Recombinant Salmonella dublin UPF0060 membrane protein ynfA (ynfA)

What is Salmonella dublin UPF0060 membrane protein ynfA?

Salmonella dublin UPF0060 membrane protein ynfA is a bacterial membrane protein found in Salmonella dublin, a zoonotic pathogen that poses significant threats to both animal and human health . The protein is part of the UPF0060 family of membrane proteins, which are conserved across various bacterial species including Escherichia coli and Shigella flexneri . The ynfA protein in Salmonella dublin (strain CT_02021853) consists of 108 amino acids and has been assigned the UniProt accession number B5FHP9 . This protein is primarily localized to the bacterial membrane and may play important roles in membrane integrity and bacterial survival, though its precise functions remain under investigation.

How conserved is the UPF0060 membrane protein ynfA across bacterial species?

UPF0060 membrane protein ynfA shows notable sequence conservation across multiple bacterial species, particularly among enterobacteria. Comparative sequence analysis reveals high similarity between Salmonella dublin ynfA and its homologs in Escherichia coli and Shigella flexneri . The table below highlights the amino acid sequence similarities among these species:

This high degree of conservation suggests the protein may have fundamental roles in bacterial physiology that have been maintained through evolution . The conservation is particularly strong in the transmembrane regions, while terminal regions show more variability, potentially reflecting species-specific functional adaptations.

What are the recommended storage conditions for recombinant Salmonella dublin UPF0060 membrane protein ynfA?

For optimal stability and activity of recombinant Salmonella dublin UPF0060 membrane protein ynfA, the recommended storage conditions are:

• Primary storage: -20°C for regular use or -80°C for extended storage

• Storage buffer: Tris-based buffer with 50% glycerol, optimized for this specific protein

• Working aliquots: Store at 4°C for up to one week to minimize freeze-thaw cycles

• Important precaution: Repeated freezing and thawing is not recommended as it may lead to protein degradation and loss of activity

When reconstituting lyophilized preparations, it is advisable to centrifuge the vial briefly before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, adding glycerol to a final concentration of 30-50% is recommended before aliquoting and storing at -20°C or -80°C . These storage conditions are critical for maintaining protein integrity and ensuring reproducible experimental results.

What methodologies are most effective for detecting Salmonella dublin in research samples?

Detection of Salmonella dublin in research samples presents significant challenges due to its intermittent shedding pattern and relatively low culture sensitivity. A multi-faceted approach is recommended:

• qPCR-based detection: Quantitative PCR targeting S. Dublin-specific genomic regions offers higher sensitivity than culture methods. Standard curves can be constructed using serial dilutions of S. Dublin ATCC 15480 DNA, with copy number calculation following the equation:

  $\text{DNA copies} = \frac{6.02 \times 10^{23} \text{ (copy/mol)} \times \text{DNA amount (g)}}{\text{DNA length (bp)} \times 660 \text{ (g/mol/bp)}}$

• Culture-based methods: Despite sensitivity limitations (16-20%), culture remains important for confirming viable bacteria . Protocol involves:

  • Pre-enrichment in non-selective media

  • Subsequent culturing on selective media

  • Biochemical confirmation tests

• Serological testing: ELISA targeting antibodies against Salmonella LPS O-antigens (1, 9, and 12) with manufacturer-recommended cut-off of 35% positivity, though some evidence suggests a 20% cut-off could be sufficient for detecting latent carriers

• Combined approach: For maximum reliability in research settings, a combined approach using both direct (PCR, culture) and indirect (serology) methods is recommended, with serial testing (multiple samples taken 60 days apart) to confidently classify latent carriers .

The relatively low sensitivity of individual tests (particularly culture methods at 16-20%) underscores the importance of using multiple detection approaches in research settings for comprehensive sample analysis .

How does Salmonella dublin UPF0060 membrane protein ynfA contribute to pathogenicity?

While direct evidence linking Salmonella dublin UPF0060 membrane protein ynfA to pathogenicity remains limited, several lines of evidence suggest potential roles:

• Membrane integrity and permeability: As a membrane protein with multiple predicted transmembrane domains, ynfA likely contributes to bacterial membrane structure and function, potentially influencing cell envelope stability and permeability to antimicrobial agents .

• Conservation across pathogenic bacteria: The high degree of conservation among pathogenic enterobacteria suggests functional importance . Similar membrane proteins in related pathogens have been implicated in stress responses and survival under hostile host conditions.

• Potential role in host-pathogen interactions: Membrane proteins often mediate interactions with host cells and environmental sensing. The composition of ynfA suggests it may participate in sensing or responding to host environmental cues .

• Possible contribution to antimicrobial resistance: Recent research has identified increasing antimicrobial resistance in S. Dublin isolates, and membrane proteins like ynfA could potentially contribute to this phenotype by altering membrane permeability or participating in efflux mechanisms .

Current research at the Quadram Institute and University of Edinburgh is exploring genetic and phenotypic variations within S. Dublin with cutting-edge genome sequencing and phenotyping techniques, which may further elucidate ynfA's specific contributions to pathogenicity .

What animal models are most appropriate for studying Salmonella dublin infection?

Calves represent the most physiologically relevant animal model for studying Salmonella dublin infection, with specific considerations for experimental design:

• Age considerations: One-week-old calves have been successfully used in experimental models, though vulnerability to infection varies with age .

• Route of administration:

  • Intravenous injection produces more consistent results than oral administration in experimental settings

  • Oral administration, while more natural, produces variable clinical outcomes

• Antibody considerations:

  • The severity of S. Dublin infection is significantly influenced by pre-existing antibody levels

  • Paradoxically, the presence of specific antibody has been observed to exacerbate disease severity in some experimental models

  • An inverse relationship between low initial antibody titers and survival period has been reported

• Recommendation for antibacterial efficacy testing: Colostrum-deprived animals are preferred for evaluating antibacterial compounds, as this approach dissociates symptoms from the effects of passively acquired antibody .

• Clinical monitoring parameters: Consistent with naturally occurring cases, experimental models should monitor fever, diarrhea, respiratory symptoms, and bacteremia, with hypersensitivity reactions (potentially anaphylactic or Arthus-type) being significant contributors to pathology .

This experimental model has been specifically developed for evaluating antibacterial compounds against S. Dublin infection and provides a physiologically relevant system that replicates the key features of natural infection .

What are the current challenges in developing effective vaccines against Salmonella dublin?

Development of effective vaccines against Salmonella dublin faces several significant challenges:

• Complex host-pathogen interaction: S. Dublin infection severity is paradoxically exacerbated by the presence of specific antibodies in some cases, complicating vaccine development strategies that rely on antibody production .

• Latent carrier state: S. Dublin can establish a latent carrier state in cattle, making complete elimination difficult. Vaccinating S. Dublin latent carrier cows at dry-off with a commercial live culture vaccine has shown promise in reducing intrauterine transmission to calves, but does not completely eliminate the carrier state .

• Genetic and phenotypic variations: There is significant genetic and phenotypic variation within S. Dublin strains, requiring vaccines to provide broad protection against diverse isolates . Current research at the Quadram Institute and University of Edinburgh is specifically targeting this challenge.

• Antimicrobial resistance concerns: Increasing antimicrobial resistance in S. Dublin isolates from both cattle and humans complicates treatment options, elevating the importance of preventive approaches like vaccination .

• Zoonotic potential: As a zoonotic pathogen that can transmit from cattle to humans (particularly through raw milk and cheese), vaccines must address both animal health and public health concerns .

Despite these challenges, research continues to progress, with a recent BBSRC-funded project (£1,159,625) specifically aimed at addressing these issues through cutting-edge genome sequencing and phenotyping techniques to aid in the development of effective surveillance, control programs, and potential vaccines .

How can recombinant UPF0060 membrane protein ynfA be utilized in diagnostic test development?

Recombinant Salmonella dublin UPF0060 membrane protein ynfA offers several promising applications for improved diagnostic test development:

• ELISA-based detection systems: Recombinant ynfA protein can serve as a specific antigen in enzyme-linked immunosorbent assays for detecting antibodies against S. Dublin . Such systems could overcome the limitations of current ELISA tests that target LPS O-antigens 1, 9, and 12, which may cross-react with other Salmonella serovars .

• Recombinant protein standards for qPCR: Purified recombinant ynfA can be used to establish standard curves for quantitative PCR assays, improving accuracy in determining bacterial load in research and clinical samples . This approach could enhance the sensitivity of detection methods beyond the current 16-20% sensitivity observed in culture-based methods .

• Development of monoclonal antibodies: Recombinant ynfA can be used to develop highly specific monoclonal antibodies for immunodiagnostic applications, potentially enabling rapid detection methods with improved specificity compared to current serological tests .

• Multiplex diagnostic platforms: Integration of recombinant ynfA-based detection with other S. Dublin biomarkers could enhance diagnostic accuracy through multiplexed platforms, addressing the challenge of intermittent shedding patterns in latent carriers .

• Point-of-care testing: With appropriate antibody development, recombinant ynfA could enable development of point-of-care diagnostic tests applicable in field settings for rapid screening of cattle herds .

Implementation of these approaches could significantly improve our ability to detect S. Dublin in both research and clinical settings, enhancing surveillance and control programs, particularly for latent carriers that represent a significant reservoir for transmission .

How might comparative analysis of ynfA across bacterial species inform evolutionary relationships?

Comparative analysis of UPF0060 membrane protein ynfA across bacterial species provides valuable insights into evolutionary relationships and functional conservation:

• Sequence homology analysis: The high sequence similarity between Salmonella dublin ynfA (UniProt: B5FHP9) and homologs in Escherichia coli (UniProt: B7NUQ7) and Shigella flexneri (UniProt: Q0T4M5) suggests recent evolutionary divergence and functional conservation . Key differences in amino acid composition can be analyzed to determine species-specific adaptations.

• Structural conservation patterns: Comparison of transmembrane topology predictions across species reveals that the membrane-spanning regions of ynfA are more highly conserved than terminal domains, suggesting evolutionary pressure to maintain specific membrane interactions while allowing terminal regions to adapt to species-specific functions .

• Genomic context analysis: Examining the genomic neighborhood of ynfA across species can reveal conserved operons or regulatory elements that have been maintained through evolution, providing insights into functional associations and regulatory networks .

• Horizontal gene transfer assessment: The pattern of conservation across enterobacteria could be analyzed to determine whether ynfA has been subject to horizontal gene transfer events, which would inform our understanding of pathogen evolution and adaptation .

This comparative approach not only illuminates evolutionary relationships among these pathogenic bacteria but may also identify species-specific adaptations in ynfA that contribute to the unique pathogenicity profiles of Salmonella dublin compared to other enteric pathogens .

What molecular mechanisms underlie the transmission of Salmonella dublin from carrier cows to calves?

Recent research has shed light on the molecular mechanisms involved in Salmonella dublin transmission from carrier cows to calves:

• Intrauterine transmission: S. Dublin latent carrier cows can transmit the pathogen to calves during pregnancy. A recent study demonstrated that vaccination of S. Dublin latent carrier cows at dry-off with a commercial live culture vaccine reduced this intrauterine transmission .

• Intermittent shedding patterns: S. Dublin carriers exhibit intermittent bacterial shedding, which complicates detection and control. This shedding pattern is thought to be influenced by stress, immunosuppression, and other physiological changes during the periparturient period .

• Role of antibody response: Paradoxically, there appears to be an inverse relationship between low initial antibody titers and survival period in calves, suggesting complex immunological interactions . The severity of S. Dublin infection can be exacerbated by the presence of specific antibody, with some symptoms consistent with anaphylactic or Arthus-type hypersensitivity .

• Maternal antibody influence: Anaphylaxis associated with S. Dublin infection has been linked to antibody derived from maternal colostrum, while Arthus hypersensitivity may be associated with antibody synthesized by the calf itself .

• Diagnostic challenges: The relatively low sensitivity of individual diagnostic tests (particularly culture methods at 16-20%) complicates detection of carrier animals . More reliable classification of latent carriers requires multiple positive tests taken 60 days apart .

Understanding these mechanisms is crucial for developing effective control strategies, including targeted vaccination programs and improved diagnostic approaches for identifying carrier animals before transmission occurs .

What are the implications of Salmonella dublin UPF0060 membrane protein ynfA for antimicrobial resistance?

While direct evidence linking Salmonella dublin UPF0060 membrane protein ynfA to antimicrobial resistance (AMR) remains limited, several properties suggest potential implications:

• Membrane location and permeability: As a membrane protein, ynfA could potentially influence bacterial membrane permeability and therefore affect the entry of antimicrobial agents into the cell . The hydrophobic amino acid composition and predicted transmembrane domains support this possibility.

• Growing AMR concerns: S. Dublin is increasingly associated with antimicrobial resistance in both cattle and human isolates, representing a significant public health concern . Current research at the Quadram Institute and University of Edinburgh specifically addresses this emerging issue.

• Potential involvement in stress responses: Membrane proteins similar to ynfA in other bacterial species have been implicated in stress response mechanisms, which can contribute to antimicrobial tolerance and persistence .

• Conservation across resistant strains: Comparative analysis of ynfA across antimicrobial-resistant and susceptible S. Dublin isolates could reveal whether specific sequence variations correlate with resistance phenotypes, potentially identifying functional domains involved in AMR.

• Target for novel antimicrobials: Conversely, the conservation of ynfA across pathogenic bacteria suggests it could potentially serve as a target for novel antimicrobial development, particularly if it proves essential for bacterial survival or virulence .

The increasing incidence of AMR in S. Dublin isolates underscores the importance of understanding the potential contributions of membrane proteins like ynfA to resistance mechanisms, particularly given the pathogen's significance in both veterinary and human medicine .

What experimental controls should be included when working with recombinant Salmonella dublin UPF0060 membrane protein ynfA?

When designing experiments with recombinant Salmonella dublin UPF0060 membrane protein ynfA, the following controls should be incorporated to ensure robust and reproducible results:

• Positive controls:

  • Purified S. Dublin ATCC 15480 isolate for PCR and genomic studies

  • Known positive serum samples for antibody detection assays

  • Commercially available recombinant UPF0060 membrane proteins from related species (E. coli, Shigella) for comparative studies

• Negative controls:

  • Buffer-only controls for protein-based assays

  • Nuclease-free water instead of template DNA for PCR-based detection

  • Samples from S. Dublin-free herds for validation of diagnostic approaches

• Expression controls:

  • Empty vector controls when expressing recombinant proteins

  • Expression of an unrelated protein using the same expression system

  • Western blot confirmation of protein expression with tag-specific antibodies

• Functional controls:

  • Inclusion of fresh and freeze-thawed protein preparations to assess stability

  • Testing of protein activity immediately after preparation and after storage

  • Dose-response experiments to determine optimal protein concentrations

• Specificity controls:

  • Testing for cross-reactivity with proteins from related Salmonella serovars

  • Validation that detection methods are specific to S. Dublin and not detecting related pathogens

These controls are essential for validating experimental findings and ensuring that observed effects are specifically attributable to the recombinant Salmonella dublin UPF0060 membrane protein ynfA rather than experimental artifacts or contaminating factors .