Recombinant Klebsiella pneumoniae subsp. pneumoniae ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase in Klebsiella pneumoniae?

ATP synthase in K. pneumoniae is a multi-subunit protein complex that catalyzes ATP synthesis using the proton gradient across the cell membrane. The enzyme consists of two major subcomplexes:

• The membrane-embedded F₀ complex (containing subunits a, b, and c)

• The cytoplasmic F₁ complex (containing α, β, γ, δ, and ε subunits)

The c-subunit (atpE) forms part of the c-ring within the F₀ complex that rotates as protons pass through the membrane, driving conformational changes in F₁ that enable ATP synthesis .

The ATP synthase structure reveals unique features in the proton-conducting a-subunit, with specific adaptations along both entry and exit pathways that are absent in mitochondrial ATP synthases . These structural differences represent potential targets for antimicrobial development.

How does ATP synthase contribute to K. pneumoniae pathogenicity?

ATP synthase plays a crucial role in K. pneumoniae pathogenicity through:

• Energy production: As the culmination point of bioenergetics, ATP synthase generates the ATP necessary for bacterial survival and virulence factor production .

• Adaptation to host environments: ATP synthesis capabilities allow the bacterium to thrive in different host niches with varying nutrient availability.

• Potential link to antibiotic resistance: Research suggests connections between energy metabolism and resistance mechanisms. For example, the RamA transcription factor, which confers multidrug resistance, regulates the MlaFEDCB ATP-binding cassette transporter that contributes to membrane stability and intrinsic resistance .

The ATP synthase operates within the broader metabolic network that includes other enzymes like citrate synthase (GltA), which enables bacterial replication in the lung and intestine , highlighting the interconnected nature of metabolism and virulence.

What expression systems are optimal for producing recombinant K. pneumoniae ATP synthase subunit c?

Based on established protocols for bacterial membrane proteins, several expression systems have proven effective:

Bacterial Expression Systems:

• E. coli BL21(DE3) with pET vector systems under T7 promoter control

• High-level expression can be achieved through fusion of short peptides to the N-terminus of the native protein, similar to techniques used for other K. pneumoniae membrane proteins

Expression Optimization Table:

The protein can be purified using affinity chromatography methods, with yields enhanced by optimizing expression conditions for inclusion body formation . Subsequent solubilization and refolding steps are critical for obtaining functional protein.

What purification strategies are most effective for isolating functional recombinant atpE protein?

Purification of recombinant K. pneumoniae ATP synthase subunit c requires specialized approaches due to its hydrophobic nature:

• Initial extraction: Solubilization from inclusion bodies or membrane fractions using detergents such as n-dodecyl-β-D-maltoside (DDM) or trans-4-(trans-4′-propylcyclohexyl)cyclohexyl-α-D-maltoside (tPCC-α-M)

• Chromatography sequence:

  • Affinity chromatography (Strep-tag or His-tag)

  • Ion exchange chromatography (MonoQ)

  • Size exclusion chromatography for final polishing

• Reconstitution options:

  • Peptidiscs for stabilization of membrane proteins

  • Nanodiscs or liposomes for functional studies

Using a protocol similar to that employed for other bacterial ATP synthases, researchers should verify protein integrity through SDS-PAGE and mass spectrometry. The molecular mass of the purified protein can be confirmed by electrospray mass spectrometry .

How does K. pneumoniae ATP synthase subunit c compare structurally with other bacterial homologs?

K. pneumoniae ATP synthase subunit c shares key structural features with other bacterial homologs while possessing unique characteristics:

Conserved Features:

• Hairpin structure with two transmembrane helices

• Conserved proton-binding glutamate residue essential for function

• Organization into a c-ring comprising multiple c-subunits

Distinctive Elements in K. pneumoniae:

• The a/c₁₀ interface shows unique structural adaptations in the proton-conducting pathways

• These distinctive features are absent in mitochondrial ATP synthases, representing potential targets for specific inhibitors

The c-ring in K. pneumoniae ATP synthase likely contains 10 c-subunits (c₁₀), similar to other gram-negative bacteria but differing from mycobacterial c-rings, which contain 9 subunits . This structural difference affects the interaction with inhibitors like bedaquiline (BDQ), explaining differential sensitivity across bacterial species.

What research approaches can determine if K. pneumoniae ATP synthase is a viable antimicrobial target?

Evaluating ATP synthase as an antimicrobial target requires multi-faceted approaches:

Target Validation Methods:

• Genetic approaches:

  • Construction of conditional knockdowns (since complete deletion is likely lethal)

  • CRISPR interference to reduce expression

  • Site-directed mutagenesis of key residues

• Pharmacological validation:

  • Screening of known ATP synthase inhibitors against K. pneumoniae

  • Structure-based design of inhibitors targeting the unique a/c₁₀ interface

  • Testing diarylquinolines (DARQs) optimized for K. pneumoniae

• In vivo efficacy studies:

  • Mouse pneumonia models similar to those used for evaluating outer membrane proteins

  • Bacterial load reduction measurements in lungs, kidneys, and spleen

The unique structural features at the a/c₁₀ interface in K. pneumoniae ATP synthase suggest that highly specific inhibitors could be developed with minimal cross-reactivity with human ATP synthase, addressing a key concern for antimicrobial development .

How might ATP synthase inhibition affect drug-resistant and hypervirulent K. pneumoniae strains?

ATP synthase inhibition represents a promising strategy against both drug-resistant and hypervirulent K. pneumoniae:

Effects on Drug-Resistant Strains:

• ATP synthase inhibitors would likely maintain efficacy against strains with resistance to conventional antibiotics, as they target a different cellular process

• The essential nature of ATP synthesis means resistance-conferring mutations would likely incur significant fitness costs

Impact on Hypervirulent Strains:

• Hypervirulent K. pneumoniae strains can infect otherwise healthy individuals in community settings and have acquired resistance to carbapenem antibiotics

• ATP synthase inhibition could potentially disrupt the enhanced metabolic capacity that may contribute to hypervirulence

• As an energy-depleting strategy, ATP synthase inhibition might reduce production of virulence factors

Recent research has identified the genetic elements responsible for converting classical K. pneumoniae into hypervirulent strains . ATP synthase inhibitors targeting conserved features could potentially address both conventional and hypervirulent variants.

What biophysical techniques are most informative for studying K. pneumoniae ATP synthase conformational states?

Multiple complementary techniques provide insights into ATP synthase dynamics:

Structural Analysis Methods:

• Cryo-electron microscopy (cryo-EM):

  • Enables visualization of ATP synthase in different conformational states

  • Has successfully resolved structures of bacterial ATP synthases in three distinct conformational states

  • Particularly valuable for membrane proteins that resist crystallization

• Mass spectrometry approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Cross-linking mass spectrometry to identify subunit interactions

  • Native mass spectrometry to study intact complexes

• Spectroscopic techniques:

  • Fluorescence resonance energy transfer (FRET) for real-time conformational changes

  • Electron paramagnetic resonance (EPR) with spin labeling for distance measurements

• Functional assays:

  • ATP synthesis/hydrolysis measurements with reconstituted enzyme

  • Proton pumping assays in liposomes or nanodiscs

These techniques have revealed self-inhibition mechanisms in the F₁ subcomplex that support unidirectional rotation, preventing wasteful ATP consumption .

How do ATP synthase inhibitors differ in their mechanism compared to conventional antibiotics?

ATP synthase inhibitors represent a distinct antimicrobial strategy:

Conventional Antibiotics vs. ATP Synthase Inhibitors:

ATP synthase inhibitors like bedaquiline (BDQ) bind with high specificity to the interface between the c-ring and a-subunit . Similar compounds could be developed for K. pneumoniae based on its unique a/c₁₀ interface. Structure-based drug design may facilitate the development of compounds targeting this interface in ESKAPE pathogens, including K. pneumoniae .

What structural features of K. pneumoniae ATP synthase can be exploited for selective inhibition?

Several unique features of K. pneumoniae ATP synthase offer opportunities for selective targeting:

Key Targetable Features:

• The a/c₁₀ interface:

  • Contains unique structural adaptations along proton-conducting pathways

  • These features are absent in human mitochondrial ATP synthase

  • Similar interfaces have been successfully targeted in mycobacterial ATP synthases

• Self-inhibition mechanism:

  • The F₁ subcomplex reveals a specific self-inhibition mechanism supporting unidirectional rotation

  • Compounds that disrupt this regulation could potentially disrupt energy homeostasis

• Species-specific residues:

  • Amino acid differences in key functional sites between bacterial and human ATP synthases

  • Could allow development of highly specific inhibitors with minimal off-target effects

Targeted screens of diarylquinoline compounds (similar to those used against mycobacteria) could yield inhibitors specific to K. pneumoniae ATP synthase . These structural differences explain why some compounds inhibit ATP synthases from particular bacterial species but not others.

How can ATP synthase inhibition be integrated with other antimicrobial strategies?

Combination approaches leveraging ATP synthase inhibition offer several advantages:

Strategic Combination Opportunities:

• Synergy with membrane-targeting agents:

  • ATP synthase inhibition could enhance the efficacy of polymyxins or other membrane-disrupting agents

  • Energy depletion may prevent active efflux of other antibiotics

• Combination with virulence inhibitors:

  • ATP synthase inhibitors could reduce energy available for virulence factor production

  • Particularly relevant for hypervirulent strains causing severe tissue-invasive infections

• Targeting multiple metabolic pathways:

  • Combined inhibition of ATP synthase and citrate synthase (GltA), which enables bacterial replication in lung and intestine

  • Could more effectively suppress bacterial energy metabolism

• Immunomodulatory approaches:

  • Potential combination with vaccines targeting outer membrane proteins like Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005, which induce protective immune responses

  • Energy-depleted bacteria may be more susceptible to immune clearance

These combination strategies could be particularly valuable against carbapenem-resistant hypervirulent strains reported by the European Centre for Disease Control and Prevention .

What genetic manipulation approaches are most effective for studying atpE function in K. pneumoniae?

Given the essential nature of ATP synthase, specialized genetic approaches are required:

Recommended Genetic Tools:

• Inducible expression systems:

  • Tetracycline-responsive promoters for controlled expression

  • Allow titration of expression levels to study partial loss of function

• Site-directed mutagenesis:

  • Target conserved residues like the essential glutamate in the c-subunit

  • Create variants with altered proton translocation efficiency

• Domain swapping:

  • Replace segments with corresponding regions from related bacteria

  • Identify determinants of species-specific inhibitor sensitivity

• Reporter fusions:

  • C-terminal fusions with fluorescent proteins to study localization

  • Split-GFP approaches to minimize functional disruption

For genetic manipulation, techniques similar to those used for creating the isogenic gltA mutant could be adapted . This would involve electroporating competent K. pneumoniae cells with a target site fragment containing a resistance cassette, followed by selection and confirmation by colony PCR.

What are the most informative in vitro and in vivo models for evaluating ATP synthase inhibitors against K. pneumoniae?

A multi-tiered approach provides comprehensive evaluation:

In Vitro Models:

• Biochemical assays:

  • Purified enzyme assays measuring ATP synthesis/hydrolysis

  • Membrane vesicle preparations for proton-pumping studies

• Cellular systems:

  • Growth inhibition in minimal media where oxidative phosphorylation is essential

  • Time-kill assays under varying nutrient conditions

  • Membrane potential measurements using fluorescent probes

In Vivo Models:

• Mouse pneumonia model:

  • Intranasal infection with K. pneumoniae followed by treatment

  • Survival rate monitoring for 144 hours

  • Particularly relevant as K. pneumoniae is a common cause of hospital-acquired pneumonia

• Systemic infection model:

  • Intravenous challenge followed by organ bacterial load assessment

  • Significant bacterial load reductions in lungs, kidneys, and spleen would indicate efficacy

  • Bacterial counts in kidneys have shown the most significant reductions in previous studies

• Specialized models for hypervirulent strains:

  • Models that recapitulate the tissue-invasive infections seen in otherwise healthy individuals

  • Assessment of both survival and tissue bacterial burden

These models have been successfully used to evaluate other potential therapeutics against K. pneumoniae and could be adapted for ATP synthase inhibitor evaluation.

How can researchers distinguish between direct ATP synthase inhibition and secondary metabolic effects?

Differentiating direct from indirect effects requires specific experimental approaches:

Differentiation Strategies:

• Molecular evidence of target engagement:

  • Thermal shift assays with purified ATP synthase

  • Competition binding assays with known inhibitors

  • Resistance mutations mapping to atpE gene

• Temporal analysis:

  • Immediate effects on membrane potential and ATP levels indicate direct ATP synthase inhibition

  • Delayed metabolic changes suggest secondary effects

• Metabolomic fingerprinting:

  • Characteristic metabolite profiles for ATP synthase inhibition vs. other targets

  • Comparison with profiles of known inhibitors

• Genetic validation:

  • Reduced sensitivity in strains with point mutations in target sites

  • Increased sensitivity in strains with reduced ATP synthase expression

• Biochemical reconstitution:

  • Demonstration of direct inhibition using purified, reconstituted ATP synthase

  • Elimination of secondary targets through defined reconstitution systems

These approaches provide multiple lines of evidence to confirm that observed antimicrobial effects result directly from ATP synthase inhibition rather than off-target effects.

What role might ATP synthase play in interactions between K. pneumoniae and host immune responses?

ATP synthase may influence host-pathogen interactions through several mechanisms:

• Energy provision for virulence:

  • ATP synthase supplies energy needed for expression of immune evasion factors

  • Inhibition could potentially enhance bacterial susceptibility to immune clearance

• Potential immunomodulatory effects:

  • ATP released by bacteria can act as a damage-associated molecular pattern (DAMP)

  • Modulation of ATP levels through ATP synthase inhibition might alter inflammatory responses

• Interaction with opsonophagocytosis:

  • Energy depletion may impair bacterial defense against phagocytosis

  • Similar to how antibodies against outer membrane proteins facilitate opsonophagocytic killing by HL-60 cells

• Impact on T-cell responses:

  • Metabolically impaired bacteria may present antigens differently

  • Could potentially enhance vaccine-induced responses like the IFN-γ-, IL-4-, and IL-17A-mediated immune responses observed with outer membrane protein vaccines

Understanding these interactions could inform combination strategies pairing ATP synthase inhibitors with immunomodulatory approaches or vaccines.

How might ATP synthase inhibitors be optimized for hypervirulent and multi-drug resistant K. pneumoniae strains?

Developing optimal ATP synthase inhibitors for emerging K. pneumoniae threats requires:

• Structure-guided optimization:

  • Focusing on the unique a/c₁₀ interface in K. pneumoniae ATP synthase

  • Designing compounds that exploit structural differences from human ATP synthase

• Activity against diverse strain types:

  • Testing against panels of clinical isolates, including hypervirulent strains

  • Ensuring efficacy against carbapenem-resistant strains

• Physicochemical optimization:

  • Ensuring adequate penetration of the gram-negative outer membrane

  • Minimizing susceptibility to efflux pumps regulated by transcription factors like RamA

• Combination potential:

  • Co-optimization with agents targeting the MlaFEDCB ATP-binding cassette transporter

  • Could simultaneously inhibit energy production and membrane maintenance

These approaches could address the increasing prevalence of hypervirulent, drug-resistant K. pneumoniae strains reported by the European Centre for Disease Control and Prevention .

What are the most promising experimental approaches for investigating the relationship between ATP synthase function and bacterial persistence?

Several experimental strategies can illuminate ATP synthase's role in persistence:

• Single-cell analysis:

  • Microfluidic devices with real-time imaging of ATP levels

  • Correlation of energy status with persistence phenotypes

• Persister formation assays:

  • Effects of sub-inhibitory ATP synthase inhibitor concentrations on persister frequency

  • Manipulation of ATP synthase expression levels and correlation with persistence

• Stress response connections:

  • Investigation of links between energy stress and activation of persistence mechanisms

  • Transcriptional profiling to identify energy-regulated persistence pathways

• In vivo persistence models:

  • Chronic infection models to assess long-term bacterial survival

  • Evaluation of ATP synthase inhibitors for clearing persistent infections

These approaches could provide insights into how energy metabolism influences bacterial persistence, potentially revealing new strategies for addressing recalcitrant K. pneumoniae infections.

How can structural biology advances further illuminate K. pneumoniae ATP synthase as a drug target?

Emerging structural biology approaches offer new opportunities:

• Time-resolved cryo-EM:

  • Capturing transient conformational states during the catalytic cycle

  • Identifying unique dynamic features that could be exploited for inhibition

• Computational approaches:

  • Molecular dynamics simulations of proton translocation

  • Virtual screening of compound libraries against specific binding sites

• Integrative structural biology:

  • Combining cryo-EM, mass spectrometry, and spectroscopic data

  • Building comprehensive models of ATP synthase function and inhibition

• Structural studies of resistant variants:

  • Determining how mutations confer resistance to ATP synthase inhibitors

  • Informing design of next-generation inhibitors that maintain activity against resistant enzymes

These structural insights could identify unique features in the proton-conducting pathways of K. pneumoniae ATP synthase that are absent in mitochondrial ATP synthases, representing attractive targets for the development of next-generation therapeutics .

What potential exists for developing ATP synthase-targeting vaccines or immunotherapeutics?

While challenging due to membrane localization, several approaches merit investigation:

• Epitope identification:

  • Mapping exposed epitopes of ATP synthase components

  • Focus on regions unique to bacterial ATP synthase

• Recombinant protein approaches:

  • Using recombinant ATP synthase subunits as vaccine antigens

  • Potentially in combination with outer membrane proteins that have shown protective effects

• Targeted antibody development:

  • Generation of monoclonal antibodies against accessible epitopes

  • Potential combination with antibiotics for enhanced clearance

• Adjuvant properties:

  • Exploration of ATP synthase components as carrier proteins

  • Similar to how the K. pneumoniae OmpA protein has shown carrier properties for other antigens