Recombinant Haemophilus influenzae ATP synthase subunit c (atpE)

What is the functional role of ATP synthase subunit c in Haemophilus influenzae?

ATP synthase subunit c in H. influenzae is a critical component of the F0 part of ATP synthase that catalyzes the production of ATP from ADP in the presence of a proton gradient. This subunit forms a cylindrical oligomer that is membrane-spanning and crucial for the flow of protons across the bacterial membrane for ATP synthesis . The subunit plays a direct role in cooperating with subunit a (Atp6-equivalent) in the proton-pumping process essential for energy production in the bacterium .

How is the atpE gene organized within the H. influenzae genome?

The atpE gene in H. influenzae is part of the ATP synthase operon. Based on comparative genomic analysis, this gene exists within conserved regions of the H. influenzae genome but shows some variability across different strains. Recent genomic research on H. influenzae isolates has revealed that the ATP synthase genes, including atpE, can be identified through whole genome sequencing and comparative analysis . Unlike some bacterial species that may have multiple copies of ATP synthase components, the H. influenzae genome typically maintains a single copy of the atpE gene within its ATP synthase operon.

How does ATP synthase function differ between pathogenic and non-pathogenic H. influenzae strains?

While the core function of ATP synthase remains consistent across H. influenzae strains, genomic analysis suggests variations that may correlate with pathogenicity. Recent comparative studies of emerging non-typeable H. influenzae strains (C1 and C2 clusters) have revealed distinct genomic features that may influence ATP synthase function in pathogenic variants . The acquisition of unique mobile cassettes and accessory genes in certain strains correlates with increased capacity for systemic infection rather than respiratory colonization. These genomic differences may influence energy metabolism pathways in which ATP synthase plays a central role, potentially contributing to the varying pathogenic potential observed between H. influenzae strains.

What expression systems are most effective for recombinant H. influenzae atpE production?

Based on research methodologies employed for similar ATP synthase components, E. coli expression systems utilizing pET vectors under T7 promoter control have proven effective for the expression of recombinant H. influenzae atpE. For optimal expression, consider the following parameters:

When expressing recombinant atpE, researchers should consider codon optimization for E. coli expression as H. influenzae has different codon usage patterns . Induction conditions should be carefully optimized, with lower temperatures (16-25°C) often yielding more properly folded membrane proteins.

What purification strategies yield functional recombinant H. influenzae atpE?

Purification of recombinant H. influenzae atpE requires specialized approaches due to its hydrophobic nature as a membrane protein. The following methodology has proven successful:

• Membrane fraction isolation: After cell lysis, differential centrifugation should be performed to isolate membrane fractions containing the recombinant protein.

• Detergent solubilization: Utilize mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin to solubilize the membrane protein while maintaining native conformation.

• Affinity chromatography: If the recombinant protein includes an affinity tag (His-tag recommended), immobilized metal affinity chromatography (IMAC) provides effective initial purification.

• Size exclusion chromatography: This final step helps separate the properly oligomerized c-subunit from aggregates and other contaminants.

For reconstitution studies, the purified protein should be incorporated into liposomes using a detergent removal method, which allows assessment of functional proton translocation activity .

How can researchers verify the structural integrity of purified recombinant atpE?

Verification of structural integrity is crucial for functional studies. Multiple complementary approaches should be employed:

• Circular dichroism (CD) spectroscopy to confirm secondary structure composition

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to assess oligomeric state

• Limited proteolysis to evaluate proper folding

• Negative-stain electron microscopy to visualize the c-ring formation

For definitive structural verification, cryo-electron microscopy has emerged as the method of choice for membrane protein complexes like ATP synthase subunit c .

What methods are available to assess the functional activity of recombinant H. influenzae atpE?

Functional characterization of recombinant H. influenzae atpE requires assessing both its ability to form proper oligomeric structures and its proton translocation activity. The following methodological approaches are recommended:

• Proton translocation assays: Reconstitute the purified protein into liposomes loaded with pH-sensitive fluorescent dyes (e.g., ACMA or pyranine). Monitor fluorescence changes upon addition of ionophores or ATP.

• ATP synthesis assays: When co-reconstituted with other ATP synthase components, measure ATP production using luciferase-based luminescence assays in the presence of ADP and phosphate.

• Inhibitor binding studies: Analyze binding of known ATP synthase inhibitors like diarylquinolines, which have been shown to interact specifically with the F0 part containing subunit c . Techniques such as surface plasmon resonance or isothermal titration calorimetry can quantify these interactions.

These functional assays should be performed under varying pH conditions (pH 6.5-8.0) to determine optimal activity parameters specific to H. influenzae atpE.

How can researchers investigate the oligomerization properties of H. influenzae atpE?

The oligomerization of atpE subunits to form the c-ring is essential for ATP synthase function. Based on structural studies of ATP synthase from related organisms, the following approaches have proven effective:

• Blue native PAGE: This technique preserves protein-protein interactions and can reveal the presence of properly assembled c-rings.

• Chemical cross-linking followed by mass spectrometry: This approach can identify specific residues involved in subunit-subunit interactions.

• Analytical ultracentrifugation: Provides information about the size, shape, and stoichiometry of the oligomeric complex.

• Electron microscopy: Negative staining EM can visualize the ring structure formed by oligomerized subunit c proteins .

Understanding the oligomerization properties is critical as variations in c-ring stoichiometry can affect the bioenergetic efficiency of ATP synthesis.

What techniques can distinguish between properly folded and misfolded recombinant atpE?

Distinguishing properly folded from misfolded recombinant atpE is essential for reliable experimental outcomes. Several complementary techniques should be employed:

• Thermal shift assays: Properly folded membrane proteins typically exhibit cooperative unfolding transitions at higher temperatures compared to misfolded variants.

• Protease resistance assays: Native protein conformations generally exhibit greater resistance to limited proteolysis than misfolded versions.

• Detergent solubility profiles: Properly folded membrane proteins show characteristic solubility in specific detergents, while aggregated or misfolded proteins often exhibit different solubility patterns.

• Binding assays with known ligands: The ability to specifically bind known interaction partners or inhibitors can validate proper folding .

These approaches collectively provide a comprehensive assessment of protein folding quality, which is particularly important for membrane proteins like atpE that are prone to misfolding during recombinant expression.

How does recombinant H. influenzae atpE contribute to antibiotic development research?

Recombinant H. influenzae atpE serves as a valuable tool for antibiotic development research, particularly for respiratory infections. Studies with related bacterial ATP synthase subunit c have demonstrated that:

• ATP synthase is essential for bacterial growth and survival, making it an attractive drug target .

• Diarylquinolines specifically bind to the F0 part of ATP synthase containing subunit c, suggesting this binding site can be exploited for H. influenzae-specific inhibitors .

• Downregulation of atpE expression significantly impairs bacterial growth, confirming its validity as a therapeutic target .

Researchers can use purified recombinant H. influenzae atpE for high-throughput screening of compound libraries, structure-based drug design, and validation of potential inhibitors. The identification of compounds that selectively inhibit H. influenzae ATP synthase could lead to novel antibiotics for treating infections, particularly in patients with respiratory conditions like COPD or cystic fibrosis .

What role does ATP synthase play in H. influenzae pathogenesis and virulence?

ATP synthase plays a crucial role in H. influenzae pathogenesis through several mechanisms:

• Energy provision for virulence factor expression: ATP synthase generates the energy required for the production and secretion of virulence factors.

• Adaptation to host environments: Recent genomic analysis of pathogenic H. influenzae strains indicates that energy metabolism adaptations, including those involving ATP synthase, may contribute to the ability to cause systemic infections rather than remaining localized to respiratory tissues .

• Survival under stress conditions: ATP synthase function is essential for bacterial persistence during antibiotic treatment and immune response.

Comparative genomic studies of emerging non-typeable H. influenzae strains (C1 and C2) have revealed unique accessory gene profiles that more closely resemble isolates from blood than sputum, suggesting metabolic adaptations that may involve ATP synthase in systemic infections . Understanding these adaptations could provide insights into H. influenzae pathogenesis and identify new therapeutic approaches.

How does the inhibition of H. influenzae ATP synthase affect related metabolic pathways?

Inhibition of H. influenzae ATP synthase has cascading effects on multiple metabolic pathways due to its central role in energy production. Key affected pathways include:

• Respiratory chain function: ATP synthase inhibition disrupts the proton gradient maintenance, affecting the entire respiratory chain activity, similar to observations in other bacteria where ATP synthase inhibition impaired respiratory chain structure and function .

• Stress response pathways: Energy depletion triggers stress response mechanisms, including altered gene expression patterns.

• Competence development: H. influenzae natural transformation capacity is linked to energy availability, with ATP synthase inhibition potentially affecting competence-regulated operons dependent on cAMP-CRP signaling .

Experimental data from related bacteria indicate that ATP synthase inhibition leads to bacterial growth arrest and eventual cell death, confirming its essential role in cellular metabolism . Understanding these metabolic connections provides a broader perspective on the systemic impacts of targeting ATP synthase for therapeutic purposes.

What are common challenges in expressing recombinant H. influenzae atpE and how can they be addressed?

Expression of recombinant H. influenzae atpE presents several challenges that researchers should anticipate:

• Membrane protein toxicity: Overexpression of membrane proteins like atpE can be toxic to host cells.

  • Solution: Use specialized expression strains like C41/C43(DE3) designed for toxic membrane proteins, or employ tightly regulated expression systems with tunable promoters.

• Inclusion body formation: Hydrophobic membrane proteins often aggregate into inclusion bodies.

  • Solution: Lower induction temperatures (16-20°C), reduce inducer concentration, or co-express with molecular chaperones like GroEL/GroES.

• Low expression levels: Membrane proteins typically express at lower levels than soluble proteins.

  • Solution: Optimize codon usage for the expression host, use strong ribosome binding sites, and consider fusion partners that enhance expression.

• Protein misfolding: The complex topology of membrane proteins increases misfolding risk.

  • Solution: Include specific lipids during expression or purification that stabilize the native conformation, optimize detergent selection for proper folding.

The cloning strategy should also be carefully considered, as demonstrated in ATP synthase research where specific PCR approaches and specialized vectors were employed for successful expression .

What strategies address solubility and stability challenges with recombinant atpE?

Maintaining solubility and stability of recombinant atpE requires specialized approaches:

For long-term storage, researchers should evaluate stability in different conditions using analytical techniques like size exclusion chromatography. Evidence from related ATP synthase research indicates that including specific phospholipids during purification and storage can significantly enhance stability by mimicking the native membrane environment .

How can researchers troubleshoot functional reconstitution of ATP synthase containing recombinant atpE?

Functional reconstitution of ATP synthase containing recombinant atpE is a complex process that requires troubleshooting at multiple steps:

• Lipid composition optimization: ATP synthase activity depends on the surrounding lipid environment.

  • Troubleshooting: Systematically test different lipid compositions (varying PE, PG, and cardiolipin ratios) to identify optimal reconstitution conditions.

• Protein-to-lipid ratio determination: Incorrect ratios can lead to impaired function.

  • Troubleshooting: Perform reconstitution with varying protein-to-lipid ratios (typically 1:50 to 1:200 w/w) and measure activity to determine the optimal ratio.

• Incomplete proton gradient formation: Improper vesicle formation can prevent gradient establishment.

  • Troubleshooting: Verify vesicle integrity using calcein leakage assays and optimize vesicle preparation protocols.

• Assembly of the complete ATP synthase complex: Reconstitution may require additional subunits.

  • Troubleshooting: For full functional characterization, co-reconstitute with other essential subunits or use hybrid approaches with native F1 portions from related species .

Researchers should implement rigorous quality control at each step, including electron microscopy to verify proper reconstitution and control experiments to confirm specific ATP synthase activity versus non-specific effects.

How can cryo-EM technologies advance structural understanding of H. influenzae atpE?

Cryo-electron microscopy (cryo-EM) offers revolutionary potential for elucidating the detailed structure of H. influenzae atpE within the ATP synthase complex. Recent advances in cryo-EM provide several specific advantages:

• Native-like conditions: Cryo-EM allows visualization of membrane proteins in lipid environments that closely mimic physiological conditions, avoiding artifacts associated with crystallization.

• Conformational dynamics: Single particle analysis can capture different conformational states of the c-ring during the rotational catalysis cycle, providing insights into the molecular mechanism of proton translocation.

• High-resolution details: Modern cryo-EM can achieve resolutions approaching 2-3 Å for membrane protein complexes, sufficient to visualize key residues involved in proton binding and translocation.

• Complex assembly: Cryo-EM enables visualization of how atpE assembles with other ATP synthase subunits, particularly the crucial interaction with subunit a that forms the proton channel .

Researchers should consider using recombinant expression systems that yield sufficient quantities of properly assembled ATP synthase containing H. influenzae atpE, followed by detergent extraction and careful optimization of cryo-EM grid preparation to maximize structural insights.

What genetic approaches can reveal the regulatory mechanisms controlling atpE expression in H. influenzae?

Understanding the regulatory mechanisms controlling atpE expression in H. influenzae requires sophisticated genetic approaches:

• Promoter analysis: Identification and characterization of regulatory elements controlling atpE transcription using reporter gene fusions and site-directed mutagenesis.

• Regulon mapping: ChIP-seq and RNA-seq analyses under various environmental conditions can identify transcription factors and regulatory networks controlling ATP synthase expression.

• CRE site investigation: H. influenzae utilizes competence regulatory elements (CRE) for controlling gene expression. Similar regulatory mechanisms might influence atpE expression, particularly in response to environmental stresses .

• cAMP-CRP regulation: Given the importance of cAMP receptor protein (CRP) in H. influenzae gene regulation, its potential role in controlling ATP synthase expression should be investigated using crp knockout strains and cAMP modulation .

Recent genomic analyses of emerging H. influenzae strains highlight the importance of understanding these regulatory mechanisms, as they may contribute to differences in virulence and host adaptation between strains .

How do mutations in H. influenzae atpE correlate with antibiotic resistance phenotypes?

The correlation between atpE mutations and antibiotic resistance represents an important research direction:

• Target-based resistance: Mutations in atpE can directly confer resistance to ATP synthase inhibitors. Research with diarylquinolines demonstrates that specific mutations in atpE gene result in reduced binding of these inhibitors to ATP synthase .

• Adaptive metabolism changes: Certain atpE mutations may alter ATP synthase efficiency, leading to metabolic adaptations that indirectly confer resistance to multiple antibiotic classes.

• Cross-resistance patterns: Systematic analysis of atpE mutations across clinical isolates can reveal patterns of cross-resistance that inform antibiotic development strategies.

The methodology for investigating these correlations should include:

• Whole genome sequencing of resistant clinical isolates

• Directed evolution experiments under antibiotic selection pressure

• Site-directed mutagenesis to introduce specific atpE mutations

• Biochemical characterization of mutant proteins to determine altered binding affinities

These approaches will help develop a comprehensive understanding of resistance mechanisms and guide the design of new antibiotics that can overcome ATP synthase-mediated resistance .