Recombinant Rickettsia akari ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Rickettsia akari and what is its function?

ATP synthase subunit c (atpE) in R. akari is a small hydrophobic protein component of the F0 sector of F-type ATP synthase, functioning as part of the membrane-embedded proton channel. This protein facilitates proton translocation across the bacterial membrane, which drives the synthesis of ATP by the F1 sector.

In Rickettsia species, including R. akari, atpE is encoded by a highly conserved gene within the bacterial genome. The protein typically consists of approximately 70-80 amino acids, with R. conorii atpE comprising 74 amino acids . The high degree of conservation observed in the R. akari genome suggests that atpE likely maintains similar structural properties and functional roles across various Rickettsia isolates .

How does the structure of R. akari atpE compare to other Rickettsia species?

Based on the information available for R. conorii atpE, we can infer that R. akari atpE likely has a similar amino acid sequence and secondary structure. The R. conorii atpE consists of 74 amino acids with the sequence: MDMVSLKFIGTGLMAIGMYGAALGVSNIFSSLLSSIARNPSATENLQRMALIGAGLAEAMGLFSFVIAMLLIFS . The protein likely forms a hairpin-like structure with a transmembrane helix-loop-helix motif that spans the bacterial membrane.

Why is recombinant R. akari atpE important for research applications?

Recombinant R. akari atpE is valuable for research applications for several reasons:

• As an obligate intracellular pathogen, R. akari is difficult to culture in large quantities, making recombinant protein production necessary for obtaining sufficient material for experimental studies.

• The protein can serve as an antigen for developing serological tests for rickettsialpox diagnosis or for generating antibodies for research purposes.

• It provides a system for studying the structure-function relationships of this essential bacterial protein, potentially revealing mechanisms that could be targeted for therapeutic intervention.

• Recombinant atpE can be used in protein-protein interaction studies to identify binding partners within the ATP synthase complex or with host factors during infection.

What expression systems are most effective for producing recombinant R. akari atpE?

Based on experience with other Rickettsia proteins, E. coli expression systems are typically most effective for producing recombinant R. akari atpE. When designing an expression strategy, consider the following approach:

• Vector selection: pET expression vectors with N-terminal His-tags facilitate purification while minimizing interference with protein function. This approach mirrors the successful expression of R. conorii atpE .

• E. coli strain: BL21(DE3) or Rosetta(DE3) strains are recommended, especially when dealing with the potential codon usage bias of Rickettsia genes.

• Expression conditions: Induction at lower temperatures (16-20°C) overnight with reduced IPTG concentrations (0.1-0.5 mM) may improve solubility of the membrane protein.

• Extraction methods: For this highly hydrophobic protein, detergent-based extraction using mild detergents like n-dodecyl β-D-maltoside (DDM) or n-octyl glucoside (OG) is recommended.

Consider that while the full-length protein includes hydrophobic transmembrane regions, expression of the soluble domains alone might improve yield if the full-length protein proves difficult to express.

What purification strategies should be employed for recombinant R. akari atpE?

Purification of recombinant R. akari atpE requires specific strategies due to its hydrophobic nature:

• Immobilized Metal Affinity Chromatography (IMAC): Using Ni-NTA resin for His-tagged protein is the recommended first step, maintaining detergent above critical micelle concentration in all buffers.

• Buffer optimization: PBS-based buffers with 6% trehalose at pH 8.0 have been successful for stabilizing similar rickettsial proteins .

• Storage and reconstitution: After purification, lyophilization preserves activity. Reconstitution should be in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage at -20°C/-80°C .

• Quality control: Verify protein purity using SDS-PAGE (>90% purity), and confirm identity through western blotting or mass spectrometry analysis.

• Avoiding aggregation: Addition of stabilizing agents such as trehalose (6%) in storage buffers can prevent aggregation of the hydrophobic protein .

How can researchers assess the functional activity of recombinant R. akari atpE?

Assessing the functional activity of recombinant R. akari atpE requires specialized techniques:

• Reconstitution into liposomes: Incorporate purified atpE into artificial liposomes to measure proton translocation activity.

• Proton flux measurements: Using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) to monitor proton movement across membranes containing reconstituted atpE.

• ATP synthesis coupling: In a more complex system, assess whether atpE can cooperate with other ATP synthase components to generate ATP.

• Structural integrity validation: Use circular dichroism (CD) spectroscopy to confirm proper secondary structure formation, particularly alpha-helical content expected for this transmembrane protein.

• Oligomerization analysis: Native PAGE or analytical ultracentrifugation to verify if the protein forms the expected c-ring oligomeric structure.

How can recombinant R. akari atpE be used to study host-pathogen interactions?

Recombinant R. akari atpE can serve as a valuable tool for studying host-pathogen interactions through several approaches:

• Surface display system: While atpE is not typically exposed on the bacterial surface, it can be used in heterologous display systems to study protein interactions with host components.

• Host immune response characterization: Recombinant atpE can be used to study specific antibody responses in infected hosts, particularly as R. akari induces a distinct cytokine profile compared to other Rickettsia species .

• Cross-reactivity studies: Determine if antibodies generated against atpE cross-react with host ATP synthase components, potentially revealing molecular mimicry mechanisms.

• Protein-protein interaction studies: Using techniques such as pull-down assays, identify potential interactions between atpE and host proteins during rickettsial infection.

• T-cell response analysis: As T-cell mediated immunity is crucial for controlling R. akari infections , recombinant atpE can be used to identify and characterize T-cell epitopes.

What structural modifications can improve the stability and solubility of recombinant R. akari atpE?

Improving stability and solubility of recombinant R. akari atpE requires strategic modifications:

• Fusion partners: Addition of solubility-enhancing tags such as MBP, SUMO, or Thioredoxin can dramatically improve expression and solubility.

• Construct optimization: For structural studies, consider expressing only the hydrophilic portions or creating chimeric constructs with more soluble homologs.

• Amphipathic environment: Utilize specialized detergents like fluorinated surfactants or nanodisc technology to maintain the native fold while improving solubility.

• Site-directed mutagenesis: Strategic replacement of certain hydrophobic residues at non-critical positions can improve solubility without compromising function.

• Co-expression with chaperones: Simultaneous expression with molecular chaperones like GroEL/GroES can facilitate proper folding and reduce aggregation.

How can researchers investigate the role of atpE in R. akari pathogenesis?

Investigating atpE's role in R. akari pathogenesis requires sophisticated approaches:

• Gene expression analysis: Quantify atpE expression during different stages of infection using RT-qPCR, similar to the expression analysis performed for Sca proteins in R. typhi .

• Inhibitor studies: Use specific inhibitors targeting ATP synthase to determine effects on bacterial viability and pathogenicity in cell culture models.

• Protein localization: Develop specific antibodies against recombinant atpE to track its localization during infection using immunofluorescence microscopy.

• Interaction with bacterial proteases: Investigate whether atpE is processed by rickettsial proteases such as APRc, which has been shown to process major surface antigens in R. conorii .

• Comparative genomic analysis: Compare atpE sequence conservation across Rickettsia species with different pathogenicity profiles to identify potential correlations with virulence.

How does R. akari atpE differ from atpE in other Rickettsia species in terms of sequence and function?

While specific sequence data for R. akari atpE is not provided in the search results, comparative analysis can be inferred based on related information:

What is the relationship between atpE and other surface proteins in the pathogenesis of R. akari?

While atpE is not classified as a surface protein in Rickettsia, understanding its relationship with established virulence factors is important:

• ATP synthase activity provides energy for the expression and function of true surface antigens like the Sca (Surface Cell Antigen) family proteins, which are known virulence factors .

• Unlike Sca proteins that show significant variation between Rickettsia species and are involved in host-pathogen interactions , atpE is likely more conserved as it serves a fundamental metabolic function.

• The presence of proteolytic pathways involving proteases like APRc that process surface proteins suggests a complex regulatory network that may indirectly involve energy metabolism proteins like atpE.

• R. akari demonstrates actin-based motility inside host cells , a process requiring energy that is dependent on ATP synthase function, thus linking atpE indirectly to pathogenesis mechanisms.

• The T-cell mediated immune response critical for controlling R. akari infections may recognize bacterial antigens including components of ATP synthase, though surface proteins are typically more immunogenic.

What are common challenges in expressing and purifying functional recombinant R. akari atpE?

Researchers working with recombinant R. akari atpE face several technical challenges:

• Protein aggregation: The hydrophobic nature of atpE makes it prone to aggregation. Addressing this requires careful optimization of expression conditions, including lower temperatures (16°C), reduced inducer concentrations, and appropriate detergent selection.

• Low expression yields: Codon optimization for E. coli expression systems can improve yields, as can fusion with solubility-enhancing tags.

• Maintaining native conformation: Ensuring the recombinant protein maintains its native structure requires careful buffer optimization. PBS-based buffers with 6% trehalose at pH 8.0 have shown success with similar proteins .

• Proteolytic degradation: Add protease inhibitors during purification and consider expressing in E. coli strains deficient in specific proteases.

• Accurate concentration determination: The high hydrophobicity can interfere with standard protein quantification methods; use multiple techniques (Bradford, BCA, and UV absorbance) for verification.

How can researchers differentiate between the functions of atpE and other membrane proteins in R. akari?

Differentiating the specific functions of atpE from other membrane proteins requires specialized approaches:

• Specific inhibitors: Use oligomycin or other F0 sector-specific inhibitors to selectively target ATP synthase function without affecting other membrane proteins.

• Complementation studies: Express R. akari atpE in heterologous bacterial systems with defective or deleted endogenous atpE to assess functional complementation.

• Site-directed mutagenesis: Create targeted mutations in conserved functional residues of atpE to establish structure-function relationships.

• Protein-protein interaction mapping: Use techniques such as cross-linking followed by mass spectrometry to identify the specific interaction partners of atpE versus other membrane proteins.

• Comparative studies with related species: Leverage the high conservation of the R. akari genome to compare with functions established in better-studied Rickettsia species.