involves:

• Cloning the YPN_2368 gene sequence into an expression vector containing an N-terminal histidine tag.

• Transforming the construct into a suitable E. coli strain optimized for membrane protein expression (e.g., C41(DE3), C43(DE3), or BL21(DE3)pLysS).

• Inducing protein expression with IPTG (isopropyl β-D-1-thiogalactopyranoside) at lower temperatures (16-25°C) to facilitate proper folding.

• Harvesting cells and disrupting membranes using detergents such as n-dodecyl β-D-maltoside (DDM) or n-octyl-β-D-glucoside (OG).

• Purifying the His-tagged protein using immobilized metal affinity chromatography (IMAC).

• Further purification via size exclusion chromatography to achieve >90% purity .

Alternative expression systems, including cell-free protein synthesis methods, have been explored for difficult-to-express membrane proteins but are less commonly used for YPN_2368 specifically. Yeast expression systems (Pichia pastoris) might represent a viable alternative when post-translational modifications are required, though this is not typically necessary for bacterial membrane proteins.

How might YPN_2368 contribute to Yersinia pestis virulence mechanisms?

While the specific function of YPN_2368 remains under investigation, several hypotheses exist regarding its potential role in Y. pestis virulence:

• Membrane integrity: As an integral membrane protein, YPN_2368 may contribute to membrane stability under varying environmental conditions encountered during host infection .

• Host-pathogen interaction: The protein could participate in adhesion to host cells or evasion of host immune responses, similar to other membrane proteins in pathogenic bacteria.

• Survival in diverse environments: Y. pestis must adapt to drastically different conditions in flea vectors and mammalian hosts. YPN_2368 may participate in environmental sensing or adaptation mechanisms .

• Potential involvement in amoebal persistence: Recent research has identified amebae as potential environmental reservoirs for Y. pestis. Membrane proteins like YPN_2368 might play roles in survival within ameboid hosts, similar to mechanisms that allow the bacterium to survive in macrophages .

• Transport functions: Many uncharacterized membrane proteins eventually prove to function as transporters for ions, nutrients, or waste products. YPN_2368 might transport molecules essential for survival during infection.

Experimental approaches to test these hypotheses include gene knockout studies, host cell interaction assays, and comparative proteomics between virulent and avirulent strains of Y. pestis under different environmental conditions.

What experimental design approaches are recommended for studying YPN_2368 function?

A systematic Design of Experiments (DOE) approach is strongly recommended for investigating YPN_2368 function. A comprehensive experimental design would include:

• Factorial Design: Test multiple factors simultaneously rather than one-factor-at-a-time (OFAT) approaches, which are inefficient and may miss important interactions .

• Sequential Screening: Begin with a fractional factorial design to identify significant factors, followed by response surface methodology to optimize conditions .

• Key Factors to Consider:

  • Temperature (both growth temperature and experimental conditions)

  • pH conditions

  • Ion concentrations (particularly calcium and magnesium)

  • Presence of specific host factors

  • Growth phase of bacteria

• Recommended Experimental Matrix:

• Randomization and Replication: Implement proper randomization of experimental runs and include at least three biological replicates to ensure statistical validity .

• Controls: Include appropriate positive and negative controls, including known membrane proteins with established functions and knockout strains.

This structured approach allows for systematic investigation of YPN_2368 function while minimizing experimental bias and maximizing information gain from each experiment.

How does YPN_2368 compare to homologous proteins in other Yersinia species?

Comparative genomic and proteomic analyses reveal important evolutionary relationships between YPN_2368 and homologous proteins in other Yersinia species:

• Sequence Conservation: YPN_2368 shares approximately 98-99% sequence identity with homologs in Y. pestis CO92 and KIM strains, indicating high conservation within the species .

• Cross-Species Comparison:

  • 85-90% identity with homologs in Y. pseudotuberculosis

  • 70-75% identity with homologs in Y. enterocolitica

  • 50-60% identity with more distant Yersinia species

• Evolutionary Significance: The higher conservation between Y. pestis and Y. pseudotuberculosis compared to Y. enterocolitica is consistent with the evolutionary history of these species, where Y. pestis evolved from Y. pseudotuberculosis relatively recently.

• Functional Divergence: Despite sequence similarity, functional differences may exist between these homologs. For example, Y. pestis contains modified versions of several membrane proteins that contribute to its unique pathogenicity compared to other Yersinia species .

• Structural Conservation: Predicted transmembrane domains appear to be more highly conserved than loop regions, suggesting functional constraints on membrane-spanning segments.

Experimental approaches to investigate functional differences include heterologous expression studies, where YPN_2368 homologs from different species are expressed in a common background strain to compare phenotypic effects.

What is the potential role of YPN_2368 in ameba reservoir interactions?

Recent research has identified environmental amebae as potential reservoirs for Y. pestis during interepizootic periods . This discovery has significant implications for understanding plague persistence and transmission cycles. YPN_2368, as a membrane protein, may play several roles in these interactions:

• Survival Mechanisms: Y. pestis has been shown to survive and replicate within certain ameba species, particularly Dictyostelium discoideum, where it exhibits 226.67% replication at 48 hours post-infection . Membrane proteins like YPN_2368 may contribute to this survival ability.

• Yersinia-Containing Vacuole (YCV) Formation: In macrophages, Y. pestis forms specialized vacuoles for intracellular survival. Similar structures may form in amebae, potentially involving membrane proteins such as YPN_2368 .

• GTPase Recruitment: Y. pestis survival in macrophages involves recruitment of Rab1b GTPases, and homologous sequences (99.8% similarity) have been identified in D. discoideum . YPN_2368 might interact with these GTPases.

• Experimental Approach to Study Ameba Interactions:

• Methodology: To study YPN_2368's role in ameba interactions, researchers should consider knockdown/knockout studies, fluorescently tagged protein localization, and comparative proteomics between ameba-exposed and unexposed bacteria .

The implications of these studies extend beyond basic research, potentially informing plague surveillance and prevention strategies by identifying environmental reservoirs.

What approaches are recommended for studying YPN_2368 structural characteristics?

Several complementary approaches can be employed to elucidate the structural characteristics of YPN_2368:

• X-ray Crystallography:

  • Challenges: Membrane proteins are notoriously difficult to crystallize due to their hydrophobic nature.

  • Recommended approach: Utilize lipidic cubic phase (LCP) crystallization methods which provide a membrane-mimetic environment.

  • Detergent screening is critical: Test a panel of at least 10 different detergents including DDM, OG, LDAO, and newer amphipols.

• Cryo-Electron Microscopy (Cryo-EM):

  • Increasingly useful for membrane proteins that resist crystallization.

  • For smaller proteins like YPN_2368 (17 kDa), consider expressing fusion constructs with larger proteins or antibody fragments to increase particle size.

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Solution NMR: Suitable for determining the structure of smaller membrane proteins in detergent micelles.

  • Solid-state NMR: Can provide structural information on membrane proteins reconstituted in lipid bilayers.

  • Requires isotopic labeling (¹⁵N, ¹³C, ²H) during protein expression.

• Circular Dichroism (CD) Spectroscopy:

  • Provides information about secondary structure content (α-helices, β-sheets).

  • Useful for monitoring structural changes under different conditions (pH, temperature, ligand binding).

• Molecular Dynamics Simulations:

  • Computational approach to model protein behavior in membrane environments.

  • Can predict structural dynamics, flexibility, and potential binding sites.

  • Should be validated with experimental data whenever possible.

Each method has strengths and limitations, and a multi-technique approach is typically required for comprehensive structural characterization of membrane proteins like YPN_2368.

How can site-directed mutagenesis be applied to study YPN_2368 function?

Site-directed mutagenesis represents a powerful approach to investigate structure-function relationships in YPN_2368:

• Key Residues for Targeted Mutagenesis:

  • Conserved residues across Yersinia species (particularly charged amino acids in transmembrane regions)

  • Potential phosphorylation sites (Ser, Thr, Tyr residues)

  • Cysteine residues that might form disulfide bonds

  • Residues predicted to face the membrane interface

• Recommended Mutagenesis Strategy:

  • Alanine scanning: Systematically replace selected residues with alanine to identify functionally important positions

  • Conservative substitutions: Replace residues with chemically similar amino acids to fine-tune functional analysis

  • Introduction of reporter residues: Insert cysteine residues for subsequent labeling experiments

• Experimental Design Considerations:

  • Use Gibson Assembly or Q5 site-directed mutagenesis kits for efficient mutagenesis

  • Maintain the His-tag for consistent purification protocols

  • Verify mutations by DNA sequencing before expression

  • Confirm protein expression and membrane localization for each mutant

• Functional Assays for Mutant Proteins:

  • Membrane integration efficiency

  • Protein stability assessments

  • Bacterial survival under stress conditions

  • Host cell interaction studies

  • Ameba survival assays as described previously

• Data Analysis Framework:

  • Compare multiple parameters for each mutant to wild-type protein

  • Cluster mutants by phenotypic similarity

  • Map mutations onto predicted structural models to identify functional domains

This systematic mutagenesis approach will help delineate the functional domains of YPN_2368 and potentially identify regions critical for pathogen survival or virulence.

What are the recommended protocols for functional assays of YPN_2368?

To comprehensively characterize YPN_2368 function, multiple complementary assays should be employed:

• Membrane Localization Assays:

  • Subcellular fractionation followed by Western blotting

  • Immunofluorescence microscopy using anti-His antibodies or custom antibodies against YPN_2368

  • Protease accessibility assays to determine topology

• Protein-Protein Interaction Studies:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Pull-down assays using His-tagged YPN_2368 as bait

  • Cross-linking studies followed by mass spectrometry

  • FRET-based interaction assays if working with fluorescently labeled proteins

• Functional Complementation:

  • Generate YPN_2368 knockout strains and assess phenotypic changes

  • Complement knockout strains with wild-type or mutant variants

  • Heterologous expression in surrogate bacterial hosts

• Environmental Response Assays:

  • Survival under various stress conditions:

• Biophysical Characterization:

  • Ion flux measurements if YPN_2368 is suspected to be a channel or transporter

  • Lipid binding assays to assess membrane interactions

  • Thermal stability assays under varying conditions

• Infection Models:

  • Macrophage infection assays comparing wild-type and YPN_2368 mutants

  • Ameba infection models as described in previous research

  • Animal models for virulence studies (if appropriate biosafety facilities are available)

These assays should be conducted using proper experimental design principles, including appropriate controls, replication, and statistical analysis .

How might YPN_2368 research contribute to plague vaccine development?

Research on YPN_2368 has several potential implications for plague vaccine development:

• Novel Antigen Identification: If YPN_2368 is surface-exposed and immunogenic, it could represent a new target for vaccine development. Unlike the current focus on F1 and V antigens , membrane proteins might provide broader protection.

• Multicomponent Vaccine Possibilities: Current research indicates that fusion proteins combining multiple antigens (like F1-V) provide superior protection against both bubonic and pneumonic plague . YPN_2368 could potentially be incorporated into such multicomponent vaccines if it proves immunogenic.

• Addressing Vaccine Escape Variants: F1-negative (F1-) strains of Y. pestis have been isolated from human cases and rodents, against which current vaccines show limited efficacy . If YPN_2368 is conserved in these variants, targeting it could provide more universal protection.

• Experimental Approach for Vaccine Potential Assessment:

  • Immunogenicity studies: Determine if recombinant YPN_2368 elicits antibody responses

  • Epitope mapping: Identify immunodominant regions within the protein

  • Protection studies: Test if YPN_2368 immunization provides protection in animal models

  • Combination studies: Evaluate YPN_2368 in combination with established antigens (F1, V)

• Potential Advantages of YPN_2368-Based Vaccines:

  • Conservation across Y. pestis strains could provide broader protection

  • Membrane proteins often represent stable, well-presented targets for immune recognition

  • Potential for cross-protection against related Yersinia species

While preliminary, this research direction could contribute to addressing limitations of current plague vaccines, particularly their ineffectiveness against pneumonic plague and F1- variant strains .

What are the challenges and solutions for studying YPN_2368 in BSL-3 environments?

Studying YPN_2368 in the context of virulent Y. pestis strains presents significant biosafety challenges, as these strains must be handled in Biosafety Level 3 (BSL-3) facilities. Several approaches can address these challenges:

• Biosafety Considerations:

  • Y. pestis is classified as a Tier 1 Select Agent requiring specialized facilities and training

  • Work with fully virulent strains requires BSL-3 containment

  • Research on certain attenuated strains may be conducted at BSL-2

• Alternative Experimental Approaches:

  • Use of attenuated Y. pestis strains: The YopP-expressing Y. pestis strain exhibits reduced virulence (at least 10⁷-fold reduction) while maintaining molecular characteristics

  • Surrogate systems: Express YPN_2368 in closely related but less pathogenic species like Y. pseudotuberculosis

  • Recombinant systems: Study the protein in heterologous expression systems (E. coli)

• Technological Solutions:

  • Automated systems that minimize direct handling

  • Microfluidic devices for miniaturized experiments

  • Advanced imaging systems compatible with BSL-3 environments

• Experimental Design Considerations:

• Collaborative Approaches:

  • Partner with institutions having established BSL-3 facilities

  • Divide experimental workflow between BSL-2 and BSL-3 components

  • Utilize core facilities with specialized BSL-3 equipment and expertise

By implementing these strategies, researchers can effectively study YPN_2368 while maintaining appropriate biosafety standards and generating meaningful data about this protein's role in virulent Y. pestis strains.

How can contradictory data about YPN_2368 function be reconciled in research settings?

When confronting contradictory data about YPN_2368 function, researchers should implement systematic approaches to reconcile discrepancies:

• Sources of Experimental Variability:

  • Strain differences: Genetic variations between Y. pestis strains may affect YPN_2368 function

  • Experimental conditions: Temperature, media composition, and growth phase significantly impact Y. pestis gene expression

  • Methodological differences: Variation in protein purification, assay conditions, and detection methods

• Systematic Reconciliation Framework:

  • Meta-analysis approach: Compile all available data with detailed experimental parameters

  • Standardization efforts: Develop consensus protocols for YPN_2368 research

  • Multi-laboratory validation: Reproduce key experiments across different laboratories

• Recommended Resolution Strategy:

  • Identify specific variables that might explain contradictions

  • Design factorial experiments specifically targeting these variables

  • Use statistical methods to quantify contributions of each variable to observed differences

  • Develop computational models that can accommodate seemingly contradictory data points

• Case Example: Resolving Conflicting Survival Data

  If different research groups report contradictory data about YPN_2368's role in bacterial survival, consider:

  • Temperature effects: Y. pestis exhibits temperature-dependent expression patterns (28°C in flea vector vs. 37°C in mammalian host)

  • Growth phase impacts: Stationary phase cells often show different protein functionality than exponential phase cells

  • Strain-specific effects: Compare results across multiple Y. pestis strains (CO92, KIM, Antiqua)

• Documentation and Reporting Practices:

  • Comprehensive methods reporting

  • Full disclosure of negative and contradictory results

  • Data sharing through repositories

  • Detailed recording of all experimental parameters

By approaching contradictions systematically rather than dismissing them, researchers can gain deeper insights into the context-dependent functions of YPN_2368 and develop more nuanced understanding of its biological roles.

What are the most promising future research directions for YPN_2368?

Based on current knowledge and research gaps, several promising directions for future YPN_2368 research emerge:

• Structural Biology Approaches: Determining the three-dimensional structure of YPN_2368 would significantly advance understanding of its function. Cryo-EM and advanced NMR techniques show particular promise for membrane protein structural studies.

• Systems Biology Integration: Investigating YPN_2368 in the context of the broader Y. pestis membrane proteome and interactome would provide insights into its functional networks and regulatory mechanisms.

• Environmental Persistence Mechanisms: Further exploration of YPN_2368's potential role in ameba interactions could reveal critical insights into plague persistence between epizootic cycles .

• Comparative Studies Across Yersinia Species: Systematic comparison of YPN_2368 homologs across pathogenic and non-pathogenic Yersinia species could illuminate evolutionary adaptations relevant to virulence.

• Immunological Studies: Characterizing host immune responses to YPN_2368 could reveal its potential as a diagnostic marker or vaccine component .

• Therapeutic Target Assessment: Evaluating YPN_2368 as a potential target for novel antimicrobials would address the need for alternative treatment options for plague.

• Technological Innovations: Development of new tools specifically designed for studying membrane proteins in highly pathogenic bacteria would benefit the broader research community.

These research directions, pursued through collaborative and multidisciplinary approaches, hold promise for transforming our understanding of YPN_2368 from an uncharacterized membrane protein to a well-defined component of Y. pestis biology with potential applications in disease diagnosis, prevention, and treatment.

How should researchers approach reproducibility challenges in YPN_2368 studies?

Ensuring reproducibility in YPN_2368 research requires deliberate methodological approaches:

• Standardized Protocols Development:

  • Establish consensus methods for protein expression, purification, and functional assays

  • Create detailed standard operating procedures (SOPs) with attention to often-overlooked variables

  • Develop reference materials and positive controls for assay validation

• Comprehensive Reporting:

  • Document all experimental parameters, including:

    • Exact strain designations and passage numbers

    • Complete media compositions

    • Precise growth conditions (temperature, aeration, humidity)

    • Detailed buffer compositions including pH measurement methods

    • Lot numbers of critical reagents

    • Equipment calibration status

• Design of Experiments Implementation:

  • Apply factorial design principles to identify critical variables affecting outcomes

  • Include positive and negative controls in every experiment

  • Ensure adequate statistical power through appropriate sample sizes

  • Implement proper randomization and blinding procedures

• Collaborative Verification:

  • Establish multi-laboratory validation networks

  • Conduct inter-laboratory comparison studies

  • Share raw data and detailed protocols through repositories

• Technological Approaches:

  • Utilize electronic laboratory notebooks with standardized templates

  • Implement automated data capture where possible

  • Develop computational pipelines for consistent data analysis