Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B UPF0266 membrane protein YE1773 (YE1773)

What is the basic structural information of YE1773 protein?

YE1773 is a membrane protein belonging to the UPF0266 family from Yersinia enterocolitica serotype O:8 / biotype 1B. It consists of 153 amino acids with a molecular weight of approximately 17.8 kDa. The complete amino acid sequence is:
MSVTDIVLIVFIALLLAYAIYDEFIMNMMKGKTRLQIQLKRKSKIDCAIFVGLIAILVYNNVMANGEPLTTYLLVGLALIAFYLSYIRWPKLLFKNTGFFYANAFIEYRRIKSMNLSEDGILVIDLEQRRLLIQVRQLDDLEKIYNFFVENQS

The protein's membrane localization suggests it may play a role in bacterial-host interactions, though its precise function remains to be fully characterized through experimental studies.

How is YE1773 classified taxonomically within bacterial proteins?

YE1773 belongs to the UPF0266 protein family, a classification of uncharacterized protein families with conserved sequences but undetermined functions. It is specifically found in Yersinia enterocolitica serotype O:8 / biotype 1B (strain NCTC 13174 / 8081). This particular serotype/biotype combination is significant as it represents a highly pathogenic variant of Y. enterocolitica associated with more severe infections in humans .

When designing experiments to elucidate function, researchers should consider comparative genomics approaches with other Yersinia species and related Enterobacteriaceae to identify potential conserved domains and functional similarities.

What expression systems are optimal for producing recombinant YE1773?

The most validated expression system for recombinant YE1773 is Escherichia coli. Based on available data, full-length YE1773 (amino acids 1-153) can be successfully expressed in E. coli with an N-terminal His-tag . When designing your expression protocol, consider the following methodological approach:

• Clone the YE1773 gene into an expression vector containing an N-terminal His-tag sequence

• Transform the construct into an E. coli strain optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3))

• Induce protein expression at lower temperatures (16-20°C) to minimize inclusion body formation

• Harvest cells and lyse using appropriate detergents to solubilize membrane proteins

• Purify using nickel affinity chromatography followed by size exclusion chromatography

Researchers should conduct optimization experiments testing multiple expression conditions, as membrane proteins can be challenging to express in their native conformation.

What are the recommended storage conditions for purified recombinant YE1773?

Purified recombinant YE1773 protein should be stored according to the following protocol to maintain stability and function:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot the protein solution to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• Store long-term aliquots at -20°C/-80°C

Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of function. When designing experiments requiring multiple uses of the protein, prepare appropriate aliquot volumes to minimize waste and maintain protein integrity.

How should researchers design experiments to elucidate the membrane topology of YE1773?

To determine the membrane topology of YE1773, a multi-method experimental approach is recommended:

• Computational prediction: Begin with in silico analysis using membrane protein topology prediction algorithms such as TMHMM, Phobius, or TOPCONS to generate initial hypotheses about transmembrane regions.

• Cysteine scanning mutagenesis: Systematically replace amino acids with cysteine residues throughout the protein sequence and assess their accessibility to membrane-impermeable thiol-reactive reagents.

• Fusion protein approach: Create fusion constructs with reporter proteins (GFP, PhoA, LacZ) at various positions to determine which segments are intracellular versus extracellular.

• Protease protection assay: Express YE1773 in membrane vesicles and treat with proteases to identify accessible regions.

• Structural analysis: If possible, employ advanced techniques such as X-ray crystallography or cryo-electron microscopy to resolve the three-dimensional structure.

When interpreting results, researchers should remember that YE1773 belongs to the UPF0266 family, which may provide comparative insights based on the limited knowledge of related proteins .

What experimental design would best determine if YE1773 interacts with host cell proteins during infection?

A rigorous experimental design to investigate potential YE1773 interactions with host proteins should follow this methodological framework:

Stage 1: Identification of potential interactions

• Perform pull-down assays using His-tagged recombinant YE1773 and host cell lysates

• Conduct yeast two-hybrid screening with YE1773 as bait against human cDNA libraries

• Use proximity labeling techniques (BioID or APEX) with YE1773 expressed in host cells

Stage 2: Validation of interactions

• Confirm direct interactions using purified proteins in in vitro binding assays

• Perform co-immunoprecipitation experiments in infected host cells

• Visualize co-localization using fluorescence microscopy

Stage 3: Functional characterization

• Generate YE1773 deletion mutants in Y. enterocolitica

• Compare wildtype and mutant strains in infection models

• Assess specific host response pathways based on the identified interactions

This approach should be designed with appropriate controls, considering that Yersinia species are known to manipulate host signaling pathways through type III secretion systems and effector proteins that target cellular processes including Rho GTPase signaling and histone modifications .

How can researchers investigate the potential role of YE1773 in Y. enterocolitica serotype O:8 virulence?

To investigate YE1773's potential role in virulence, researchers should implement a comprehensive experimental design:

Step 1: Generate and validate YE1773 mutants

• Create YE1773 deletion mutants using allelic exchange methods

• Construct complemented strains expressing YE1773 from a plasmid

• Verify mutation and complementation by PCR, sequencing, and Western blot

Step 2: In vitro virulence assays

• Assess bacterial adhesion and invasion using epithelial cell lines

• Measure survival within macrophages over time

• Evaluate cytotoxicity toward different host cell types

• Analyze secretion of virulence factors using proteomics approaches

Step 3: In vivo infection models

• Compare colonization ability in mouse infection models

• Measure bacterial dissemination to different organs

• Assess inflammatory responses and tissue damage

• Monitor survival rates of infected animals

When interpreting results, researchers should consider that Yersinia enterocolitica serotype O:8 is associated with more severe infections and may persist latently in healthy carriers . Additionally, investigators should assess whether YE1773 influences known Yersinia virulence mechanisms such as type III secretion or modulation of host chromatin states .

What techniques can be used to determine if YE1773 affects host chromatin modifications similar to other Yersinia effectors?

Given that Yersinia enterocolitica can alter host chromatin states to reprogram gene expression , researchers investigating if YE1773 has similar effects should employ the following methodological approach:

Stage 1: Preliminary assessment

• Infect human macrophages with wildtype and YE1773-deficient Y. enterocolitica

• Perform Western blot analysis for key histone modifications (H3K4me3, H3K27ac)

• Assess nuclear morphology and chromatin condensation by microscopy

Stage 2: Genome-wide analysis

• Conduct ChIP-seq for relevant histone modifications in infected versus uninfected cells

• Compare the histone modification patterns between cells infected with wildtype bacteria versus YE1773 mutants

• Perform RNA-seq to correlate histone modification changes with gene expression alterations

Stage 3: Mechanistic studies

• Determine if purified YE1773 directly interacts with histones or chromatin-modifying enzymes

• Investigate if YE1773 localizes to the nucleus during infection

• Assess if YE1773 influences the activity of other known Yersinia effectors like YopP

This experimental design draws on the finding that Yersinia effectors reorganize histone modifications at approximately 14,500 loci in promoters and enhancers, with the effector YopP responsible for about half of these modulatory activities .

How can researchers design experiments to determine if YE1773 affects Rho GTPase signaling pathways?

Given that Yersinia effectors are known to target Rho GTPase pathways and that altered histone modifications affect 61% of all known Rho GTPase pathway genes , a sophisticated experimental approach to investigate YE1773's potential role would include:

Stage 1: Biochemical interaction studies

• Conduct pull-down assays using purified YE1773 and GTPases (RhoA, Rac1, Cdc42)

• Perform in vitro GTPase activity assays to determine if YE1773 affects GTP hydrolysis

• Assess binding of YE1773 to GTPase regulators (GEFs, GAPs, GDIs)

Stage 2: Cellular signaling analysis

• Express YE1773 in host cells and monitor Rho GTPase activation using FRET-based biosensors

• Investigate cytoskeletal rearrangements using live-cell imaging

• Compare phosphorylation states of downstream effectors in control vs. YE1773-expressing cells

Stage 3: Infection context

• Compare Rho GTPase activity in cells infected with wildtype vs. YE1773-deficient bacteria

• Assess if YE1773 deletion affects other Yersinia effectors that target Rho GTPases

• Evaluate if YE1773 and YpkA (a known Yersinia protein kinase targeting Galphaq signaling ) have synergistic effects

This experimental framework recognizes the interconnected nature of bacterial virulence mechanisms and considers that YE1773 may function either directly or indirectly in modulating host cell signaling pathways.

What methodological approaches can resolve contradictory findings related to UPF0266 family proteins across different bacterial species?

When confronted with contradictory findings about UPF0266 family proteins (to which YE1773 belongs) across different bacterial species, researchers should implement a systematic approach to resolve discrepancies:

• Structural homology modeling:

  • Generate 3D models of YE1773 and homologs from different species

  • Identify conserved domains and divergent regions

  • Correlate structural differences with observed functional variations

• Cross-species complementation experiments:

  • Generate deletion mutants of UPF0266 family proteins in multiple bacterial species

  • Express YE1773 in these heterologous hosts and assess functional complementation

  • Identify species-specific versus conserved functions

• Domain swapping and chimeric protein analysis:

  • Create chimeric proteins combining domains from UPF0266 family members of different species

  • Test these constructs for function in various experimental systems

  • Map functional domains to specific protein regions

• Evolutionary analysis:

  • Conduct comprehensive phylogenetic analysis of UPF0266 family proteins

  • Correlate protein sequence divergence with host adaptation and pathogenicity

  • Apply selection pressure analysis to identify rapidly evolving regions

• Multi-omics approach:

  • Compare transcriptome, proteome, and metabolome data from wildtype and mutant strains across species

  • Identify common and distinct pathways affected by UPF0266 family proteins

  • Generate integrated models explaining species-specific observations

This methodological framework accounts for the possibility that UPF0266 family proteins may have evolved different functions in various bacterial species despite sequence homology, which could explain contradictory findings in the literature.