Recombinant Streptococcus pneumoniae ATP synthase subunit delta (atpH)

What is the role of ATP synthase in Streptococcus pneumoniae pathogenesis?

ATP synthase is a crucial enzyme complex for energy production in S. pneumoniae, directly affecting its growth, survival, and virulence capabilities. The F0F1-ATP synthase complex generates ATP through oxidative phosphorylation, providing energy for numerous cellular processes essential for bacterial pathogenesis.

Research indicates that proper assembly and function of F0F1-ATP synthase components, including the delta subunit, are vital for normal pneumococcal physiology. Disruptions in this complex can significantly impact virulence as demonstrated in mouse models of bacteremia and pneumonia . The ATP synthase complex appears to be less dependent on the Signal Recognition Particle (SRP) pathway in S. pneumoniae compared to other streptococcal species, suggesting unique regulatory mechanisms that may be exploited for targeted interventions .

What expression systems are most effective for producing recombinant S. pneumoniae atpH?

For optimal recombinant production of S. pneumoniae atpH, E. coli-based expression systems utilizing strategically optimized culture media compositions have proven most effective. While no system is universally superior, methodological optimization is essential.

The expression methodology should incorporate:

• Selection of an appropriate E. coli strain (commonly BL21(DE3) or derivatives)

• Codon optimization for the target gene

• Fusion tags selection (His-tag, MBP, or GST) for improved solubility and purification

• Culture media optimization focusing on key nutritional factors

Media optimization studies have shown that glucose, NH4Cl, and yeast extract concentrations significantly impact recombinant protein yields in E. coli . Through systematic optimization using Response Surface Methodology (RSM) and Artificial Neural Network (ANN) approaches, researchers have achieved significant improvements in recombinant protein production.

Table 1. Optimal Media Composition for Recombinant Protein Production in E. coli

Implementation of this optimized media formulation has demonstrated an 88.4% increase in recombinant protein yield compared to basic media formulations .

What purification strategies are recommended for recombinant S. pneumoniae atpH?

A systematic multi-step purification approach is recommended for isolating high-purity recombinant S. pneumoniae atpH:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged recombinant atpH

• Intermediate Purification: Ion-exchange chromatography to separate based on charge differences

• Polishing: Size-exclusion chromatography to achieve final purity

For optimal results, buffer conditions should be carefully optimized with consideration for:

• pH stability range (typically 7.0-8.0 for ATP synthase components)

• Salt concentration (150-300 mM NaCl)

• Addition of glycerol (10-15%) to enhance stability

• Potential inclusion of reducing agents (1-5 mM DTT or β-mercaptoethanol)

The purification protocol must be adapted based on specific construct design and experimental requirements. Verification of protein identity and purity should be performed using SDS-PAGE, Western blotting, and mass spectrometry.

How does the YidC2 insertase influence the membrane integration of ATP synthase components in S. pneumoniae?

The YidC2 insertase plays a critical role in the membrane integration and assembly of F0F1-ATP synthase components in S. pneumoniae. Research has demonstrated that F0F1-ATP synthase is a known substrate of YidC2 and depends on this insertase for efficient and correct assembly .

Unlike in Streptococcus mutans, where both the Signal Recognition Particle (SRP) pathway and YidC2 are collectively required for proper F0F1-ATP synthase function under acidic conditions, S. pneumoniae appears to have a more balanced relationship between these two protein targeting systems . This is likely due to the different environmental pressures faced by the two species—S. pneumoniae does not encounter the same acidic stress in its nasopharyngeal niche as S. mutans does in the oral cavity.

Interestingly, proteomic analysis has shown that the amounts of membrane-localized F0F1-ATP synthase subunits alpha, beta, and gamma were not significantly different between the wild-type strain D39 and a ΔscRNA mutant (deficient in the RNA component of the SRP pathway) . This suggests that in S. pneumoniae, YidC2 is adequate for proper F0F1-ATP synthase assembly and function, with the SRP pathway playing a less critical role than in other streptococci.

This differential dependence on protein targeting pathways represents a unique aspect of S. pneumoniae physiology that could potentially be exploited for species-specific therapeutic interventions.

What experimental models are most effective for studying atpH function in relation to S. pneumoniae virulence?

Multiple complementary experimental models should be employed to comprehensively investigate atpH function in S. pneumoniae virulence:

In vitro cellular models:

• Alveolar epithelial cell lines (A549) have been effectively used to study S. pneumoniae-host interactions, particularly in examining pneumococcal effects on cellular signaling pathways

• Primary alveolar epithelial cells provide a more physiologically relevant system for studying host-pathogen interactions

• Temperature-dependent effects on bacterial-host interactions should be carefully controlled, as they can significantly influence experimental outcomes

In vivo animal models:

• Mouse models of bacteremia and pneumonia have proven valuable for assessing S. pneumoniae virulence factors and their contribution to pathogenesis

• These models allow for evaluation of bacterial survival, dissemination, and host inflammatory responses

Genetic approaches:

• Construction of targeted gene deletion mutants (ΔatpH) using allelic replacement techniques

• Complementation studies to confirm phenotype specificity

• Site-directed mutagenesis to investigate specific functional domains

Biochemical approaches:

• ATP synthesis/hydrolysis assays to directly measure enzymatic function

• Membrane potential measurements to assess the impact on proton motive force

• Protein-protein interaction studies to map the interaction network of atpH

Integrating data from these complementary approaches provides the most comprehensive understanding of atpH function in pneumococcal virulence. Recent studies have successfully employed mouse models of bacteremia and pneumonia to demonstrate that disruptions in key cellular processes significantly attenuate S. pneumoniae virulence, highlighting the utility of these models for studying factors like ATP synthase components .

How do pH changes affect ATP synthase function and assembly in S. pneumoniae?

The relationship between pH and ATP synthase function in S. pneumoniae reveals distinct adaptations compared to other streptococci. Unlike S. mutans, which faces significant acid stress in the oral cavity, S. pneumoniae inhabits the relatively neutral pH environment of the nasopharynx, resulting in different evolutionary pressures on ATP synthase regulation .

Research indicates that S. pneumoniae does not exhibit the same heavy dependence on F0F1-ATP synthase for acid tolerance as observed in S. mutans. In S. mutans, none of the ΔscRNA, Δffh, ΔftsY, and ΔyidC2 mutants (affecting protein targeting pathways) could grow at pH 5.0, indicating that both SRP and YidC2 pathways are essential for ATP synthase assembly under acidic conditions . S. pneumoniae has likely evolved a more balanced utilization of these protein targeting pathways.

When S. pneumoniae encounters acidic environments during infection, several adaptive responses occur:

• Alterations in membrane composition and integrity

• Induction of stress response systems

• Changes in ATP synthase activity to maintain essential energy production

These findings suggest that while ATP synthase function is important for S. pneumoniae under various conditions, the regulatory mechanisms governing its assembly and activity under acid stress differ from those in other streptococci, reflecting adaptation to different ecological niches.

What optimization approaches can enhance recombinant S. pneumoniae atpH production?

Recombinant production of S. pneumoniae atpH can be significantly enhanced through systematic optimization approaches that integrate statistical modeling with machine learning techniques. A multi-stage optimization strategy is recommended:

• Initial Screening: Plackett-Burman design to identify key variables affecting protein yield

  • In recombinant protein production studies, glucose, NH4Cl, and yeast extract were identified as the most significant variables affecting yield

• Path of Steepest Ascent: Determine the direction of optimization based on initial screening results

  • Step sizes for significant factors should be based on coefficient estimates (e.g., step sizes of 2, 3, and 4 for glucose, NH4Cl, and yeast extract respectively)

• Response Surface Methodology (RSM): Further refine optimization with Box-Behnken Design

  • Establish quadratic regression equations relating variables to protein yield

  • Generate three-dimensional response surfaces to visualize variable interactions

• Artificial Neural Network (ANN) Modeling: Capture complex non-linear relationships

  • Use a back-propagation method with tansig and purelin transfer functions for hidden and output layers

  • Employ the trainbr algorithm with appropriate allocation ratios for training, testing, and validation datasets

• Genetic Algorithm (GA) Optimization: Use with ANN model to determine precise optimal conditions

Table 2. Performance Comparison of Optimization Models

The ANN-GA combined approach has demonstrated superior regression accuracy in both prediction and optimization processes compared to RSM alone . Implementation of optimized conditions derived from ANN-GA modeling has achieved up to 88.4% improvement in recombinant protein yield compared to basic medium formulations.

How can proteomics approaches be used to study S. pneumoniae ATP synthase assembly and regulation?

Proteomics approaches offer powerful tools for investigating ATP synthase assembly and regulation in S. pneumoniae. These methodologies allow researchers to examine protein expression, modifications, interactions, and localization under various conditions.

Quantitative Proteomics Strategies:

• Comparative Membrane Proteomics:

  • Studies comparing wild-type and mutant strains have revealed important insights into membrane protein assembly

  • Analysis of the membrane proteome of wild-type D39 versus ΔscRNA mutant showed that levels of F0F1-ATP synthase subunits alpha, beta, and gamma remained unchanged despite disruption of the SRP pathway

  • This approach identified cellular adaptation mechanisms that maintain critical energy-generating complexes

• Interaction Proteomics:

  • Affinity purification coupled with mass spectrometry to identify protein-protein interactions

  • Crosslinking mass spectrometry to capture transient interactions between ATP synthase subunits

  • Proximity labeling techniques (BioID, APEX) to identify proteins in the vicinity of atpH in living cells

• Post-translational Modification Analysis:

  • Phosphoproteomics to identify potential regulatory phosphorylation sites

  • S. pneumoniae utilizes serine/threonine kinase StkP for stress response regulation, which could potentially affect ATP synthase components

• Spatial Proteomics:

  • Subcellular fractionation coupled with proteomics to track protein localization

  • Tracking of ATP synthase assembly intermediates in different cellular compartments

• Time-resolved Proteomics:

  • Pulse-chase experiments to monitor protein synthesis and turnover rates

  • Analysis of ATP synthase assembly kinetics under different environmental conditions

These proteomic approaches have revealed that S. pneumoniae employs distinct protein targeting mechanisms for assembling membrane proteins like ATP synthase compared to other streptococci, with potentially important implications for bacterial physiology and pathogenesis .

What are the critical parameters for optimizing recombinant S. pneumoniae atpH solubility?

Enhancing the solubility of recombinant S. pneumoniae atpH requires systematic optimization of multiple parameters across the expression and purification workflow:

Expression System Design:

• Fusion partner selection: Solubility-enhancing tags (MBP, SUMO, Thioredoxin) often outperform simple affinity tags

• Codon optimization: Adjusting rare codons to match E. coli usage while maintaining mRNA secondary structure

• Vector selection: Low-copy number vectors may reduce protein aggregation through controlled expression rates

Expression Conditions:

• Temperature: Lower temperatures (16-25°C) generally enhance solubility by slowing protein synthesis

• Induction parameters: IPTG concentration (typically 0.1-0.5 mM) and induction timing (mid-log phase)

• Media composition: The optimal concentrations identified for recombinant protein production (glucose at 6.488 g/L, NH4Cl at 5.653 g/L, and yeast extract at 14.807 g/L) provide a starting point

Additives and Co-factors:

• Osmolytes: Addition of glycerol (5-10%), sucrose (5-10%), or arginine (50-100 mM)

• Specific ions: Mg²⁺ (1-5 mM) may enhance stability of ATP binding proteins

• Co-expression with chaperones: GroEL/GroES, DnaK/DnaJ/GrpE systems

Buffer Optimization:

• pH screening: Typically in 0.5 unit increments around physiological pH

• Salt concentration: Ionic strength testing (typically 50-500 mM NaCl)

• Reducing agents: DTT or TCEP (1-5 mM) to prevent disulfide bond formation

Purification Strategy:

• Gentle lysis methods: Enzymatic lysis (lysozyme) over mechanical disruption when possible

• Rapid processing: Minimizing time between cell lysis and initial purification steps

• Stabilizing additives: Carry forward successful additives identified during expression

Implementing a systematic approach to test these parameters through factorial design experiments can significantly improve recombinant atpH solubility and yield. The optimization of culture media components alone has been shown to increase recombinant protein yields by up to 88.4% in E. coli expression systems .

How can researchers verify the functional integrity of purified recombinant S. pneumoniae atpH?

Verifying the functional integrity of purified recombinant S. pneumoniae atpH requires a multi-faceted approach addressing structural integrity, binding capabilities, and enzymatic function:

Structural Verification:

• Circular Dichroism (CD) Spectroscopy

  • Analyze secondary structure composition (α-helices, β-sheets)

  • Compare spectra with theoretically predicted secondary structure

  • Monitor thermal stability through temperature-dependent CD measurements

• Thermal Shift Assays

  • Assess protein stability using fluorescence-based thermal denaturation

  • Compare melting temperatures (Tm) under different buffer conditions

  • Evaluate stabilizing effects of potential ligands or interaction partners

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

  • Determine oligomerization state in solution

  • Verify monodispersity of the purified protein

  • Detect potential aggregation

Functional Verification:

• Binding Assays

  • Surface Plasmon Resonance (SPR) to measure interactions with other ATP synthase subunits

  • Microscale Thermophoresis (MST) to quantify binding affinities

  • Pull-down assays to confirm interactions with physiological partners

• Assembly Reconstitution

  • In vitro reconstitution with other purified ATP synthase components

  • Analysis of complex formation by native PAGE or analytical ultracentrifugation

  • Electron microscopy to visualize reconstituted complexes

• Enzymatic Activity

  • ATP hydrolysis assays (if applicable to the delta subunit)

  • Proton transport measurements in reconstituted liposomes

  • Coupling efficiency determinations

The integrity of F0F1-ATP synthase components is particularly important for proper function, as demonstrated by studies showing that correct assembly is dependent on specific protein targeting pathways in S. pneumoniae . Therefore, comprehensive functional verification is essential for ensuring that recombinant atpH can serve as a valid model for the native protein in subsequent experiments.

What bioinformatic approaches can predict interaction sites between atpH and other ATP synthase components?

Advanced bioinformatic approaches offer powerful tools for predicting interaction interfaces between S. pneumoniae atpH and other ATP synthase components:

Sequence-Based Methods:

• Multiple Sequence Alignment (MSA) Analysis

  • Identification of conserved residues across bacterial species

  • Coevolution analysis to detect correlated mutations using methods like Direct Coupling Analysis (DCA) or GREMLIN

  • Conservation mapping to identify functionally important surfaces

• Interface Prediction Algorithms

  • SPPIDER, WHISCY, or ProMate to predict protein-protein interaction sites

  • Prediction based on physicochemical properties, evolutionary conservation, and structural features

  • Machine learning approaches integrating multiple features for improved accuracy

Structure-Based Methods:

• Homology Modeling

  • Construction of S. pneumoniae atpH 3D model based on homologous proteins

  • Refinement using energy minimization and molecular dynamics simulations

  • Quality assessment using tools like PROCHECK, VERIFY3D, and QMEANDisCo

• Molecular Docking

  • Protein-protein docking using tools like HADDOCK, ClusPro, or ZDOCK

  • Integration of experimental constraints (if available) to guide docking

  • Ensemble docking approaches to account for protein flexibility

• Molecular Dynamics Simulations

  • Analysis of binding interface stability in explicit solvent models

  • Identification of key intermolecular interactions that stabilize the complex

  • Free energy calculations to estimate binding affinity

Integrated Approaches:

• Network Analysis

  • Construction of protein interaction networks

  • Identification of hub proteins and critical interaction nodes

  • Prediction of functional modules within the ATP synthase complex

• AlphaFold2 Multimer

  • Deep learning-based structure prediction of protein complexes

  • Generation of accurate models of interacting proteins without template structures

  • Confidence metrics to assess prediction reliability

These computational approaches can guide experimental designs by identifying specific residues likely involved in atpH interactions with other ATP synthase components. The resulting predictions can inform targeted mutagenesis studies to experimentally validate the role of specific residues in complex assembly and function.

Applied to S. pneumoniae ATP synthase, these methods can help elucidate the unique features that distinguish pneumococcal ATP synthase from those of other bacterial species, potentially revealing species-specific interaction patterns that could be exploited for antimicrobial development.