Recombinant Renibacterium salmoninarum ATP synthase subunit beta (atpD)

What is Renibacterium salmoninarum and what disease does it cause?

Renibacterium salmoninarum is a Gram-positive, slow-growing diplobacillus that functions as a facultative intracellular bacterium, replicating primarily within fish macrophages. It is the causative agent of Bacterial Kidney Disease (BKD) in salmonids and other fish species. The bacterium has a preferred growth temperature of 15°C and is characterized by its intracellular lifestyle, which presents significant challenges for treatment and prevention . BKD is economically significant in aquaculture, particularly affecting Atlantic salmon and other salmonid species in both freshwater and marine environments .

How is recombinant R. salmoninarum ATP synthase subunit beta typically produced?

The recombinant production of R. salmoninarum ATP synthase subunit beta is typically achieved through heterologous expression in yeast systems. According to available protocols, the protein can be purified to >85% purity using SDS-PAGE verification methods. For experimental use, the lyophilized protein is recommended to be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage at -20°C or -80°C to prevent degradation . The shelf life of the liquid form is approximately 6 months, while the lyophilized form can remain stable for up to 12 months when properly stored.

What diagnostic methods are available for detecting R. salmoninarum in fish samples?

R. salmoninarum detection employs several complementary methods:

• ELISA (Enzyme-Linked Immunosorbent Assay): Targets the p57 protein of R. salmoninarum, offering relatively rapid detection with moderate sensitivity.

• PCR Methods: Primarily target the msa2 gene, providing high sensitivity for bacterial detection even at low concentrations.

• Bacterial Culture: Utilizes selective SKDM (Selective Kidney Disease Medium) agar, though this method is time-consuming due to the slow growth of R. salmoninarum (requiring up to 12 weeks for positive identification) .

• RNA-based TaqMan qPCR Assays: Used to quantify bacterial load in infected tissues, as reported in experimental infection studies where head kidney samples were analyzed for infection levels .

These techniques can be employed sequentially for comprehensive diagnostic assessment, with molecular methods providing faster results but culture techniques remaining the gold standard for confirmed diagnosis.

How can ATP synthase genes be utilized for molecular identification of R. salmoninarum?

While ATP synthase genes are not the primary targets for routine R. salmoninarum detection, atpD sequences can serve as conserved markers for species identification and phylogenetic analysis. The atpD gene exhibits sufficient sequence conservation to reliably identify bacterial species while containing variable regions that enable strain differentiation. Specific primers targeting the unique regions of R. salmoninarum atpD could potentially be designed for PCR-based identification methods to complement existing diagnostic approaches targeting the msa genes. Sequence alignments from multiple bacterial species show that R. salmoninarum atpD shares approximately 45% identity with homologous genes from other bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum, providing sufficient divergence for species-specific detection .

What experimental animal models have been established for R. salmoninarum infection studies?

Several fish models have been validated for R. salmoninarum infection studies:

• Atlantic salmon (Salmo salar): The primary natural host, extensively used for studying BKD pathogenesis and host immune responses.

• Rainbow trout: Commonly used in challenge studies and vaccine development research.

• Lumpfish (Cyclopterus lumpus): Recently demonstrated to be susceptible to R. salmoninarum, exhibiting chronic infection patterns similar to salmonid species .

Experimental infection protocols typically involve injection of standardized bacterial doses (e.g., 2 × 10^8 CFU per fish of R. salmoninarum H-2 strain), with subsequent sampling of tissues such as head kidney at defined time points post-infection . Alternative challenge methods using hydrogen peroxide as a pre-stressor before bath challenge have also been developed to simulate natural infection routes .

How does the transcriptomic profile change during R. salmoninarum infection?

Transcriptomic profiling using microarray and RNA sequencing has revealed distinctive immune response patterns during R. salmoninarum infection:

Table 1: Key immune response genes altered during R. salmoninarum infection in lumpfish

The infection initially induces immune suppression (28 days post-infection), followed by the development of a cell-mediated adaptive immune response during chronic infection stages (98 days post-infection). This biphasic pattern suggests that R. salmoninarum initially evades host defenses by suppressing cell-mediated immunity before eventually triggering a delayed adaptive response . These findings provide important insights for designing therapeutic interventions and vaccines that could potentially accelerate protective immune responses.

What are the potential applications of recombinant R. salmoninarum ATP synthase subunit beta in vaccine development?

Recombinant ATP synthase subunit beta (atpD) shows promise as a vaccine component against BKD. Studies have investigated the use of formalin-killed recombinant E. coli cells expressing ATP synthase beta subunit (atpD) as potential vaccine candidates . This approach leverages the immunogenicity of bacterial proteins while avoiding the risks associated with live attenuated vaccines.

Potential vaccine applications include:

• Subunit vaccines: Purified recombinant atpD could be formulated as a protein subunit vaccine.

• Component in polyvalent vaccines: atpD could be combined with other R. salmoninarum antigens for broader protection.

• DNA vaccines: Plasmids encoding atpD could be developed for direct expression in fish tissues.

• Recombinant vector vaccines: Attenuated bacterial or viral vectors expressing atpD could be engineered.

Research has shown that formalin-killed whole-cell polyvalent vaccines against other fish pathogens can achieve significant protection (RPS of 84%), suggesting similar approaches might be effective for R. salmoninarum using atpD as an antigenic component .

How does R. salmoninarum ATP synthase subunit beta compare with homologous proteins from other bacteria?

Sequence homology analyses reveal significant conservation patterns across bacterial species:

Table 2: Sequence homology comparison of ATP synthase beta subunit across bacterial species

This conservation pattern suggests that while the core functional domains are preserved across species, there are species-specific variations that might relate to functional adaptations. Seven conserved regions have been identified through sequence alignment of these homologous proteins , which likely correspond to essential functional elements of the enzyme.

What are the challenges in expressing and purifying functional recombinant ATP synthase subunits?

Researchers face several technical challenges when working with recombinant ATP synthase subunits:

• Maintaining structural integrity: ATP synthase subunits typically function within a complex, and isolation of individual subunits may affect their folding and stability.

• Expression system selection: Yeast systems have proven effective for R. salmoninarum atpD expression, but optimization of expression conditions is critical for proper protein folding.

• Purification complexity: Achieving >85% purity while preserving functionality requires careful optimization of purification protocols.

• Storage stability: As noted in protocols, repeated freeze-thaw cycles should be avoided, and glycerol addition is recommended for maintaining long-term stability .

• Functional assessment: Evaluating ATP synthase activity of recombinant subunits outside their native complex presents methodological challenges.

Overcoming these challenges requires careful optimization of expression constructs, purification protocols, and storage conditions specific to R. salmoninarum atpD.

How might ATP synthase function contribute to R. salmoninarum pathogenicity and intracellular survival?

As a facultative intracellular pathogen, R. salmoninarum must adapt to varying energy availability within host macrophages. The ATP synthase complex likely plays a crucial role in this adaptation through several potential mechanisms:

• Energy production flexibility: The ability to utilize ATP synthase for both ATP synthesis and hydrolysis may help the bacterium adjust to changing intracellular conditions.

• Maintenance of membrane potential: Beyond ATP synthesis, ATP synthase can contribute to membrane potential homeostasis, which is essential for bacterial survival.

• Adaptation to acidic phagosomes: Within macrophage phagosomes, pH changes may modulate ATP synthase function, potentially contributing to bacterial persistence.

• Metabolic modulation during chronic infection: ATP synthase activity may be regulated differently during acute versus chronic infection phases, affecting bacterial growth rates and visibility to the host immune system.

These hypotheses warrant further investigation through targeted gene disruption or modification studies to elucidate the specific contributions of atpD to R. salmoninarum virulence.

What methodological approaches can be used to study ATP synthase activity in R. salmoninarum?

Several complementary approaches can be employed to investigate ATP synthase functionality:

• Enzymatic activity assays: Measuring ATP synthesis/hydrolysis rates using purified enzyme or membrane preparations.

• Membrane potential measurements: Using fluorescent probes to assess proton gradient formation and dissipation.

• Genetic manipulation: Creating targeted atpD mutations or knockdowns to assess phenotypic effects.

• Structural biology approaches: X-ray crystallography or cryo-EM to determine the three-dimensional structure of the R. salmoninarum ATP synthase complex.

• Inhibitor studies: Using specific ATP synthase inhibitors to probe functional roles in bacterial physiology.

• Transcriptomic and proteomic analyses: Examining expression patterns under different environmental conditions to understand regulatory mechanisms.

These approaches can provide complementary insights into how ATP synthase functions within the context of R. salmoninarum physiology and pathogenesis.

How can our understanding of R. salmoninarum ATP synthase inform novel therapeutic strategies?

ATP synthase represents a potential target for novel therapeutic approaches against BKD:

• Specific inhibitors: Development of compounds that selectively target bacterial ATP synthase without affecting host enzymes.

• Attenuated strains: Engineering R. salmoninarum strains with modified ATP synthase activity for potential live attenuated vaccines.

• Immune targeting: Utilizing ATP synthase subunits as targets for antibody-mediated clearance of infection.

• Combination approaches: Targeting ATP synthase alongside other bacterial systems to enhance treatment efficacy.

• Biomarker development: Using ATP synthase activity levels as indicators of bacterial metabolic state to guide treatment timing and duration.

These approaches could complement existing antibiotic treatments, which have shown variable effectiveness against R. salmoninarum infections due to its intracellular lifestyle and slow growth characteristics.

What are the optimal storage and handling conditions for recombinant R. salmoninarum ATP synthase subunit beta?

Based on established protocols, the following conditions are recommended for working with recombinant atpD:

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL.

• Glycerol addition: Add glycerol to a final concentration of 5-50% (50% is commonly used) for cryoprotection.

• Storage temperature: Store at -20°C for short-term use or -80°C for extended storage.

• Aliquoting: Prepare working aliquots to avoid repeated freeze-thaw cycles.

• Working storage: Store working aliquots at 4°C for up to one week.

• Centrifugation: Briefly centrifuge vials prior to opening to bring contents to the bottom .

These precautions help maintain protein stability and activity, ensuring reliable experimental results when working with this recombinant protein.

How can experimental infection models be optimized for reproducible R. salmoninarum challenge studies?

To achieve consistent and reproducible infection models, researchers should consider:

• Bacterial strain selection: Use well-characterized R. salmoninarum strains such as ATCC 33209 or H-2 strain with known virulence properties.

• Inoculum standardization: Precisely quantify bacterial suspensions (e.g., 2 × 10^8 CFU per fish) to ensure consistent challenge dose.

• Infection route: Consider the natural infection pathway; while injection ensures consistent dosing, bath challenges better mimic natural infection routes.

• Pre-stressor application: Use of hydrogen peroxide as a pre-stressor before bath challenge can enhance infection success and reproducibility.

• Temperature control: Maintain water temperature at 15°C, the preferred growth temperature for R. salmoninarum.

• Infection verification: Employ RNA-based TaqMan qPCR assays to quantify bacterial load and confirm successful infection.

• Time-point selection: Plan sampling at both early (e.g., 28 days) and chronic (e.g., 98 days) infection stages to capture the dynamic host response .

These methodological considerations can significantly improve experimental consistency and facilitate meaningful comparisons between different studies.