Recombinant Legionella pneumophila ATP synthase subunit beta (atpD)

What is the role of ATP synthase subunit beta (atpD) in Legionella pneumophila metabolism?

ATP synthase subunit beta (atpD) is a critical component of the F1F0-ATP synthase complex in L. pneumophila, which plays a fundamental role in energy metabolism. AtpD contains the catalytic sites for ATP synthesis and is essential for the bacterium's energy production through oxidative phosphorylation. In L. pneumophila, ATP synthesis is particularly important during intracellular replication phases, as the bacterium must adapt to the host environment. Studies have shown that genes involved in ATP binding are significantly regulated during intracellular growth, suggesting that energy metabolism coordination is crucial for successful infection and replication within host cells .

What genetic regulation controls atpD expression during different growth phases of L. pneumophila?

The expression of atpD in L. pneumophila is tightly regulated based on the bacterium's growth phase and environmental conditions. During intracellular growth, L. pneumophila undergoes significant transcriptional reprogramming. Gene expression studies have revealed that many genes categorized under 'ATP binding' are differentially expressed during intracellular growth compared to extracellular conditions . This regulation likely involves both global transcriptional regulators and specific response systems that detect changes in nutrient availability and host cell conditions. The transition between replicative and transmissive phases of L. pneumophila's lifecycle is particularly important for atpD expression regulation, as energy requirements differ substantially between these phases.

What expression systems are most effective for producing recombinant L. pneumophila AtpD with optimal folding and activity?

For recombinant L. pneumophila AtpD production, several expression systems have been evaluated, with yeast and E. coli-based systems showing particular promise. For complex proteins like ATP synthase subunits, eukaryotic expression systems such as yeast may provide advantages for proper protein folding . The methodology typically involves:

• Gene cloning into an appropriate expression vector containing a promoter compatible with the selected expression system

• Incorporation of affinity tags (commonly His-tag) for purification

• Optimization of expression conditions (temperature, induction parameters, and growth media)

• Cell lysis under conditions that preserve protein structure and activity

Selection of the expression system should consider the specific experimental requirements, including protein yield, folding complexity, and downstream applications.

What are the critical factors for successful solubilization and purification of recombinant AtpD from L. pneumophila?

Successful solubilization and purification of recombinant L. pneumophila AtpD requires attention to several critical factors:

• Lysis buffer composition: Buffers containing mild detergents (0.5-1% Triton X-100 or n-dodecyl β-D-maltoside) help solubilize the protein without denaturation

• pH optimization: Maintaining pH between 7.0-8.0 generally preserves AtpD structure and function

• Affinity chromatography: His-tagged AtpD can be purified using nickel or cobalt affinity resins

• Salt concentration: Moderate salt concentrations (150-300 mM NaCl) help maintain protein solubility while minimizing non-specific interactions

• Reducing agents: Addition of DTT or β-mercaptoethanol (1-5 mM) helps prevent oxidation of cysteine residues

The purification protocol should include steps to remove contaminants while preserving the native conformation of AtpD, which is essential for functional studies.

How can researchers verify the proper folding and activity of recombinant L. pneumophila AtpD after purification?

Verification of proper folding and activity of recombinant L. pneumophila AtpD can be accomplished through multiple complementary approaches:

• Enzymatic activity assays: Measuring ATP hydrolysis activity using colorimetric phosphate detection methods

• Circular dichroism spectroscopy: Analyzing secondary structure content to confirm proper folding

• Thermal shift assays: Assessing protein stability under various buffer conditions

• Size exclusion chromatography: Confirming the oligomeric state and homogeneity of the purified protein

• Limited proteolysis: Properly folded proteins typically show resistance to limited proteolytic digestion compared to misfolded variants

These analytical methods, when used in combination, provide comprehensive evaluation of the structural integrity and functional capacity of the purified recombinant AtpD protein.

What are the key functional domains of L. pneumophila AtpD and how do they contribute to ATP synthesis?

L. pneumophila AtpD contains several key functional domains essential for its role in ATP synthesis:

• Nucleotide-binding domains: These regions bind ATP and ADP and contain the Walker A and Walker B motifs, which are highly conserved sequences involved in nucleotide binding and hydrolysis

• Catalytic sites: Located at the interface between alpha and beta subunits, these sites undergo conformational changes during the catalytic cycle

• DELSEED region: This conserved sequence participates in energy transduction between the F0 and F1 portions of ATP synthase

• Alpha/beta interface regions: These domains facilitate interaction with alpha subunits to form the catalytic hexamer structure

Each domain contributes to the coordinated conformational changes that couple proton translocation through the F0 sector to ATP synthesis at the catalytic sites within the F1 sector. The precise coordination of these domains enables the rotary mechanism of ATP synthesis essential for bacterial energy production .

How does the ATP-binding pocket of L. pneumophila AtpD compare with that of human mitochondrial ATP synthase, and what implications does this have for drug targeting?

The ATP-binding pocket of L. pneumophila AtpD shares structural similarities with human mitochondrial ATP synthase beta subunit but exhibits specific differences that can be exploited for selective drug targeting:

These structural differences enable the rational design of inhibitors that selectively target the bacterial enzyme while minimizing effects on human mitochondrial ATP synthesis. Such selectivity is critical for developing antimicrobial agents that disrupt L. pneumophila energy metabolism without causing mitochondrial toxicity in human cells .

What post-translational modifications have been identified in native L. pneumophila AtpD, and how do these affect function?

Several post-translational modifications (PTMs) have been identified in native L. pneumophila AtpD that significantly impact its function:

• ADP-ribosylation: L. pneumophila effector proteins, specifically Lpg0080 (an ADP ribosyltransferase), can modify AtpD through ADP-ribosylation, potentially altering its activity during infection

• Phosphorylation: Phosphorylation at specific serine and threonine residues may regulate AtpD activity in response to environmental conditions

• Acetylation: N-terminal acetylation and lysine acetylation have been detected, potentially influencing protein stability and interactions

• Oxidative modifications: Cysteine oxidation may occur under stress conditions, affecting protein function

These PTMs constitute a complex regulatory network that allows L. pneumophila to fine-tune ATP synthase activity in response to environmental cues and host cell conditions. The reversible nature of modifications like ADP-ribosylation, which can be removed by ARH enzymes like Lpg0081, provides a sophisticated mechanism for temporal regulation of energy metabolism during infection .

How does L. pneumophila modulate AtpD function during different stages of intracellular infection?

L. pneumophila employs sophisticated mechanisms to modulate AtpD function throughout the intracellular infection cycle:

• Early infection stage: Upon entry into host cells, L. pneumophila may initially downregulate ATP synthase activity to avoid excessive energy consumption while establishing the Legionella-containing vacuole (LCV)

• Replicative phase: During intracellular replication, L. pneumophila upregulates energy metabolism genes, including those related to ATP synthase function, to support rapid bacterial multiplication

• Transmissive phase: As nutrients become limited, the bacterium transitions to the transmissive phase, with corresponding changes in AtpD regulation

This modulation involves both transcriptional regulation of atpD expression and post-translational modifications. L. pneumophila effector proteins, such as Lpg0080 and Lpg0081, which function as an ADP ribosyltransferase and an ADP ribosylhydrolase respectively, may coordinately regulate the chemical modification of ATP synthase components, including AtpD . This reversible modification system allows L. pneumophila to rapidly adapt its energy metabolism to changing conditions within the host cell.

What is the relationship between ATP synthase activity and virulence in L. pneumophila clinical isolates?

The relationship between ATP synthase activity and virulence in L. pneumophila clinical isolates reveals a complex interplay between energy metabolism and pathogenicity:

• Clinical isolates with enhanced virulence often show optimized regulation of ATP synthase activity, allowing them to efficiently manage energy resources during infection

• Transcriptional analysis of intracellular L. pneumophila reveals differential expression of genes categorized under 'ATP binding,' suggesting a correlation between energy metabolism adaptation and successful infection

• The ability to modulate ATP production in response to host cell conditions correlates with enhanced intracellular survival and replication rates

This relationship highlights the importance of metabolic adaptation in L. pneumophila pathogenesis. Strains that can efficiently regulate ATP synthase activity maintain optimal energy levels throughout the infection cycle, potentially enhancing their virulence and persistence in both environmental hosts (like Acanthamoeba) and human macrophages.

How do host cellular defenses target L. pneumophila ATP synthase during infection, and what countermeasures does the bacterium employ?

Host cellular defenses employ multiple strategies to target bacterial ATP synthase during L. pneumophila infection:

• Reactive oxygen species (ROS): Host-generated ROS can damage bacterial ATP synthase complexes, disrupting energy production

• Phagosomal acidification: Lowered pH in phagosomes can inhibit optimal ATP synthase function

• Nutritional immunity: Host restriction of essential ions and cofactors can limit ATP synthase assembly and function

• Autophagy: Host autophagy machinery can target bacterial components, including ATP synthase complexes

L. pneumophila counters these defenses through several mechanisms:

• Antioxidant systems: Upregulation of alkylhydroperoxidases (like lpg2349 and lpg2350, which were found to be upregulated 5-fold and 11-fold respectively during intracellular growth) to neutralize ROS and protect ATP synthase function

• pH regulation: Manipulation of LCV pH to maintain optimal conditions for ATP synthase activity

• Effector proteins: Secretion of effectors via the Dot/Icm type IV secretion system that modify host processes and protect bacterial energy metabolism

• Metabolic flexibility: Ability to utilize alternative energy sources when ATP synthesis is compromised

These countermeasures reflect the evolutionary adaptation of L. pneumophila to intracellular environments and the central importance of maintaining energy production during infection.

How can recombinant L. pneumophila AtpD be used as a tool for studying host-pathogen interactions?

Recombinant L. pneumophila AtpD serves as a valuable tool for studying host-pathogen interactions through multiple experimental approaches:

• Interaction studies: Purified recombinant AtpD can be used to identify host proteins that interact with this bacterial component during infection

• Immunological investigations: As a conserved bacterial protein, recombinant AtpD can be used to study host immune recognition and response mechanisms

• Trafficking studies: Fluorescently-tagged recombinant AtpD can track the localization and movement of bacterial components within host cells

• PTM analysis: Recombinant AtpD substrates can be used to study the activity of bacterial effectors like Lpg0080 (ADP ribosyltransferase) and Lpg0081 (ADP ribosylhydrolase) that target ATP synthase components

• Structural biology: High-resolution structural studies of recombinant AtpD can reveal binding interfaces with host factors

These applications provide insights into how L. pneumophila establishes infection, modulates host responses, and maintains energy homeostasis during intracellular growth.

What experimental approaches can be used to study the interaction between L. pneumophila AtpD and host mitochondrial proteins?

Several experimental approaches can effectively study interactions between L. pneumophila AtpD and host mitochondrial proteins:

• Co-immunoprecipitation (Co-IP): Using antibodies against recombinant His-tagged AtpD to pull down interacting host mitochondrial proteins

• Proximity labeling: Techniques like BioID or APEX2 fused to AtpD can identify proximal proteins in the mitochondrial environment

• Microscopy techniques:

  • Confocal microscopy with fluorescently labeled AtpD to visualize co-localization with mitochondrial markers

  • Super-resolution microscopy for nanoscale interaction mapping

  • FRET (Förster Resonance Energy Transfer) to detect direct protein-protein interactions

• Surface plasmon resonance (SPR): Quantitative measurement of binding kinetics between purified AtpD and candidate mitochondrial proteins

• Crosslinking mass spectrometry: Identification of specific residues involved in interactions between AtpD and mitochondrial proteins

These approaches, particularly when used in combination, can reveal how L. pneumophila AtpD interacts with host mitochondrial components, potentially including ADP/ATP translocases (ANTs) that are targeted by Legionella effector proteins .

What are the advantages and limitations of using recombinant AtpD versus whole-cell systems for studying L. pneumophila energy metabolism?

Using recombinant AtpD versus whole-cell systems for studying L. pneumophila energy metabolism presents distinct advantages and limitations:

The optimal approach depends on the specific research question. For detailed biochemical and structural studies, recombinant systems offer precision and control. For understanding physiological relevance and complex regulation, whole-cell systems provide the necessary biological context. Many researchers employ both approaches complementarily to develop a comprehensive understanding of L. pneumophila energy metabolism .

How can systems biology approaches integrate AtpD function into broader metabolic networks of L. pneumophila during infection?

Systems biology approaches offer powerful frameworks for integrating AtpD function into the broader metabolic networks of L. pneumophila during infection:

• Multi-omics integration: Combining transcriptomics (like the gene expression data from intracellular Legionella ), proteomics, metabolomics, and fluxomics to create comprehensive metabolic models

• Constraint-based modeling: Development of genome-scale metabolic models that incorporate ATP synthase function and energy constraints during different infection phases

• Network analysis: Identification of metabolic hubs and regulatory nodes that coordinate with ATP synthase activity

• Temporal dynamics: Mapping changes in metabolic flux distribution throughout the infection cycle

Implementation typically involves:

• Construction of a core metabolic network model based on genomic information

• Integration of experimental data, such as the differential expression of genes categorized under 'ATP binding' during intracellular growth

• Validation using experimental measurements of metabolic outputs

• Iterative refinement to improve model predictions

These approaches can reveal how AtpD function is coordinated with other metabolic processes, such as the Entner-Doudoroff pathway (with upregulated edd gene observed during intracellular growth ), to optimize bacterial fitness during infection.

What are the challenges in developing high-resolution structural models of the complete L. pneumophila ATP synthase complex?

Developing high-resolution structural models of the complete L. pneumophila ATP synthase complex presents several significant challenges:

• Membrane protein complexes: The F0 portion of ATP synthase is embedded in the membrane, making it difficult to extract and purify while maintaining native structure

• Complex assembly: ATP synthase consists of multiple subunits (including AtpD) that must assemble correctly, requiring specialized conditions to maintain the intact complex

• Dynamic nature: The rotary mechanism involves conformational changes that are challenging to capture in static structural techniques

• Size limitations: The complete complex exceeds the size limits of some structural biology methods

• Heterogeneity: Conformational heterogeneity in the complex poses challenges for techniques like cryo-EM

Current approaches to address these challenges include:

• Combining X-ray crystallography of individual components with cryo-EM of the complete complex

• Using nanodiscs or amphipols to stabilize the membrane-embedded portions

• Employing cross-linking and mass spectrometry to validate subunit arrangements

• Developing time-resolved structural methods to capture different conformational states

Advances in these techniques are gradually enabling more detailed structural understanding of the complete ATP synthase complex, including the integration of AtpD within the functional assembly.

How might directed evolution approaches be used to engineer L. pneumophila AtpD variants with altered properties for research applications?

Directed evolution offers powerful strategies for engineering L. pneumophila AtpD variants with modified properties for specialized research applications:

• Library generation methods:

  • Error-prone PCR to introduce random mutations throughout the atpD gene

  • Site-saturation mutagenesis targeting specific functional domains

  • DNA shuffling between AtpD homologs from different bacterial species

  • CRISPR-based systems for in vivo mutagenesis

• Selection/screening strategies:

  • Growth-based selection in ATP synthase-deficient bacterial strains

  • Activity-based screening using ATP production assays

  • Binding affinity screening for variants with altered interaction properties

  • Stability selection under challenging conditions

• Potential engineered properties:

  • Enhanced thermostability for structural studies

  • Modified substrate specificity

  • Altered regulatory responses

  • Increased resistance to inhibitors

  • Engineered sensitivity to specific modulators

  • Improved expression in recombinant systems

• Applications of engineered variants:

  • Studying the molecular basis of ATP synthase function

  • Developing reporter systems for monitoring bacterial metabolism

  • Creating tools for selective inhibition studies

  • Designing protein-based biosensors for ATP metabolism

This approach has successfully generated protein variants with novel properties in other systems and could be applied to AtpD to develop specialized research tools for studying L. pneumophila energy metabolism and pathogenesis.

What are the most common challenges in expressing recombinant L. pneumophila AtpD, and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant L. pneumophila AtpD, along with effective solutions:

• Inclusion body formation:

  • Challenge: AtpD may form insoluble aggregates in E. coli expression systems

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

    • Consider alternative expression hosts like yeast systems

    • Add low concentrations of mild detergents to lysis buffer

• Proteolytic degradation:

  • Challenge: Recombinant AtpD may be susceptible to proteolysis

  • Solutions:

    • Add protease inhibitors during purification

    • Use protease-deficient expression strains

    • Optimize buffer conditions to enhance stability

    • Minimize purification time

• Low expression levels:

  • Challenge: Poor yield of recombinant protein

  • Solutions:

    • Codon optimization for expression host

    • Test different promoter systems

    • Optimize induction parameters (timing, inducer concentration)

    • Screen multiple expression strains

• Improper folding:

  • Challenge: Expressed protein lacks native conformation

  • Solutions:

    • Co-express with chaperones

    • Include stabilizing additives (glycerol, specific ions)

    • Express as individual domains if full-length proves challenging

    • Consider cell-free expression systems

By systematically addressing these challenges, researchers can significantly improve the yield and quality of recombinant L. pneumophila AtpD for downstream structural and functional studies.

How can researchers distinguish between effects on AtpD function versus broader metabolic changes when studying L. pneumophila mutants?

Distinguishing between direct effects on AtpD function versus broader metabolic changes in L. pneumophila mutants requires a multi-faceted experimental approach:

• Complementation studies:

  • Generate point mutations in specific AtpD functional domains

  • Perform genetic complementation with wild-type atpD

  • Create chimeric AtpD proteins with domain swaps to isolate functional regions

• Direct activity measurements:

  • Measure ATP synthase activity in isolated bacterial membranes

  • Compare ATP synthesis/hydrolysis rates between mutant and wild-type strains

  • Assess proton pumping activity in membrane vesicles

• Metabolic profiling:

  • Perform comparative metabolomics to identify altered metabolic pathways

  • Measure intracellular ATP/ADP ratios and energy charge

  • Track carbon flux through central metabolic pathways using labeled substrates

  • Analyze expression of genes involved in alternative energy generating pathways

• Specific inhibitor studies:

  • Use ATP synthase-specific inhibitors to phenocopy atpD mutations

  • Determine if mutant phenotypes can be rescued by metabolic bypasses

These approaches help researchers differentiate between phenotypes directly attributable to ATP synthase dysfunction versus secondary metabolic adaptations, providing clearer insights into AtpD's specific role in L. pneumophila physiology and pathogenesis.

What controls and validation experiments are essential when studying post-translational modifications of L. pneumophila AtpD?

When studying post-translational modifications (PTMs) of L. pneumophila AtpD, several essential controls and validation experiments ensure reliable and reproducible results:

• Sample preparation controls:

  • Parallel processing of wild-type and mutant samples

  • Inclusion of phosphatase/deacetylase inhibitors to preserve PTMs

  • Preparation of artificially modified standards for positive controls

  • Use of PTM-deficient mutants as negative controls

• Analytical validation:

  • Multiple orthogonal detection methods (e.g., Western blot and mass spectrometry)

  • Use of PTM-specific antibodies with appropriate specificity controls

  • Site-directed mutagenesis of modified residues to confirm specificity

  • Quantitative analysis with internal standards

• Functional validation:

  • Comparison of enzymatic activity between modified and unmodified AtpD

  • Activity assays in the presence of modifying and demodifying enzymes (like Lpg0080 and Lpg0081)

  • Structure-function analysis of modified sites

  • Time-course studies to correlate modification status with functional changes

• Physiological relevance:

  • Verification of modifications in different growth conditions

  • Correlation of modification status with infection stages

  • Assessment in clinically relevant strains

  • Examination in different host cell types

These controls and validation experiments are crucial for establishing the authentic nature of PTMs and their functional significance in L. pneumophila AtpD, particularly given the complex regulatory mechanisms employed during host infection, such as the ADP-ribosylation system identified in the search results .

What emerging technologies might advance our understanding of L. pneumophila AtpD structure-function relationships?

Several emerging technologies show promise for advancing our understanding of L. pneumophila AtpD structure-function relationships:

• Cryo-electron tomography (cryo-ET):

  • Enables visualization of ATP synthase in its native cellular environment

  • Provides insights into spatial organization and interactions with other cellular components

  • Allows study of conformational states under physiologically relevant conditions

• Time-resolved structural methods:

  • X-ray free-electron lasers (XFELs) for capturing transient structural states

  • Time-resolved cryo-EM to visualize the ATP synthesis catalytic cycle

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for mapping dynamic regions

• Integrative structural biology approaches:

  • Combining multiple experimental techniques with computational modeling

  • Molecular dynamics simulations to understand energy transduction mechanisms

  • Utilizing AlphaFold2 and other AI-based structure prediction tools to model complex assemblies

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to track conformational changes

  • Optical tweezers to measure mechanical forces during ATP synthesis

  • Single-molecule localization microscopy for in situ visualization

These technologies will provide unprecedented insights into how AtpD functions within the ATP synthase complex, how its activity is regulated during infection, and how structural changes correlate with the functional adaptations observed in gene expression studies of intracellular L. pneumophila .

How might cross-disciplinary approaches combining structural biology, microbiology, and immunology enhance therapeutic targeting of L. pneumophila AtpD?

Cross-disciplinary approaches that integrate structural biology, microbiology, and immunology offer promising pathways for therapeutic targeting of L. pneumophila AtpD:

• Structure-based drug design pipeline:

  • High-resolution structures of AtpD to identify unique binding pockets

  • Virtual screening of compound libraries against bacterial-specific features

  • Rational design of inhibitors that exploit structural differences from human homologs

  • Iterative optimization based on biochemical and microbiological feedback

• Microbiology-informed therapeutic strategies:

  • Identification of growth conditions where AtpD function is most critical

  • Exploitation of bacterial metabolism vulnerabilities during infection

  • Development of combination approaches targeting multiple aspects of energy metabolism

  • Understanding resistance mechanisms through experimental evolution studies

• Immunological considerations:

  • Exploration of AtpD as a potential vaccine antigen

  • Investigation of host immune recognition of ATP synthase components

  • Development of antibody-drug conjugates targeting surface-exposed ATP synthase regions

  • Understanding how immune responses modulate bacterial energy metabolism

• Translational research pathway:

  • Initial target validation using recombinant proteins and biochemical assays

  • Progression to cellular infection models with clinical isolates

  • Animal model testing of lead compounds or immunization strategies

  • Biomarker development to monitor therapeutic efficacy

This integrated approach allows researchers to leverage the strengths of each discipline, accelerating the development of novel therapeutics that target L. pneumophila energy metabolism through AtpD inhibition, potentially providing alternatives to conventional antibiotics for treating Legionnaires' disease.

What are the potential applications of AtpD-based biosensors for studying L. pneumophila pathogenesis in real-time?

AtpD-based biosensors offer innovative approaches for studying L. pneumophila pathogenesis in real-time, with several promising applications:

• Metabolic state monitoring:

  • Conformational sensors that report on ATP synthase activity states

  • FRET-based sensors to detect changes in ATP/ADP ratios within bacteria

  • Reporters that track post-translational modifications of AtpD during infection

  • Nanobody-based sensors that recognize specific AtpD conformational states

• Host-pathogen interaction visualization:

  • Split fluorescent protein systems to detect AtpD interactions with host factors

  • Sensors reporting on effector-mediated modifications of ATP synthase components

  • Real-time tracking of energy metabolism adaptation during infection stages

  • Monitoring bacterial responses to host defense mechanisms

• High-throughput screening applications:

  • Drug discovery platforms using AtpD-based activity reporters

  • Identification of host factors that modulate bacterial energy metabolism

  • Screening for compounds that synergize with host defense mechanisms

  • Discovery of small molecules that alter ATP synthase regulation

• In vivo infection dynamics:

  • Animal models with bacteria expressing AtpD-based reporters

  • Non-invasive imaging of bacterial metabolic states in tissues

  • Correlation of energy metabolism with bacterial spread and replication

  • Evaluation of therapeutic interventions targeting bacterial metabolism

These biosensor applications would provide unprecedented insights into the dynamic regulation of L. pneumophila energy metabolism during infection, potentially revealing new therapeutic targets and intervention strategies. They would build upon current understanding of how L. pneumophila modulates its energy production during different phases of intracellular growth, as suggested by the differential expression of ATP binding genes observed in previous studies .