Recombinant Brucella melitensis biotype 2 ATP synthase subunit alpha (atpA), partial

What is ATP synthase alpha subunit (atpA) and what is its significance in Brucella melitensis?

ATP synthase is a critical enzyme complex involved in energy production, consisting of multiple subunits including the alpha (atpA) subunit. In Brucella melitensis, ATP synthase plays an essential role in energy metabolism through oxidative phosphorylation. The alpha subunit contains nucleotide binding sites and contributes to the catalytic function of the enzyme.

When Brucella faces limitations in energy production, particularly during intracellular infection, the bacterium reinforces alternate metabolic pathways such as glycolysis to synthesize ATP through substrate-level phosphorylation . This adaptation is crucial for Brucella's survival within host cells, where ATP production through the ATP synthase complex may be impaired due to environmental stressors including iron limitation .

How does the structure of Brucella ATP synthase compare to other bacterial species?

The ATP synthase complex in Brucella shows significant homology with ATP synthase in other alphaproteobacteria, including non-pathogenic relatives like Ochrobactrum anthropi, Sinorhizobium meliloti, and Mesorhizobium loti . This homology explains the cross-reactivity observed in immunological studies, where antibodies against Brucella antigens recognize antigens from non-pathogenic alphaproteobacteria and vice versa .

What expression systems are most effective for producing recombinant Brucella ATP synthase subunits?

Recombinant Brucella ATP synthase subunits can be effectively produced in several expression systems, with E. coli being the most commonly used platform due to its high yield and relatively simple protocols. The expression process typically involves:

• Gene cloning into suitable expression vectors with appropriate tags (His-tag, GST, etc.)

• Transformation into expression hosts

• Induction of protein expression (commonly with IPTG for T7-based systems)

• Cell lysis and protein purification

The recombinant proteins are typically stored in Tris-based buffers with 50% glycerol at -20°C for short-term storage or -80°C for extended storage . For functional studies, care must be taken to ensure proper folding of the expressed protein, which may require specific buffer conditions optimized for ATP synthase components.

What purification techniques yield the highest purity of recombinant Brucella ATP synthase subunits?

Purifying recombinant Brucella ATP synthase subunits requires a multi-step approach:

• Initial capture: Affinity chromatography using the tag system incorporated into the recombinant protein (Ni-NTA for His-tagged proteins)

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing: Size exclusion chromatography to achieve highest purity and remove aggregates

For structural studies requiring ultra-pure preparations, additional steps may include:

• Hydrophobic interaction chromatography

• Tag removal using specific proteases

• Second affinity step to remove uncleaved protein

Proper storage is critical for maintaining functionality. The recommended storage conditions include Tris-based buffer with 50% glycerol at -20°C, avoiding repeated freeze-thaw cycles .

What analytical methods are most informative for characterizing Brucella ATP synthase subunits?

For comprehensive characterization, iTRAQ labeling coupled with LC-MS/MS has proven effective for quantitative proteomic analysis of Brucella proteins, allowing comparison between different strains or growth conditions . This approach involves trypsin digestion of proteins, iTRAQ labeling, and subsequent LC-MS/MS analysis, providing both identification and relative quantification of proteins .

How can researchers effectively evaluate the immunogenicity of recombinant Brucella ATP synthase subunits?

Evaluating immunogenicity of Brucella ATP synthase subunits involves a systematic approach:

• In silico epitope prediction: Using bioinformatics tools to predict B and T cell epitopes before experimental validation

• Antibody response assessment: Measuring specific IgG levels in serum of immunized animals using ELISA

• Cellular immunity evaluation: Analyzing T cell responses through:

  • Cytokine profiling (especially IFN-γ and IL-6)

  • Flow cytometry to measure CD3, CD4, and CD8 T cell frequencies

• Protection studies: Challenge experiments in animal models (typically mice) followed by bacterial load determination in spleens

Protection efficacy can be quantified by calculating the reduction in splenic CFU counts compared to control groups, typically expressed in log units. For example, immunization with whole Brucella typically provides 1.5-2.0 log units of protection, while cross-protective antigens from related bacteria may provide 0.4-0.8 log units of protection .

How does iron availability affect ATP synthase expression and function in Brucella?

Iron limitation significantly impacts Brucella metabolism and ATP synthase function through complex regulatory networks:

During iron deprivation, such as within macrophages, Brucella undergoes extensive proteome reconfiguration . This includes upregulation of the iron response regulator (Irr), which controls numerous iron-dependent processes. Intracellular Brucella shows reduced oxidative phosphorylation capacity, which affects ATP synthase function.

To compensate for reduced ATP production through oxidative phosphorylation:

• Glycolytic enzymes are upregulated, including fructose-1,6-bisphosphate aldolase, phosphoglycerate kinase, enolase, and pyruvate kinase

• Substrate-level phosphorylation is enhanced to maintain ATP production

• NAD+ regeneration pathways are activated, including upregulation of zinc-containing alcohol dehydrogenase (BAB1_0128)

These adaptations allow Brucella to maintain energy homeostasis despite impaired electron transport chain function during iron limitation, highlighting the metabolic flexibility that contributes to pathogen persistence.

What role does ATP synthase play in Brucella virulence and intracellular survival?

ATP synthase plays a multifaceted role in Brucella virulence and intracellular survival:

• Energy provision: ATP synthase provides the energy necessary for virulence factor expression and adaptive responses within the host cell

• pH homeostasis: The F₀ component can function in reverse to pump protons out of the bacterial cell, helping maintain cytoplasmic pH in acidic environments

• Metabolic adaptation: During intracellular growth, Brucella shifts its metabolism, affecting ATP synthase activity

• Stress response: ATP synthase function is linked to bacterial responses to oxidative stress encountered within macrophages

Proteomic analyses of intracellular Brucella reveal significant remodeling of metabolic pathways, including changes in pyruvate dehydrogenase complex expression (with all subunits upregulated) . This metabolic adaptation is crucial for generating energy and precursor metabolites needed for intracellular survival and replication.

How can ATP synthase components be utilized in vaccine development against brucellosis?

ATP synthase components show potential as vaccine candidates against brucellosis for several reasons:

• Conservation: ATP synthase is highly conserved among Brucella species, potentially providing cross-protection

• Immunogenicity: The protein contains multiple B and T cell epitopes

• Accessibility: Some portions of the ATP synthase complex are surface-exposed

Effective vaccine development strategies include:

• Epitope-based vaccines: Using bioinformatics tools to predict and select immunogenic epitopes from ATP synthase subunits for inclusion in multi-epitope vaccines

• Subunit vaccines: Developing recombinant protein vaccines containing ATP synthase components along with appropriate adjuvants

• Cross-protective immunization: Utilizing homologous proteins from non-pathogenic alphaproteobacteria as safer alternatives to live Brucella vaccines

Animal studies have shown that immunization with related bacteria can provide partial protection against Brucella challenge. For instance, subcutaneous immunization with heat-killed Ochrobactrum anthropi provided 0.80 log units of protection against Brucella abortus challenge, while its cytosolic extract provided 0.63 log units of protection .

What are the most significant challenges in structural studies of Brucella ATP synthase complexes?

Structural studies of Brucella ATP synthase complexes face several technical challenges:

• Membrane protein complexity: The F₀ portion contains hydrophobic subunits that are difficult to express and purify in functional form

• Multi-subunit assembly: Achieving proper assembly of the complete ATP synthase complex in vitro is challenging

• Stability issues: Maintaining the integrity of the complex during purification and crystallization attempts

• Expression systems: Selecting appropriate heterologous expression systems that can produce functional bacterial ATP synthase components

• Lipid environment requirements: The need to maintain proper lipid environments for functional and structural studies

Researchers typically approach these challenges through:

• Detergent screening to identify optimal solubilization conditions

• Nanodiscs or lipid cubic phase crystallization for membrane protein structural studies

• Cryo-electron microscopy as an alternative to X-ray crystallography

• Expression of individual subunits or subcomplexes when complete complex expression is not feasible

How do ATP synthase subunits from Brucella compare with those from related alphaproteobacteria?

ATP synthase subunits show significant conservation across alphaproteobacteria, which has important implications for research and immunological cross-reactivity:

Research has demonstrated that Brucella antigens and those from non-pathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system . This cross-recognition extends to ATP synthase components due to their high sequence and structural homology.

The evolutionary relationship between Brucella and other alphaproteobacteria like Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, and Agrobacterium tumefaciens provides opportunities for comparative studies . These relationships are particularly valuable for:

• Vaccine development: Using safer NPAP as immunogens that provide cross-protection against Brucella

• Structural predictions: Modeling Brucella ATP synthase based on better-characterized homologs

• Functional studies: Understanding conserved and divergent aspects of ATP synthase function

Immunization studies with NPAP have shown varying degrees of protection against Brucella challenge, with O. anthropi (the closest relative to Brucella) providing the highest level of protection among tested NPAP (0.80 log units reduction in splenic CFU) .

What methodological approaches can identify ATP synthase modifications during Brucella infection cycles?

Tracking ATP synthase modifications during infection requires sophisticated methodological approaches:

• Temporal proteomic analysis: Using techniques like iTRAQ labeling and LC-MS/MS to quantify protein expression changes at different stages of infection

• Post-translational modification mapping: Phosphoproteomic and other PTM analyses to identify regulatory modifications

• In vivo crosslinking: To capture transient protein-protein interactions involving ATP synthase during infection

• Transcriptome-proteome correlation: Combining RNA-seq with proteomics to understand regulatory mechanisms

• Reporter systems: Using fluorescent tags to track ATP synthase localization and expression in live bacteria during infection

Detailed protocols for quantitative proteomic analysis include:

• Sample preparation from intracellular bacteria (e.g., from infected macrophages)

• Protein extraction and quantification

• Trypsin digestion followed by iTRAQ labeling

• LC-MS/MS analysis for protein identification and quantification

These approaches have revealed significant remodeling of metabolic pathways, including energy production systems, when Brucella transitions to intracellular life .

What emerging technologies show promise for ATP synthase research in Brucella?

Several cutting-edge technologies are advancing ATP synthase research in Brucella:

• CRISPR-Cas9 genome editing: For precise genetic manipulation to study structure-function relationships

• Single-molecule techniques: Including FRET and optical tweezers to study ATP synthase dynamics

• Cryo-electron microscopy: For high-resolution structural determination without crystallization

• Systems biology approaches: Integrating multiple omics datasets to understand ATP synthase in the context of global metabolic networks

• Microfluidic devices: For real-time monitoring of ATP synthase activity in varying environmental conditions

These technologies will enable researchers to address key questions about ATP synthase function during different phases of Brucella infection, potentially identifying new therapeutic targets or vaccine candidates.

How might ATP synthase components contribute to next-generation brucellosis vaccines?

ATP synthase components hold significant potential for next-generation vaccine development:

The ideal brucellosis vaccine would provide strong protection without the risks associated with current live attenuated vaccines, which can cause abortions in pregnant animals and potentially infect humans . ATP synthase components could contribute to this goal through:

• Multi-epitope vaccines: Combining immunogenic epitopes from ATP synthase with other protective antigens to create recombinant multi-epitope vaccines

• Subunit vaccine formulations: Using purified recombinant ATP synthase components with appropriate adjuvants

• Cross-protective approach: Utilizing homologous proteins from non-pathogenic alphaproteobacteria as safer alternatives

• Novel delivery systems: Incorporating ATP synthase antigens into liposomes, nanoparticles, or viral vectors for enhanced immunogenicity

Current research supports that bioinformatically designed multi-epitope vaccines can induce both humoral and cell-mediated immune responses, with Th1-Th2 mixed responses characterized by high levels of specific IgG, elevated IFN-γ and IL-6, and increased CD3, CD4, and CD8 T cell frequencies .