Recombinant Yersinia pseudotuberculosis serotype O:1b UPF0266 membrane protein YpsIP31758_2371 (YpsIP31758_2371)

What is the genomic context of UPF0266 membrane protein YpsIP31758_2371 in Y. pseudotuberculosis?

The UPF0266 membrane protein YpsIP31758_2371 is encoded within the genome of Y. pseudotuberculosis strain IP31758, which has been fully sequenced and contains more than 260 strain-specific genes compared to other Y. pseudotuberculosis strains . When investigating this protein, researchers should examine its genomic neighborhood to identify potential operons or functionally related genes. This contextual analysis might reveal associations with virulence factors or metabolic pathways specific to the IP31758 strain associated with Far East scarlet-like fever (FESLF) .

Methodologically, researchers should:

• Perform comparative genomic analysis with other Y. pseudotuberculosis strains

• Identify conserved domains through bioinformatic tools

• Analyze promoter regions for regulatory elements

• Examine codon usage patterns to assess potential horizontal gene transfer

How does the classification as UPF0266 influence research approaches?

The UPF (Uncharacterized Protein Family) designation indicates that YpsIP31758_2371 belongs to a protein family with limited functional characterization. This classification should guide initial research approaches toward:

• Structural prediction and modeling using homology-based tools

• Functional prediction based on conserved domains

• Comparative analysis with other UPF0266 family members across bacterial species

• Expression pattern analysis under various environmental conditions

When approaching uncharacterized membrane proteins, researchers should implement a multifaceted strategy combining computational prediction and experimental validation, prioritizing techniques that can provide insights into membrane localization, topology, and potential binding partners.

What expression systems are most appropriate for recombinant production of YpsIP31758_2371?

For recombinant production of YpsIP31758_2371, researchers should consider several expression systems with the following methodological considerations:

The selection should be guided by the specific research goals. For structural studies, E. coli with specialized membrane protein expression vectors might be optimal, while functional studies might benefit from expression in the native organism under controlled conditions.

How might YpsIP31758_2371 contribute to the unique pathogenicity profile of Y. pseudotuberculosis strain IP31758?

Y. pseudotuberculosis IP31758 is associated with Far East scarlet-like fever, which presents with distinct clinical manifestations including erythematous skin rash, desquamation, and toxic shock syndrome . Research approaches to investigate YpsIP31758_2371's potential role in this unique pathogenicity profile should include:

• Generation of deletion mutants using CRISPR-Cas9 or allelic exchange methods

• Complementation studies to confirm phenotypic changes

• Infection models with appropriate cell lines and animal models

• Transcriptomic and proteomic analyses comparing wild-type and mutant strains

The methodology should emphasize comparing the IP31758 strain with classical Y. pseudotuberculosis strains like IP32953 to identify strain-specific phenotypes. Given that IP31758 contains a type IVB secretion system similar to intracellular pathogens , researchers should investigate whether YpsIP31758_2371 interacts with this system or other virulence determinants.

What techniques are most effective for determining the membrane topology of YpsIP31758_2371?

Determining membrane topology is essential for understanding protein function. For YpsIP31758_2371, researchers should employ a combination of the following methodological approaches:

• Computational prediction using algorithms like TMHMM, TOPCONS, and PredictProtein

• Experimental validation through:

  • Reporter fusion techniques (PhoA/LacZ fusions at various positions)

  • Cysteine accessibility methods (SCAM)

  • Protease protection assays

  • Epitope tagging at predicted loops followed by immunofluorescence

When conducting these experiments, it's critical to maintain the protein in a native-like membrane environment. Researchers should consider using nanodiscs or liposomes derived from bacterial membranes to preserve functional conformations during analysis.

How does YpsIP31758_2371 interact with the host immune system during infection?

Considering that Y. pseudotuberculosis employs various mechanisms to evade host immunity, including the recruitment of complement regulators by outer membrane proteins like Ail , researchers investigating YpsIP31758_2371's potential immune interactions should:

• Assess binding to host immune components using:

  • Pull-down assays with purified protein

  • Surface plasmon resonance for kinetic analysis

  • Co-immunoprecipitation from infected cells

• Evaluate effects on immune signaling pathways:

  • NF-κB activation assays

  • Inflammasome activation studies

  • Cytokine profiling

• Compare immune responses between wild-type and YpsIP31758_2371-deficient strains in:

  • Macrophage infection models

  • Dendritic cell activation assays

  • Complement resistance tests

The methodological approach should control for the influence of other known immune evasion factors, potentially using multiple knockout combinations to identify synergistic effects.

What are the optimal conditions for inducing and detecting YpsIP31758_2371 expression in Y. pseudotuberculosis?

Researchers should consider that Y. pseudotuberculosis differentially regulates gene expression in response to environmental conditions. For studying YpsIP31758_2371, the following methodological considerations are important:

When designing experiments to detect expression patterns, researchers should employ epitope tagging or fluorescent protein fusions at the C-terminus to minimize disruption of membrane insertion signals typically found at the N-terminus.

How should researchers approach structural studies of YpsIP31758_2371?

For structural characterization of YpsIP31758_2371, researchers should consider a hierarchical approach:

The methodological challenges include maintaining protein stability in detergent or membrane mimetics. Researchers should test multiple extraction and purification conditions, possibly incorporating stabilizing mutations or antibody fragments to facilitate crystallization.

What strategies should be employed to identify potential binding partners of YpsIP31758_2371?

Identifying interaction partners is crucial for functional characterization. Researchers should implement:

• Unbiased screening approaches:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling techniques (BioID, APEX)

  • Chemical cross-linking coupled with mass spectrometry

• Candidate-based approaches:

  • Direct binding assays with purified components

  • Bacterial surface display for host factor screening

  • Yeast two-hybrid with membrane protein adaptations

When performing these experiments, appropriate controls must include non-specific membrane proteins of similar size and topology. For Y. pseudotuberculosis specifically, researchers should investigate potential interactions with known virulence factors such as the type IVB secretion system components found in strain IP31758 .

How should researchers distinguish between direct and indirect effects in functional studies of YpsIP31758_2371?

When analyzing phenotypes of YpsIP31758_2371 mutants, researchers must consider:

• Complementation strategies:

  • Expression of wild-type protein from various promoters

  • Site-directed mutagenesis of key residues

  • Conditional expression systems

• Control experiments:

  • Multiple independent mutant strains

  • Whole-genome sequencing to confirm absence of secondary mutations

  • Transcriptomics to identify compensatory changes

• Approaches to establish causality:

  • Time-course experiments with inducible systems

  • Dose-response relationships with tunable expression

  • Epistasis analysis with related pathway components

What bioinformatic approaches are most valuable for predicting the function of YpsIP31758_2371?

For uncharacterized proteins like YpsIP31758_2371, computational analysis can provide valuable functional insights:

• Sequence-based approaches:

  • Profile hidden Markov models to identify distant homologs

  • Co-evolution analysis to predict interaction interfaces

  • Genomic context (gene neighborhood) analysis

• Structure-based approaches:

  • Threading algorithms for fold recognition

  • Binding site prediction based on surface electrostatics

  • Molecular docking with potential ligands

• Systems-based approaches:

  • Co-expression network analysis across conditions

  • Phylogenetic profiling to identify functional associations

  • Integration with pathogen-host interaction databases

Researchers should critically evaluate computational predictions by assessing confidence scores and validating key predictions experimentally, particularly when the protein belongs to an uncharacterized family with limited reference data.

How might YpsIP31758_2371 contribute to bacterial adaptation during host infection?

Research into adaptation mechanisms should consider:

• Experimental approaches:

  • In vivo passage experiments with recovery and sequencing

  • Single-cell gene expression analysis during infection

  • Fitness measurements in different host microenvironments

• Comparative studies:

  • Analysis across Y. pseudotuberculosis isolates from different clinical presentations

  • Expression patterns relative to other virulence factors

  • Contribution to fitness in competition assays

Given that Y. pseudotuberculosis IP31758 causes distinctive clinical symptoms compared to classical strains , researchers should investigate whether YpsIP31758_2371 contributes to this unique pathogenicity profile, particularly in relation to the strain's capacity to cause Far East scarlet-like fever.

What are the challenges and solutions in developing antibodies or other detection reagents for YpsIP31758_2371?

For researchers developing detection tools, the following methodological considerations are important:

• Antigen design strategies:

  • Identification of extracellular loops based on topology predictions

  • Synthetic peptides versus recombinant protein fragments

  • Conformational epitopes preserved in detergent micelles

• Validation approaches:

  • Testing specificity across related Yersinia species

  • Confirmation using knockout strains

  • Assessment of sensitivity in complex samples

• Alternative detection strategies:

  • Aptamer development through SELEX

  • Nanobody generation using camelid immunization

  • Activity-based probes if functional activity is identified

The development of high-quality detection reagents is critical for subsequent localization studies, quantification during infection, and potential diagnostic applications.