Recombinant Yersinia pseudotuberculosis serotype O:1b UPF0299 membrane protein YpsIP31758_2460 (YpsIP31758_2460)

What is UPF0299 membrane protein YpsIP31758_2460 and what is its significance?

YpsIP31758_2460 is a 135-amino acid membrane protein belonging to the UPF0299 protein family found in Yersinia pseudotuberculosis serotype O:1b (strain IP 31758). This strain is particularly significant as it's associated with Far East scarlet-like fever (FESLF), a severe infection with symptoms including erythematous skin rash, desquamation, exanthema, hyperhemic tongue, and toxic shock syndrome . The UPF0299 family remains functionally uncharacterized, making YpsIP31758_2460 an interesting target for researchers investigating Y. pseudotuberculosis pathogenicity mechanisms. The protein has a molecular weight of approximately 15.3 kDa and likely plays a role in membrane-associated processes, though specific functions remain to be elucidated through experimental approaches .

How does Y. pseudotuberculosis serotype O:1b (strain IP 31758) differ from other Y. pseudotuberculosis strains?

Y. pseudotuberculosis IP31758 demonstrates significant genomic diversity compared to other strains like IP32953. Genomic analysis has revealed that IP31758 possesses more than 260 strain-specific genes that confer unique physiological capabilities and virulence determinants . Many of these genes were likely acquired through horizontal gene transfer from Enterobacteriaceae and soil-dwelling bacteria. The strain notably contains two novel plasmids phylogenetically unrelated to other Yersinia plasmids, with the larger plasmid harboring an icm/dot type IVB secretion system otherwise found only in intracellular pathogens of the Legionellales order . Additionally, IP31758 lacks the Yersinia High Pathogenicity Island (HPI) present in other Y. pseudotuberculosis strains, which typically encodes the yersiniabactin siderophore biosynthetic pathway important for systemic spread .

What is known about the amino acid sequence and predicted structure of YpsIP31758_2460?

The YpsIP31758_2460 protein consists of 135 amino acids with the following sequence: MRNMMSLCWQYLRAFTIIYLCLWAGKALALLLPIVIPGSIIGMLILFVLLTLQILPSPWVKPSCQLLIRYMALLFVPIGVGVMQYYEQLTKQFGPIVVSCFISTLIVMLVVAYSSHYVHRDRKVISPSTPTEGEK . Analysis of this sequence suggests it contains multiple hydrophobic regions consistent with a membrane protein topology. The high proportion of hydrophobic amino acids (leucine, isoleucine, valine, phenylalanine) indicates multiple potential membrane-spanning domains. While no crystal structure is currently available, computational prediction methods would likely suggest a structure with multiple transmembrane helices characteristic of integral membrane proteins of the UPF0299 family .

How does YpsIP31758_2460 fit into the broader context of Y. pseudotuberculosis evolution?

YpsIP31758_2460 exists within the complex evolutionary history of Y. pseudotuberculosis. Multilocus sequence analysis has revealed that Y. pseudotuberculosis represents a complex of four populations: Y. pseudotuberculosis sensu stricto (s.s.), Y. pestis, Y. similis, and a fourth population referred to as the "Korean group" . Genetic diversification within Y. pseudotuberculosis s.s. is approximately equally divided between recombination and mutation, whereas Y. pestis is more genetically monomorphic with no demonstrated recombination . The presence of specific membrane proteins like YpsIP31758_2460 may contribute to the unique characteristics of serotype O:1b strain IP 31758, potentially playing a role in its distinctive pathogenicity profile compared to other Y. pseudotuberculosis lineages .

What expression systems are optimal for recombinant YpsIP31758_2460 production?

For optimal expression of YpsIP31758_2460, E. coli-based systems represent the most accessible approach, though challenges exist due to its membrane protein nature. Based on related membrane protein studies, researchers should consider the following expression strategies:

How can researchers address inclusion body formation during YpsIP31758_2460 expression?

Inclusion body (IB) formation represents a significant challenge when expressing membrane proteins like YpsIP31758_2460. Studies with Y. pseudotuberculosis OmpF porin demonstrated that lower growth temperatures affect IB properties and protein structure . Inclusions formed at 18°C (IB-18) showed higher solubility in denaturants and contained a greater proportion of native-like protein conformation compared to those formed at 30°C (IB-30) .
To manage inclusion bodies effectively, researchers should consider:

• Reducing expression temperature to 18°C, which promotes higher native-like conformation retention within IBs

• Using mild solubilization conditions (e.g., 2-4M urea) rather than harsh denaturants

• Implementing a step-wise refolding protocol with decreasing denaturant concentrations

• Adding membrane-mimetic environments (detergents, lipids) during the refolding process

• Employing fusion partners that enhance solubility (e.g., MBP, SUMO, or thioredoxin)
  Spectroscopic analysis techniques (CD spectroscopy, fluorescence) should be used to monitor secondary structure content during solubilization and refolding to ensure proper protein conformation is achieved .

What purification strategies yield functional YpsIP31758_2460?

Purification of membrane proteins like YpsIP31758_2460 requires specialized approaches to maintain structural integrity and function. Based on related membrane protein research, an effective purification workflow would include:

• Membrane Isolation: Differential centrifugation following cell lysis to isolate membrane fractions

• Detergent Solubilization: Screening multiple detergents (DDM, LDAO, OG) at various concentrations to identify optimal solubilization conditions

• Affinity Chromatography: If using His-tagged constructs (recommended based on OmpF porin research ), employ IMAC with nickel or cobalt resins

• Size Exclusion Chromatography: To separate monomeric protein from aggregates and remove detergent micelles

• Quality Assessment: Use dynamic light scattering, CD spectroscopy, and fluorescence to verify proper folding and homogeneity
  For inclusion body-derived protein, a refolding step must be incorporated following solubilization in urea or SDS, with careful monitoring to prevent misfolding and aggregation. The refolding buffer should contain appropriate detergents or lipids to stabilize the membrane domains of YpsIP31758_2460.

How can proper folding and activity of purified YpsIP31758_2460 be verified?

Verifying proper folding of YpsIP31758_2460 is essential for functional studies. Based on approaches used with other Y. pseudotuberculosis membrane proteins, researchers should employ multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure content and compare with computational predictions based on the amino acid sequence

• Tryptophan Fluorescence: To evaluate tertiary structure formation through monitoring of intrinsic fluorescence

• Protease Resistance Assays: Properly folded membrane proteins typically show increased resistance to limited proteolysis

• Thermal Stability Analysis: Using differential scanning calorimetry or thermal shift assays with membrane-compatible dyes

• Reconstitution into Liposomes: Functional incorporation into artificial membranes suggests proper folding

• Binding Assays: If potential interaction partners are identified, binding assays can confirm functional activity
  Research with Y. pseudotuberculosis OmpF porin demonstrates that spectroscopic analysis effectively differentiates between native-like conformations and misfolded states in recombinant membrane proteins .

What experimental approaches can determine the membrane topology of YpsIP31758_2460?

Determining the membrane topology of YpsIP31758_2460 requires specialized techniques to identify transmembrane segments and their orientation. Based on membrane protein research methodologies, the following approaches are recommended:

• Cysteine Scanning Mutagenesis: Systematically replacing residues with cysteine and assessing accessibility to membrane-impermeable sulfhydryl reagents

• Protease Protection Assays: Using proteases to digest exposed regions while membrane-embedded segments remain protected

• Fusion Reporter Techniques: Creating fusions with reporters like alkaline phosphatase or GFP at various positions to determine cytoplasmic vs. periplasmic localization

• Epitope Insertion and Antibody Accessibility: Inserting epitope tags at predicted loop regions and determining their accessibility

• Computational Prediction Validation: Comparing experimental results with predictions from tools like TMHMM, Phobius, or TOPCONS
  These methods should be employed complementarily, as each has limitations. For instance, cysteine scanning provides detailed topological information but requires generating and analyzing numerous mutants, while reporter fusions offer faster results but may disrupt protein folding.

How can researchers investigate potential roles of YpsIP31758_2460 in bacterial physiology?

Investigating YpsIP31758_2460's physiological role requires multiple complementary approaches:

• Gene Deletion/Knockdown: Creating ΔYpsIP31758_2460 mutants to observe phenotypic changes in growth, stress response, or virulence

• Controlled Expression Studies: Using inducible promoters to modulate expression levels and observe dose-dependent effects

• Transcriptional Profiling: RNA-seq analysis comparing wild-type and mutant strains to identify affected pathways

• Stress Response Assays: Exposing mutants to various stressors (pH, temperature, osmotic pressure) to identify conditional phenotypes

• Metabolic Analysis: Comparing metabolite profiles between wild-type and mutant strains

• Protein-Protein Interaction Studies: Pull-down assays or bacterial two-hybrid screening to identify interaction partners
  Similar approaches have been successful in characterizing other Y. pseudotuberculosis membrane proteins. For example, research on the type III secretion system has utilized gene deletion mutants (e.g., ΔyopK ΔyopJ) to characterize protein functions in pathogenicity .

How might YpsIP31758_2460 contribute to Y. pseudotuberculosis virulence?

While specific information about YpsIP31758_2460's role in virulence is limited, we can propose investigative approaches based on Y. pseudotuberculosis pathogenicity research:

• Infection Models: Compare wild-type and ΔYpsIP31758_2460 mutant strains in appropriate infection models (e.g., cell culture invasion assays or mouse models)

• Virulence Factor Expression: Assess whether YpsIP31758_2460 affects expression or function of known virulence factors such as the Yersinia adhesion pathogenicity island (YAPI) components or type III secretion system effectors

• Host Response Analysis: Measure host immune responses (cytokine production, inflammasome activation) to wild-type vs. mutant strains

• Intracellular Survival Assays: Determine if YpsIP31758_2460 affects bacterial survival within host cells

• Transcriptional Coordination: Investigate if YpsIP31758_2460 expression correlates with virulence factor expression under infection-mimicking conditions
  The unique genomic content of Y. pseudotuberculosis IP31758 contributes to its distinctive pathogenicity profile causing Far East scarlet-like fever . As a membrane protein unique to this strain, YpsIP31758_2460 could potentially contribute to the strain's virulence through mechanisms like host cell interaction, stress resistance, or regulation of virulence factor expression.

What protein-protein interaction methods are appropriate for YpsIP31758_2460 studies?

Investigating protein-protein interactions involving membrane proteins like YpsIP31758_2460 presents unique challenges requiring specialized approaches:

How conserved is YpsIP31758_2460 across Yersinia species and strains?

YpsIP31758_2460 is part of a complex evolutionary landscape within the Yersinia genus. Multilocus sequence analysis has revealed that Y. pseudotuberculosis represents a complex of four populations with different levels of genetic diversity and pathogenicity potential . To assess YpsIP31758_2460 conservation, researchers should:

• Perform BLAST searches against sequenced Yersinia genomes to identify homologs

• Calculate sequence identity/similarity percentages across identified homologs

• Use phylogenetic analysis to map the evolutionary relationships of YpsIP31758_2460 homologs

• Identify conserved domains or motifs that might indicate functional importance

• Compare genomic context to detect synteny or gene neighborhood conservation
  Y. pseudotuberculosis IP31758 possesses approximately 261 genes not found in strain IP32953 , suggesting YpsIP31758_2460 may be part of the strain-specific gene repertoire contributing to IP31758's unique pathogenicity profile. Researchers should particularly examine conservation patterns across clinical versus environmental isolates to identify correlations with pathogenicity.

What genomic context surrounds YpsIP31758_2460 and how might it inform function?

Genomic context analysis provides valuable insights into potential functions and regulatory networks involving YpsIP31758_2460:

• Operon Structure Analysis: Determine if YpsIP31758_2460 is part of an operon through transcriptional analysis and promoter prediction

• Gene Neighborhood Conservation: Compare genomic neighborhoods across related species to identify functionally linked genes

• Regulatory Element Identification: Analyze upstream regions for transcription factor binding sites or other regulatory elements

• Mobile Genetic Element Association: Determine if YpsIP31758_2460 is associated with mobile genetic elements such as pathogenicity islands or horizontally acquired regions

• Codon Usage Analysis: Compare codon usage patterns with the core genome to identify potential horizontal gene transfer
  Y. pseudotuberculosis IP31758 contains numerous horizontally acquired genes from Enterobacteriaceae and soil-dwelling bacteria , so determining whether YpsIP31758_2460 is part of the core genome or accessory genome would provide important evolutionary context. The strain's unique plasmids and pathogenicity islands should be investigated for potential functional relationships with YpsIP31758_2460.

What genetic approaches can elucidate YpsIP31758_2460 function in vivo?

Several genetic approaches are suitable for studying YpsIP31758_2460 function in vivo:

• Clean Deletion Mutants: Creating precise gene deletions (ΔYpsIP31758_2460) to assess phenotypic changes

• Complementation Studies: Reintroducing wild-type or mutated versions to confirm phenotype specificity

• Reporter Fusions: Creating transcriptional/translational fusions to study expression patterns

• Conditional Expression Systems: Using inducible promoters to control expression timing and level

• Site-Directed Mutagenesis: Targeting conserved residues to identify functionally important amino acids

• Suppressor Screens: Identifying genetic interactions through suppressor mutation analysis
  The recombinant attenuated Y. pseudotuberculosis strain engineering approach described in the search results demonstrates the feasibility of genetic manipulation in this species. When working with YpsIP31758_2460, researchers should consider using the ΔyopK ΔyopJ Δasd triple mutation background to create an attenuated strain suitable for in vivo studies , particularly if there are biosafety concerns regarding Y. pseudotuberculosis IP31758.

How can comparative genomics inform our understanding of YpsIP31758_2460?

Comparative genomics provides powerful tools for understanding YpsIP31758_2460 in evolutionary and functional contexts:

• Pan-genome Analysis: Determine if YpsIP31758_2460 belongs to the core, accessory, or strain-specific genome

• Selection Pressure Analysis: Calculate dN/dS ratios to identify sites under positive or purifying selection

• Structural Homology Modeling: Identify structurally similar proteins with known functions

• Domain Architecture Comparison: Compare domain organization with related proteins across species

• Phylogenetic Profiling: Correlate presence/absence patterns with particular phenotypes or ecological niches

• Synteny Analysis: Identify conserved gene clusters that might indicate functional relationships
  Y. pseudotuberculosis demonstrates significant genomic diversity, with clear evidence of recombination within Y. pseudotuberculosis s.s. as well as genetic imports from Y. similis and the Korean group . This complex evolutionary history should be considered when interpreting comparative genomic data related to YpsIP31758_2460.

How can structural studies of YpsIP31758_2460 contribute to antimicrobial development?

Structural studies of YpsIP31758_2460 could potentially contribute to antimicrobial development through several pathways:

• Target Validation: If YpsIP31758_2460 proves essential for Y. pseudotuberculosis viability or virulence, its structure could serve as a foundation for rational drug design

• Binding Pocket Identification: Structural analysis may reveal druggable pockets suitable for small molecule binding

• Structure-Based Virtual Screening: Computational methods could identify potential inhibitors based on the protein structure

• Fragment-Based Drug Discovery: Structural data would enable fragment screening and optimization approaches

• Epitope Mapping: For vaccine development, structural data could identify surface-exposed regions suitable as epitopes
  The research challenges include obtaining sufficient quantities of properly folded protein and determining its structure using X-ray crystallography, cryo-EM, or NMR spectroscopy. Techniques developed for Y. pseudotuberculosis OmpF porin could provide methodological foundations, though membrane proteins remain challenging targets for structural biology.

What advanced imaging techniques can visualize YpsIP31758_2460 localization and dynamics?

Several advanced imaging techniques can be applied to study YpsIP31758_2460 localization and dynamics:

How can systems biology approaches integrate YpsIP31758_2460 into broader cellular networks?

Systems biology approaches can place YpsIP31758_2460 within broader cellular networks through:

• Transcriptomic Analysis: RNA-seq comparing wild-type and mutant strains under various conditions to identify genes co-regulated with YpsIP31758_2460

• Proteomics: Quantitative proteomics to detect changes in protein abundance or post-translational modifications dependent on YpsIP31758_2460

• Metabolomics: Metabolite profiling to identify metabolic pathways affected by YpsIP31758_2460 function

• Interactome Mapping: Comprehensive protein-protein interaction screening to place YpsIP31758_2460 in interaction networks

• Computational Network Analysis: Integration of multiple data types to predict functional relationships and system-level effects

• Flux Balance Analysis: For metabolic modeling if YpsIP31758_2460 affects metabolic processes
  The complex genomic landscape of Y. pseudotuberculosis, with significant differences between strains like IP31758 and IP32953 , necessitates strain-specific systems biology approaches. Researchers should focus on conditions relevant to infection processes, as Y. pseudotuberculosis IP31758 causes a distinctive clinical syndrome (FESLF) .

What are the challenges in developing antibodies against YpsIP31758_2460?

Developing antibodies against membrane proteins like YpsIP31758_2460 presents several challenges:

• Limited Immunogenic Epitopes: Membrane-embedded regions are poorly accessible for antibody binding

• Conformational Dependence: Native protein conformation may be essential for relevant epitope recognition

• Purification Difficulties: Obtaining sufficient quantities of properly folded protein for immunization

• Cross-Reactivity Risks: Potential cross-reactivity with host membrane proteins

• Accessibility In Vivo: Determining if antibodies can access the target in intact bacteria
  To address these challenges, researchers should:

• Identify surface-exposed loops based on topology prediction and accessibility analysis

• Synthesize peptides corresponding to these regions for immunization

• Consider using genetic immunization approaches (DNA vaccines encoding YpsIP31758_2460)

• Employ phage display to select antibodies that recognize native conformations

• Validate antibody specificity using knockout strains (ΔYpsIP31758_2460) as negative controls
  Researchers working with Y. pseudotuberculosis membrane proteins have developed various expression and purification strategies that could be adapted for generating immunogens for antibody development against YpsIP31758_2460.

What key research gaps remain in our understanding of YpsIP31758_2460?

Despite advances in Y. pseudotuberculosis research, numerous knowledge gaps remain regarding YpsIP31758_2460:

• Functional Characterization: The fundamental biological function remains unknown

• Structural Information: No experimentally determined structure is available

• Regulation Mechanisms: Expression patterns and regulatory networks are uncharacterized

• Evolutionary History: Precise origin and selective pressures shaping its evolution are unclear

• Role in Pathogenesis: Potential contributions to Y. pseudotuberculosis virulence are not established

• Interaction Partners: Protein-protein interactions and their functional significance remain to be determined
  Addressing these gaps requires integrative approaches combining structural biology, genetic manipulation, functional genomics, and infection models. The strain-specific nature of YpsIP31758_2460 suggests it may contribute to the unique pathogenicity profile of Y. pseudotuberculosis IP31758, potentially including its association with Far East scarlet-like fever .

How can emerging technologies advance YpsIP31758_2460 research?

Emerging technologies offer new opportunities to address challenges in YpsIP31758_2460 research:

• Cryo-EM for Membrane Proteins: Recent advances enable structure determination of smaller membrane proteins

• AlphaFold and Related AI Tools: For accurate structural prediction when experimental structures are unavailable

• Single-Cell Technologies: To study heterogeneity in YpsIP31758_2460 expression during infection

• CRISPR-Based Techniques: For precise genome editing and CRISPRi-based expression modulation

• Nanopore Sequencing: For real-time transcriptional profiling during infection processes

• Organoid Models: More physiologically relevant infection models to study pathogen-host interactions
  These technologies can overcome traditional limitations in membrane protein research and provide unprecedented insights into the function and role of YpsIP31758_2460 in Y. pseudotuberculosis biology and pathogenesis. Combining computational predictions with experimental validation will be particularly powerful for advancing understanding of this challenging protein.

What collaborative research approaches would accelerate YpsIP31758_2460 characterization?

Interdisciplinary collaboration would significantly accelerate YpsIP31758_2460 research:

• Structural Biology and Biochemistry: For protein expression, purification, and structure determination

• Microbial Genetics: For creating and characterizing mutant strains

• Systems Biology: For integrating YpsIP31758_2460 into broader cellular networks

• Immunology: For studying host responses to wild-type versus mutant strains

• Computational Biology: For structure prediction, evolutionary analysis, and network modeling

• Clinical Microbiology: For understanding relevance to human infections Collaborative platforms sharing standardized protocols, strains, and reagents would facilitate comparative studies across laboratories. Given the potential relevance to pathogenicity, coordination between basic research and public health institutions studying Y. pseudotuberculosis outbreaks could provide valuable epidemiological context for laboratory findings.