Recombinant Yersinia pseudotuberculosis serotype IB Fumarate reductase subunit C (frdC)

What are the synonyms for the frdC gene in Y. pseudotuberculosis?

The frdC gene in Y. pseudotuberculosis is also known by several alternate designations:

• YpsIP31758_3669

• Fumarate reductase 15 kDa hydrophobic protein

• Quinol-fumarate reductase subunit C

• QFR subunit C

When conducting literature searches or database queries, researchers should use all these alternative names to ensure comprehensive retrieval of relevant information.

What expression systems are recommended for producing recombinant Y. pseudotuberculosis frdC?

E. coli is the preferred expression system for recombinant Y. pseudotuberculosis frdC protein production. Based on established protocols, the protein is typically expressed with an N-terminal His tag to facilitate purification . This approach has several advantages:

• E. coli provides high yield and efficient expression of bacterial membrane proteins

• N-terminal His tagging allows for single-step affinity purification

• The expression constructs can be designed to include the full-length protein (amino acids 1-130)

For optimal expression, consider using E. coli strains specifically designed for membrane protein expression, such as C41(DE3) or C43(DE3), as frdC is a membrane-associated subunit of fumarate reductase.

What are the recommended storage and reconstitution protocols for recombinant frdC protein?

Storage recommendations:

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

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots may be kept at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended: 50%)

• Aliquot for long-term storage at -20°C/-80°C

The addition of glycerol helps maintain protein stability during freeze-thaw cycles by preventing the formation of ice crystals that can damage protein structure.

How does frdC contribute to Y. pseudotuberculosis metabolism and pathogenesis?

The frdC protein forms part of the fumarate reductase complex, which plays a crucial role in anaerobic respiration by catalyzing the reduction of fumarate to succinate. This metabolic pathway is particularly important for Y. pseudotuberculosis during:

• Oxygen-limited conditions encountered in host tissues

• Adaptation to different environmental niches during infection

• Energy generation under anaerobic conditions

Research has revealed that the pyruvate metabolism and tricarboxylic acid cycle (TCA) are significantly perturbed in virulence regulator mutants of Y. pseudotuberculosis . The fumarate reductase complex, including frdC, is involved in this metabolic node that appears to be a focal point for controlling host colonization. Mutants with genetic perturbations in this metabolic branch point showed significant reduction of virulence in oral mouse infection models .

This connection between core metabolism and virulence represents a potential target for novel therapeutic approaches.

How can systems biology approaches be applied to understand frdC's role in Y. pseudotuberculosis?

Systems biology provides powerful tools for understanding frdC's position within the metabolic network of Y. pseudotuberculosis:

• 13C-based fluxome analysis: This technique allows for precise quantification of metabolic fluxes through pathways involving frdC. Research has successfully employed this method to track carbon flow through central metabolism in Y. pseudotuberculosis under different conditions .

• Integration with transcriptome data: Combining fluxome data with gene expression profiles enables researchers to correlate metabolic states with virulence control. This approach has revealed that the absence of specific virulence regulators particularly perturbs fluxes and gene expression of pyruvate metabolism and the TCA cycle .

• Continuous culture systems with temperature control: Since temperature is an important infection parameter for Y. pseudotuberculosis, advanced bioreactor systems can be employed to mimic the infection process. This allows for examining how frdC expression and activity changes during the transition from environmental to host temperature .

What biosafety considerations are necessary when working with Y. pseudotuberculosis components?

Working with Y. pseudotuberculosis components, including recombinant proteins like frdC, requires appropriate biosafety measures:

• Laboratory containment: Work should be conducted in properly engineered facilities with appropriate controls .

• Disinfection protocols: Y. pseudotuberculosis is susceptible to:

  • 2-5% phenol

  • 1% sodium hypochlorite

  • Other standard laboratory disinfectants

• Primary laboratory hazards:

  • Creation of splashes or aerosols

  • Exposure to mucous membranes

  • Ingestion

  • Exposure to contaminated sharps

• Personal protective equipment: Staff must be trained in the proper use of required PPE when in spaces containing the agent .

• Training requirements: All personnel must be adequately trained in safe laboratory practices, universal precautions, and proper surface and equipment disinfection before initiating any work with this agent .

What steps should be taken in case of accidental exposure to Y. pseudotuberculosis or its components?

In case of accidental exposure to Y. pseudotuberculosis or its components, including recombinant frdC:

For needlestick, animal bite, or laceration:

• Wash area thoroughly with soap and running water

• Do not apply disinfectant directly to the skin

For mucous membrane exposure (eyes, nose, mouth):

• Flush the eyes for 10-15 minutes if exposed to splash or spray containing bacteria

Additional important considerations:

• Immunocompromised individuals and those with hemochromatosis or any disease with a potential for iron overload are especially susceptible to infections with Yersinia species

• Medical attention should be sought, particularly for those in at-risk groups (children, young adults, immunocompromised individuals)

How can frdC be used to study metabolic adaptation in Y. pseudotuberculosis under different environmental conditions?

The frdC protein plays a key role in metabolic adaptation, making it a valuable target for studying how Y. pseudotuberculosis responds to environmental changes:

• Temperature adaptation studies: Y. pseudotuberculosis transitions between environmental temperatures and host body temperature during infection. The virulence regulator RovA responds to temperature, affecting metabolic pathways including those involving frdC . Researchers can design experiments using continuous culture with advanced temperature control to mimic this transition.

• Oxygen availability models: As frdC is involved in anaerobic respiration, it can serve as a marker for metabolic shifts between aerobic and anaerobic conditions. Experimental setups can include:

  • Controlled oxygen gradients in bioreactors

  • Microfluidic devices simulating tissue microenvironments

  • In vitro tissue models with defined oxygen tensions

• Nutrient limitation experiments: Examining frdC expression and activity under different nutrient conditions can reveal how Y. pseudotuberculosis prioritizes metabolic pathways during infection.

What role does frdC play in the interaction between Y. pseudotuberculosis and host immune cells?

The metabolic functions of frdC contribute to Y. pseudotuberculosis interactions with host immune cells, particularly phagocytes:

• Survival in macrophages: Y. pseudotuberculosis can survive within macrophages, requiring metabolic adaptation. The fumarate reductase complex may support bacterial survival under the oxygen-limited conditions within phagosomes .

• Immune modulation: Y. pseudotuberculosis proteins affect immune cell function, potentially including metabolic interactions. Recent evidence shows that both plasmid-encoded and chromosome-encoded toxins contribute to bacterial defense against phagocytes, with metabolic enzymes potentially playing supporting roles .

• Macrophage polarization: Y. pseudotuberculosis can induce macrophage polarization towards the M2 phenotype, which is more permissive for bacterial survival. Metabolic pathways involving frdC may contribute to this process by altering the bacterial metabolic state in ways that influence immune cell polarization .

To study these interactions, researchers can employ:

• Macrophage infection models with wild-type and frdC-mutant Y. pseudotuberculosis

• Metabolomic analysis of infected vs. uninfected macrophages

• Transcriptomic profiling of both bacteria and host cells during infection

What are promising new approaches for studying frdC function in Y. pseudotuberculosis?

Several cutting-edge approaches show promise for deepening our understanding of frdC function:

• CRISPR-Cas9 genome editing: Precise modification of frdC and related genes can reveal functional relationships and regulatory networks.

• Single-cell analysis: Examining frdC expression at the single-cell level can reveal heterogeneity in bacterial populations that may contribute to survival strategies.

• In vivo imaging: Development of reporter systems linked to frdC expression could allow for real-time tracking of metabolic adaptation during infection.

• Host-pathogen metabolic interaction studies: Dual-species metabolomics approaches can reveal how frdC-dependent metabolic pathways interact with host metabolism during infection.

• Structural biology: Determining the detailed structure of the fumarate reductase complex, including frdC, could provide insights into its function and potential as a drug target.

How might understanding frdC contribute to novel antimicrobial strategies?

The central role of frdC in Y. pseudotuberculosis metabolism presents opportunities for novel therapeutic approaches:

• Metabolic vulnerability targeting: The pyruvate-TCA cycle node has been identified as a focal point for controlling host colonization . Compounds that disrupt this metabolic node, including fumarate reductase function, could reduce bacterial virulence.

• Anti-virulence strategies: Rather than killing bacteria outright, targeting metabolic pathways that support virulence could reduce pathogenicity while minimizing selective pressure for resistance.

• Host-directed therapies: Understanding how bacterial metabolism interacts with host metabolic processes could reveal opportunities to modify host responses in ways that disfavor bacterial survival.

• Combination approaches: Targeting both traditional antibiotic targets and metabolic vulnerabilities like those associated with frdC could enhance treatment efficacy and reduce the emergence of resistance.

Such approaches require detailed understanding of metabolic networks and their relationship to virulence, underscoring the importance of continued research on frdC and related metabolic components.