Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B Protein AaeX (aaeX)

What is Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B Protein AaeX (aaeX)?

Recombinant AaeX is a protein derived from the highly pathogenic Yersinia enterocolitica serotype O:8 / biotype 1B strain. The AaeX protein (aa 1-67) is typically produced through recombinant technology using expression systems such as E. coli, yeast, baculovirus, or mammalian cells . This protein is part of the bacterial membrane and may play a role in pathogenicity. The biotype 1B strain is significant as it belongs to the high pathogenicity category of Y. enterocolitica, containing specific virulence factors that contribute to its disease-causing potential .

What is the amino acid sequence of the AaeX protein?

The AaeX protein from Yersinia pseudotuberculosis serotype O:3, which shares similarities with Y. enterocolitica, has the following amino acid sequence: MSLLPVMVIFGLSFPPIFLELLISLALFFVVRRILQPTGIYEFVWHPALFNTALYCCLFYLTSRLFS . The Y. enterocolitica serotype O:8 / biotype 1B AaeX protein would have a similar but not identical sequence. Researchers working with this protein should verify the specific sequence for their strain of interest, as variations can affect protein function and antigenic properties.

How does Yersinia enterocolitica serotype O:8/biotype 1B differ from other strains?

Y. enterocolitica strains are classified into biotypes based on biochemical characteristics and serotypes based on lipopolysaccharide O-antigens. The biotype 1B/serotype O:8 strain is considered highly pathogenic compared to other biotypes . This strain contains the Yersinia high pathogenicity island, as confirmed by genomic analysis . In experimental studies, biotype 1B strains demonstrate different patterns of protein expression and regulation compared to lower pathogenicity strains like biotype 2/O:9. For example, research has shown differences in urease expression regulation between these strains, with the regulator OmpR having strain-specific effects .

What expression systems are recommended for producing recombinant AaeX protein?

For recombinant production of Y. enterocolitica AaeX protein, several expression systems can be utilized:

• E. coli expression system: Most commonly used due to its simplicity and high yield. Optimal for initial characterization studies .

• Yeast expression system: Useful when post-translational modifications may be important.

• Baculovirus expression system: Provides a eukaryotic environment that can be advantageous for certain applications.

• Mammalian cell expression system: Most suitable when studying host-pathogen interactions in a context closer to natural infection .

The choice of expression system should be guided by the specific research questions and downstream applications. For immunological studies or vaccine development, preserving the native conformation of the protein is crucial, which might require eukaryotic expression systems.

What methods are effective for detecting AaeX-expressing Y. enterocolitica strains?

Several molecular methods have been developed for detecting Y. enterocolitica strains:

• PCR-based detection: Using primers designed from invasion genes present only in pathogenic strains. This approach has been adapted for direct use with clinical samples, including tissues and feces .

• Nested PCR assay: This modification improves sensitivity when analyzing pig tissues, overcoming initial inhibition issues encountered with direct PCR .

• Pre-PCR enrichment: This step enhances detection in fecal samples by allowing bacterial multiplication before PCR analysis .

• Pulsed field gel electrophoresis (PFGE): Useful for strain typing and epidemiological studies, allowing identification of potential reservoirs and assessment of strain heterogeneity .

When specifically targeting AaeX-expressing strains, primers designed from the aaeX gene sequence can be incorporated into these protocols for selective detection.

What protocols are recommended for studying AaeX protein function?

To investigate AaeX protein function, consider the following methodological approaches:

• Gene knockout studies: Creating aaeX deletion mutants through homologous recombination using suicide vectors (similar to the technique used for ompB deletion) . This allows comparison of wild-type and mutant strains to identify phenotypic differences.

• Complementation assays: Reintroducing the aaeX gene via plasmid to confirm that observed phenotypic changes are specifically due to aaeX deletion.

• Protein-protein interaction studies: Techniques such as pull-down assays, yeast two-hybrid systems, or co-immunoprecipitation can identify potential interaction partners of AaeX.

• Cellular localization studies: Using fluorescently tagged AaeX to determine its localization within bacterial cells under various conditions.

• Heterologous expression: Expressing AaeX in non-pathogenic bacteria to study which virulence characteristics it might confer.

How does AaeX contribute to the immune response in Yersinia infections?

While specific information about AaeX's immunological role is limited in the provided search results, we can draw parallels from studies of other Yersinia antigens:

In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components . Research has identified several immunodominant T cell antigens in Y. enterocolitica, including the 19-kd urease beta subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and heat-shock protein 60 (hsp60) .

To investigate AaeX's potential role as an immunodominant antigen:

• Lymphocyte proliferation assays using synovial fluid mononuclear cells from patients with suspected Yersinia infections can be performed with purified recombinant AaeX.

• T cell clones specific for AaeX can be generated and characterized for their cytokine secretion patterns to determine if they exhibit Th1 or Th2-like responses.

• Cross-reactivity studies can assess whether AaeX-specific T cells recognize human proteins, which would suggest a potential role in autoimmunity.

How can recombinant AaeX be used in developing diagnostic tools for Y. enterocolitica infections?

Recombinant Y. enterocolitica AaeX protein has potential applications in diagnostic tool development:

• Serological assays: ELISA tests using recombinant AaeX as the capture antigen can detect anti-AaeX antibodies in patient sera, potentially indicating current or past Y. enterocolitica infection.

• T cell-based diagnostics: Lymphocyte proliferation assays using recombinant AaeX can identify Yersinia-specific T cell responses in patients, similar to the approach used with other Yersinia antigens in patients with Yersinia ReA .

• Multiplex antigen panels: Combining AaeX with other immunodominant Yersinia antigens could enhance diagnostic sensitivity and specificity.

• Lateral flow assays: Development of rapid point-of-care tests using recombinant AaeX for field diagnostics in both medical and veterinary settings.

The effectiveness of these approaches depends on determining whether AaeX elicits a consistent and measurable immune response during Y. enterocolitica infections.

How is AaeX expression regulated in different Y. enterocolitica strains?

While specific information about AaeX regulation is not detailed in the search results, insights can be drawn from studies of other Y. enterocolitica proteins:

The expression of various proteins in Y. enterocolitica is regulated by factors such as:

• Temperature: Many virulence factors show temperature-dependent expression. For example, urease activity in strain Ye9N (biotype 2/O:9) is higher at 26°C than at 37°C .

• Transcriptional regulators: OmpR has been shown to regulate the expression of multiple proteins in Y. enterocolitica. Proteomic analysis identified that OmpR positively regulates the production of two urease structural subunits (UreA and UreC) in strain Ye9N .

• Strain-specific regulation: The same regulator can have different effects in different Y. enterocolitica strains. For example, OmpR plays different roles in controlling urease expression in low pathogenic (biotype 2/O:9) versus highly pathogenic (biotype 1B/O:8) strains .

To investigate AaeX regulation, similar approaches could be applied:

• Comparing AaeX expression at different temperatures (26°C vs. 37°C) using qRT-PCR or Western blotting.

• Creating knockout mutants of potential regulators (like OmpR) and assessing their impact on AaeX expression.

• Performing promoter analysis to identify regulatory elements in the aaeX gene.

What role might AaeX play in acid survival of Y. enterocolitica?

Y. enterocolitica strains require mechanisms to survive acidic environments encountered during infection. While AaeX's specific role in acid survival is not directly mentioned in the search results, we can consider potential involvement based on information about acid survival mechanisms in Y. enterocolitica:

• The regulator OmpR has been shown to be necessary for survival of Y. enterocolitica strains of biotypes 1B/O:8 and 2/O:9 in acidic conditions . When exposed to pH 4.0 for 90 minutes, OmpR-deficient mutants displayed significantly decreased survival compared to wild-type strains .

• OmpR also regulates urease expression, which can contribute to acid resistance by hydrolyzing urea to produce ammonia that neutralizes acid .

To investigate if AaeX contributes to acid survival:

• Create aaeX deletion mutants and assess their survival under acidic conditions.

• Determine if AaeX expression is upregulated in acidic environments.

• Investigate potential interactions between AaeX and known acid-resistance systems.

• Assess whether AaeX is co-regulated with urease or other acid-resistance factors.

How can genome sequencing enhance our understanding of AaeX function?

Genome sequencing approaches have been valuable in characterizing Y. enterocolitica strains and can be applied to understand AaeX function:

• Whole genome sequencing: The Y. enterocolitica biotype 1B strain genome has been sequenced using technologies like Illumina NextSeq 500 with the Nextera XT protocol . Similar approaches can be used to:

  • Identify variations in the aaeX gene across different strains

  • Analyze the genetic context of aaeX to understand potential operons or regulatory elements

  • Perform comparative genomics to identify species-specific features of aaeX

• De novo assembly: As demonstrated with strain IP41365 , de novo assembly of sequenced genomes can help identify genomic islands and other mobile genetic elements that might influence aaeX function.

• In silico genomic analysis: This approach can confirm the presence of pathogenicity islands and virulence determinants that might interact with AaeX .

• Transcriptomic analysis: RNA-seq can identify conditions that influence aaeX expression and co-regulated genes, providing insights into its functional role.

What experimental designs are most effective for studying AaeX protein-host interactions?

To investigate AaeX protein-host interactions, consider the following experimental approaches:

• Cell culture models:

  • Infection of epithelial cell lines with wild-type and aaeX mutant Y. enterocolitica

  • Assessment of adhesion, invasion, and intracellular survival

  • Analysis of host cell cytokine responses using ELISA or multiplex cytokine arrays

• T cell response studies:

  • Stimulation of synovial fluid mononuclear cells with recombinant AaeX

  • Generation of AaeX-specific T cell clones

  • Characterization of cytokine secretion patterns (Th1 vs. Th2) using ELISA

• Animal infection models:

  • Comparison of wild-type and aaeX mutant strains in mouse infection models

  • Analysis of bacterial dissemination and persistence in tissues

  • Assessment of tissue pathology and immune responses

• Protein-protein interaction studies:

  • Yeast two-hybrid screens to identify host proteins that interact with AaeX

  • Pull-down assays using tagged recombinant AaeX

  • Immunoprecipitation followed by mass spectrometry to identify interaction partners

What is the potential relationship between AaeX and urease activity in Y. enterocolitica?

While a direct link between AaeX and urease activity is not established in the search results, investigating potential relationships could be valuable since urease is an important virulence factor:

• Urease plays a role in acid resistance and is differentially regulated in different Y. enterocolitica strains . The ureolytic activity varies by:

  • Strain: Higher activity in low pathogenic strain Ye9N than highly pathogenic strain Ye8N

  • Temperature: Higher activity at 26°C than 37°C in strain Ye9N

  • Regulatory factors: Positively regulated by OmpR in strain Ye9N but not significantly affected in strain Ye8N

• To investigate potential relationships between AaeX and urease:

  • Compare urease activity in wild-type and aaeX mutant strains using Christensen's urea agar method

  • Perform quantitative RT-PCR to determine if aaeX deletion affects transcription of urease genes

  • Use proteomic analysis to assess if AaeX affects the production of urease subunits

  • Investigate if AaeX and urease are co-regulated under various environmental conditions

What novel therapeutic approaches might target AaeX protein?

Based on our understanding of Y. enterocolitica pathogenesis, several therapeutic approaches targeting AaeX could be explored:

• Inhibitory peptides or small molecules: If AaeX plays a role in virulence, designing molecules that specifically bind to and inhibit its function could reduce pathogenicity.

• Vaccine development: Recombinant AaeX protein could serve as a vaccine antigen if it proves to be immunogenic and protective. Research has already shown that recombinant Y. enterocolitica antigens can be useful for vaccine development .

• Targeted antibody therapy: If AaeX is surface-exposed, antibodies targeting this protein could potentially neutralize Y. enterocolitica or enhance opsonization and clearance.

• Combination approaches: Targeting AaeX alongside other virulence factors, such as urease, might have synergistic effects in preventing or treating Yersinia infections.

• CRISPR-Cas systems: Developing phage-delivered CRISPR-Cas systems targeting the aaeX gene could provide a novel antimicrobial approach.

How might comparative analysis of AaeX across Yersinia species inform evolutionary understanding?

Comparative analysis of AaeX across Yersinia species can provide insights into bacterial evolution and adaptation:

• Sequence conservation analysis: Comparing aaeX sequences from Y. enterocolitica, Y. pseudotuberculosis , and other Yersinia species can identify conserved domains that might be functionally important.

• Structural prediction and comparison: Using bioinformatic tools to predict protein structures and comparing these across species can highlight structural conservation despite sequence variations.

• Expression pattern analysis: Investigating whether aaeX expression patterns differ across Yersinia species in response to environmental conditions could reveal adaptive strategies.

• Functional complementation studies: Testing whether AaeX from one Yersinia species can complement an aaeX deletion in another species would indicate functional conservation.

• Evolutionary pressure analysis: Calculating the ratio of non-synonymous to synonymous substitutions in aaeX across species can determine if the gene is under positive, negative, or neutral selection.

This evolutionary perspective could provide context for understanding AaeX's role in virulence and identify potential targets for broad-spectrum interventions against Yersinia infections.