Recombinant Salmonella paratyphi B ATP synthase subunit c (atpE)

What is the role of ATP synthase in Salmonella paratyphi B pathogenesis?

ATP synthase plays a critical role in energy metabolism for Salmonella species, including S. paratyphi B. The F1Fo ATP synthase is responsible for the synthesis of the majority of adenosine triphosphate (ATP) in the bacterial cell, which is essential for pathogen survival within host environments . Unlike some other metabolic enzymes, ATP synthase in Salmonella has evolved to function specifically within the challenging environments encountered during infection, including the acidic conditions found within macrophages. The proper functioning of ATP synthase helps maintain cytoplasmic pH near 7 when experiencing mildly acidic pH inside host cells, which is crucial for bacterial persistence .

How does ATP synthase subunit c (atpE) differ structurally from subunit a (atpB)?

ATP synthase subunit c (atpE) and subunit a (atpB) are both components of the membrane-embedded Fo sector of the F1Fo ATP synthase but serve distinct structural and functional roles:

The specific sequence of atpB from S. paratyphi C begins with "MASENMTPQEYIGHHLNNLQLDLRTFSLVDPQNPPATFWTLNIDSMFFSVVLGLLFLVMF..." as identified in recombinant protein studies .

What are the optimal expression conditions for recombinant S. paratyphi B atpE protein in E. coli systems?

For optimal expression of recombinant S. paratyphi B atpE protein in E. coli systems, researchers should consider:

• Expression vector selection: pET vectors with T7 promoter systems typically yield high expression for membrane proteins like atpE.

• E. coli strain optimization: BL21(DE3) derivatives specialized for membrane protein expression (such as C41/C43(DE3)) often perform better than standard strains.

• Induction parameters:

  • Temperature: Lower temperatures (16-25°C) generally improve proper folding

  • IPTG concentration: 0.1-0.5 mM typically optimal

  • Duration: Extended expression periods (overnight) at lower temperatures

• Buffer composition for protein extraction:

  • Base buffer: Tris/PBS-based buffer (pH 8.0)

  • Additives: 6% Trehalose as a stabilizer

  • Detergents: n-Dodecyl-β-D-maltoside (DDM) or CHAPS at concentrations above CMC

• Purification strategy:

  • Initial capture: IMAC for His-tagged constructs

  • Further purification: Size exclusion chromatography

Similar approaches have been successfully employed for other ATP synthase subunits from Salmonella, with appropriate modifications for the highly hydrophobic nature of subunit c .

How can researchers effectively study the interaction between MgtC virulence protein and ATP synthase in Salmonella paratyphi B?

To study the interaction between MgtC virulence protein and ATP synthase in Salmonella paratyphi B, researchers should employ a multi-faceted approach:

• Genetic approaches:

  • Create precise gene deletions of mgtC and ATP synthase components using λ-Red recombination

  • Generate double mutants (e.g., mgtC atpB or mgtC atpE) to assess genetic interactions

  • Employ complementation studies with wild-type and mutated versions of the genes

• Biochemical interaction studies:

  • Co-immunoprecipitation using epitope-tagged proteins

  • Bacterial two-hybrid assays to confirm direct protein interactions

  • Surface plasmon resonance to determine binding kinetics

• Functional assays:

  • ATP synthesis measurement in membrane vesicles

  • Proton transport assays using fluorescent probes

  • Cytoplasmic pH measurements using pH-sensitive GFP variants

• Structural studies:

  • Cryo-EM analysis of the ATP synthase complex with and without MgtC

  • Protein cross-linking followed by mass spectrometry

Research has established that MgtC inhibits F1Fo ATP synthase activity in Salmonella, affecting bacterial survival within macrophages. The virulence protein MgtC directly interacts with the ATP synthase, reducing ATP levels and affecting cytoplasmic pH regulation, which is crucial for intramacrophage survival . Similar methodologies can be applied for S. paratyphi B, with appropriate controls for serovar-specific variations.

What techniques are most effective for measuring the impact of atpE mutations on proton translocation and ATP synthesis in Salmonella?

For measuring the impact of atpE mutations on proton translocation and ATP synthesis in Salmonella, several complementary techniques provide comprehensive analysis:

When designing atpE mutations, researchers should focus on conserved residues involved in:

• c-ring formation and stability

• Proton binding sites (critical acidic residues)

• Interface regions with other subunits, particularly subunit a

The experimental approach should include complementation studies with wild-type atpE to confirm phenotypes and careful controls for potential polar effects when creating chromosomal mutations .

How does ATP synthase activity in S. paratyphi B influence its persistence within human macrophages compared to S. Typhi?

ATP synthase activity significantly impacts Salmonella persistence within human macrophages, with important differences between serovars:

S. Typhi and S. Paratyphi can persist within human macrophages, whereas S. Typhimurium rapidly induces apoptotic macrophage cell death . This differential behavior is linked to the regulation of ATP synthesis and energy metabolism. In S. Typhi and S. Paratyphi, persistence correlates with:

• Regulated ATP synthase activity: Controlled ATP production helps maintain appropriate cytoplasmic pH despite the acidic macrophage environment

• MgtC-mediated inhibition: The virulence factor MgtC inhibits the F1Fo ATP synthase, reducing ATP levels when appropriate for intracellular survival

• NF-κB signaling effects: ATP synthase activity indirectly influences NF-κB-dependent responses in host cells. Pharmacologic inhibition of NF-κB affects the persistence of both S. Typhi and S. Paratyphi A within macrophages

• Apoptosis regulation: Unlike S. Typhimurium, typhoidal serovars like S. Typhi and S. Paratyphi lack specific SPI2 effectors with pro-apoptotic functions, contributing to their ability to persist within macrophages rather than triggering cell death

These differences in ATP synthase regulation contribute to the distinct clinical manifestations between typhoidal and non-typhoidal Salmonella infections, with S. paratyphi B showing intermediate characteristics depending on the specific strain being studied.

What is the relationship between atpE expression levels and antimicrobial resistance in clinical isolates of S. paratyphi B?

The relationship between atpE expression levels and antimicrobial resistance in S. paratyphi B clinical isolates is complex and multifaceted:

• Energy-dependent efflux systems: Altered ATP synthase activity impacts the energy available for efflux pump systems, which are major contributors to multidrug resistance. Changes in atpE expression can indirectly affect the efficiency of these pumps.

• Membrane potential modulation: ATP synthase contributes to membrane potential maintenance, which affects the uptake and efficacy of many antimicrobials, particularly aminoglycosides and polymyxins.

• Adaptive response to stress: Clinical isolates showing reduced atpE expression often display:

  • Slower growth rates

  • Enhanced stress tolerance

  • Increased persistence under antibiotic pressure

  • Modified biofilm formation capacity

• Compensatory mutations: Reduced ATP synthase function due to atpE mutations may be compensated by mutations in other energy-generating pathways, creating complex resistance phenotypes.

Research utilizing transcriptomic and proteomic approaches has revealed that antimicrobial exposure can trigger adaptive responses involving ATP synthase expression modulation. Studies of clinical isolates with varying resistance profiles would benefit from including atpE expression analysis as part of a comprehensive characterization of resistance mechanisms.

How does the CigR anti-virulence protein affect ATP synthase function in different Salmonella serovars?

The CigR anti-virulence protein regulates ATP synthase function through an intricate interaction network that varies among Salmonella serovars:

CigR acts as an anti-virulence protein by binding to and inhibiting the virulence protein MgtC, which normally inhibits F1Fo ATP synthase . This creates a regulatory cascade affecting ATP production and bacterial persistence:

• Regulatory mechanism: CigR competes with the F1Fo ATP synthase subunit AtpB for binding to MgtC, preventing MgtC from inhibiting ATP synthase activity

• Expression control: The cigR gene is expressed both constitutively and from a PhoP-dependent promoter shared with mgtC, creating a threshold of CigR protein that MgtC must overcome to initiate its virulence program

• Serovar differences:

  • In S. Typhimurium, a cigR mutant exhibits lower ATP levels and ATPase activity than wild-type, opposite to the phenotypes of an mgtC mutant

  • The balance between CigR and MgtC varies among serovars, potentially contributing to differences in virulence

• pH regulation impact: CigR indirectly affects cytoplasmic pH by modulating MgtC's inhibition of ATP synthase. A cigR mutant maintains higher cytoplasmic pH than wild-type Salmonella both during growth in defined media and inside macrophages

This regulatory network illustrates how fine-tuning of ATP synthase activity through the CigR-MgtC interaction contributes to Salmonella's metabolic adaptation during infection. The conservation and variation of this system across serovars may contribute to their differential pathogenicity.

What structural features of ATP synthase subunit c (atpE) make it a potential target for antimicrobial development?

ATP synthase subunit c (atpE) possesses several structural features that make it an attractive target for antimicrobial development:

• Essential function: As a component of the ATP synthase complex, atpE is essential for bacterial energy metabolism and survival

• Surface accessibility: Portions of the c-ring are accessible from the periplasmic space, providing potential binding sites for inhibitors

• Structural conservation: Key functional residues are highly conserved across bacterial species, allowing for broad-spectrum targeting

• Unique features compared to mammalian counterparts:

  • Different stoichiometry of c-subunits in the c-ring

  • Variations in specific amino acid residues at the proton binding site

  • Distinct interactions with other ATP synthase subunits

• Known binding sites for existing inhibitors:

  • The natural product bedaquiline binds to mycobacterial ATP synthase c-subunit

  • Various other inhibitors demonstrate the druggability of this target

• Oligomeric structure: The c-ring's oligomeric nature provides multiple identical binding sites, potentially enhancing inhibitor efficacy

Researchers designing inhibitors targeting atpE should focus on compounds that:

• Disrupt c-ring assembly

• Interfere with the essential proton translocation mechanism

• Block rotation of the c-ring relative to subunit a

• Target interfaces between c-subunits or between the c-ring and other subunits

The high conservation of ATP synthase across bacterial species makes it a promising broad-spectrum target, while structural differences from human ATP synthase provide a basis for selectivity.

How can CRISPR-Cas9 genome editing be optimized for studying atpE function in Salmonella paratyphi B?

Optimizing CRISPR-Cas9 genome editing for studying atpE function in Salmonella paratyphi B requires careful consideration of several technical aspects:

• Delivery system optimization:

  • Plasmid-based: Use temperature-sensitive plasmids for transient expression

  • Phage-based: Leverage modified P22 phage for efficient delivery

  • Conjugation: Employ tri-parental mating with appropriate helper strains

• Guide RNA design considerations:

  • Target unique regions to avoid off-target effects

  • Verify PAM site accessibility in the native chromosome

  • Design guides targeting both coding and regulatory regions

  • Account for GC content and secondary structure

• Repair template design:

  • For point mutations: 40-60 bp homology arms

  • For deletions/insertions: ≥500 bp homology arms

  • Include silent mutations in PAM or seed region to prevent re-cutting

  • Consider incorporating screening markers (antibiotic resistance cassettes with FRT sites for later removal)

• Special considerations for atpE:

  • Essential gene manipulation requires conditional approaches

  • Design strategies for creating point mutations rather than null mutations

  • Include complementation strategies (ectopic expression under inducible promoter)

  • Consider polar effects on downstream genes in the ATP synthase operon

• Screening methods:

  • MASC-PCR for high-throughput mutation screening

  • Deep sequencing for complex mutant libraries

  • Growth-based screening in conditions requiring ATP synthase function

The efficacy of CRISPR-Cas9 editing can be verified using ATP synthase activity assays similar to those used in previous studies of ATP synthase function in Salmonella .

What emerging technologies show promise for real-time monitoring of ATP synthase activity during Salmonella infection?

Several emerging technologies show exceptional promise for real-time monitoring of ATP synthase activity during Salmonella infection:

• Genetically encoded biosensors:

  • ATP-sensitive FRET-based sensors (ATeam)

  • pH-sensitive fluorescent proteins (pHluorin variants)

  • Membrane potential indicators (PROPS, QuasAr)

  • Integration into Salmonella chromosome for stable expression

• Advanced microscopy approaches:

  • Light-sheet microscopy for 3D visualization with reduced phototoxicity

  • Super-resolution microscopy (STED, PALM/STORM) for nanoscale localization

  • Multi-photon intravital microscopy for in vivo infection monitoring

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

• Microfluidics and single-cell technologies:

  • Droplet microfluidics for high-throughput single bacterium analysis

  • Microfluidic devices mimicking host cell environments

  • Real-time monitoring of single-cell metabolism through integrated sensors

• Metabolomic approaches:

  • Stable isotope labeling to track ATP synthesis rates

  • Mass spectrometry imaging for spatiotemporal metabolite analysis

  • NMR-based metabolic flux analysis

• Host-pathogen interface monitoring:

  • Split reporter systems spanning bacterial and host cell components

  • Resonance energy transfer between labeled host and bacterial proteins

  • CRISPR-based recording systems for capturing transient interactions

Implementation challenges include maintaining physiological relevance, achieving sufficient sensitivity for detecting changes in ATP synthase activity, and integrating multiple measurement modalities. These technologies can provide unprecedented insights into how ATP synthase function dynamically changes during the infection process, particularly during the transition from intestinal invasion to systemic spread that characterizes Salmonella paratyphi infections .

How might atpE mutations contribute to the differential pathogenesis observed between typhoidal and non-typhoidal Salmonella?

Mutations in atpE potentially contribute to the differential pathogenesis between typhoidal and non-typhoidal Salmonella through several mechanisms:

• Energy metabolism adaptation:

  • Typhoidal Salmonella like S. Typhi and S. Paratyphi show distinct adaptations for persistent infection compared to non-typhoidal serovars

  • Subtle variations in atpE may optimize ATP synthase function for different host environments

  • Evidence indicates that typhoidal serovars maintain different ATP homeostasis within macrophages

• Interaction with virulence regulators:

  • Typhoidal and non-typhoidal serovars differ in their virulence factor repertoires

  • ATP synthase components in typhoidal Salmonella may have evolved different interactions with regulators like MgtC and CigR

  • The balance between ATP synthase inhibition by MgtC and the counteraction by CigR likely varies between serovars

• Host immune response modulation:

  • ATP synthase activity influences bacterial persistence within macrophages

  • S. Typhi and S. Paratyphi persist within human macrophages, while S. Typhimurium rapidly induces apoptotic cell death

  • NF-κB-dependent responses are differently affected by typhoidal versus non-typhoidal Salmonella, with ATP homeostasis playing a role

• Clinical manifestation correlation:

  • Typhoidal Salmonella cause prolonged systemic illness (enteric fever)

  • Non-typhoidal Salmonella typically cause acute gastroenteritis

  • These distinct clinical pictures correlate with different bacterial persistence mechanisms, in which ATP synthase regulation plays a key role

Understanding the subtleties of atpE variations across serovars may reveal how evolutionary adaptations in basic metabolic machinery contribute to pathogenic specialization. Research combining comprehensive genomic comparison with functional analyses of ATP synthase activity in different serovars would help elucidate how seemingly minor changes in this essential enzyme complex might influence the dramatically different disease presentations.

What are the most effective methods for purifying functional recombinant ATP synthase complexes containing atpE from Salmonella?

Purifying functional recombinant ATP synthase complexes containing atpE from Salmonella requires a carefully optimized protocol to maintain structural integrity and enzymatic activity:

• Expression system considerations:

  • Bacterial expression: Modified E. coli strains (C43(DE3)) optimized for membrane protein expression

  • Controlled expression: Tunable promoters to prevent toxicity from overexpression

  • Co-expression: Complete ATP synthase operon or selected subunits with proper stoichiometry

• Membrane extraction and solubilization:

  Detergent	Concentration	Advantages	Limitations
  n-Dodecyl-β-D-maltoside (DDM)	1-2%	Preserves activity, mild	Expensive
  Digitonin	1-2%	Maintains native complexes	Very expensive
  CHAPS	8-10 mM	Good for preliminary extraction	Variable results
  Lauryl maltose neopentyl glycol (LMNG)	0.5-1%	High stability, lower CMC	Recent adoption

• Purification strategy:

  • Initial capture: Affinity chromatography (His-tag on non-critical subunit)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography in appropriate detergent

  • Alternative: Density gradient ultracentrifugation

• Activity preservation considerations:

  • Buffer composition: Tris/PBS-based buffer, pH 8.0

  • Stabilizers: 6% Trehalose

  • Lipid supplementation: E. coli polar lipids or synthetic mixtures

  • Storage: Avoid repeated freeze-thaw cycles, store working aliquots at 4°C

• Functional validation:

  • ATP synthesis activity measurement

  • ATP hydrolysis assays

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Structural integrity verification via electron microscopy

This approach can be applied to recombinant Salmonella paratyphi B ATP synthase, leveraging techniques successfully employed for other ATP synthase components like the AtpB subunit from S. paratyphi C .

How can researchers overcome the challenges of studying membrane protein interactions involving atpE in Salmonella?

Researchers facing challenges in studying membrane protein interactions involving atpE in Salmonella can employ several advanced strategies:

• In vivo crosslinking approaches:

  • Photo-crosslinking with genetically incorporated unnatural amino acids

  • Chemical crosslinking with membrane-permeable reagents

  • Advantages: Captures interactions in native environment

  • Applications: Demonstrated success in studying MgtC interactions with ATP synthase components

• Advanced microscopy techniques:

  • Single-molecule localization microscopy (PALM/STORM)

  • Förster resonance energy transfer (FRET)

  • Bioluminescence resonance energy transfer (BRET)

  • Fluorescence correlation spectroscopy (FCS)

  • Implementation: Requires careful fusion protein design to maintain function

• Membrane mimetic systems:

  System	Composition	Advantages	Limitations
  Nanodiscs	Membrane scaffold proteins + lipids	Native-like environment, monodisperse	Complex preparation
  Liposomes	Synthetic or native lipids	Functional assays possible	Heterogeneous
  Bicelles	Phospholipids + detergents	Compatible with NMR	Limited stability
  Amphipols	Amphipathic polymers	High stability	Less native-like

• Genetic approaches:

  • Suppressor mutation analysis

  • Deep mutational scanning

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Comprehensive application: Combine with biochemical validation of identified interactions

• Computational methods to guide experimental design:

  • Molecular dynamics simulations of membrane protein complexes

  • Coevolutionary analysis to predict interaction interfaces

  • Integrative modeling combining low-resolution experimental data

These approaches have been successfully applied to study interactions between the virulence protein MgtC and ATP synthase components, revealing how MgtC inhibits ATP synthase activity by binding to the ATP synthase Fo sector . Similar methodologies can be adapted to investigate specific interactions involving atpE in Salmonella paratyphi B, particularly in the context of understanding how these interactions affect pathogenesis and bacterial persistence within host cells.

What are the most promising directions for developing ATP synthase-targeted therapeutics against Salmonella paratyphi B infections?

Several promising directions for developing ATP synthase-targeted therapeutics against Salmonella paratyphi B infections have emerged:

• Subunit c (atpE) specific inhibitors:

  • Small molecules targeting the c-ring rotation

  • Compounds disrupting c-ring assembly

  • Peptide-based inhibitors targeting the proton channel

  • Potential for broad-spectrum activity against multiple bacterial pathogens

• MgtC-ATP synthase interaction modulators:

  • Compounds enhancing MgtC's natural inhibition of ATP synthase

  • Peptide mimetics of the MgtC binding interface

  • Small molecules stabilizing the MgtC-ATP synthase complex

  • Advantage: Targeting a virulence-specific regulation mechanism

• Proton translocation pathway disruptors:

  • Compounds blocking the critical a/c subunit interface

  • Proton channel blockers

  • Conformation-trapping molecules preventing rotational catalysis

  • Demonstrated precedent: Bedaquiline's success against mycobacterial ATP synthase

• Delivery strategies for enhanced efficacy:

  • Nanoparticle encapsulation for targeted delivery

  • Prodrug approaches to enhance permeability

  • Siderophore conjugation for active transport

  • Exploitation of Salmonella's unique uptake mechanisms

• Combination approaches:

  • ATP synthase inhibitors with conventional antibiotics

  • Multi-target inhibitors affecting both ATP synthase and related systems

  • Host-directed therapies combined with ATP synthase inhibition

  • Strategy: Simultaneous targeting of energy production and utilization pathways

Research shows that ATP synthase inhibition affects Salmonella's ability to maintain cytoplasmic pH and survive within macrophages . These findings suggest that ATP synthase inhibitors could be particularly effective against the persistent infections characteristic of typhoidal Salmonella like S. paratyphi B. The development of such therapeutics would benefit from structural studies of the specific ATP synthase components from S. paratyphi B to identify unique targeting opportunities.

How might research on atpE contribute to understanding the evolutionary divergence of Salmonella serovars?

Research on atpE can provide valuable insights into the evolutionary divergence of Salmonella serovars through multiple approaches:

• Comparative sequence analysis:

  • Phylogenetic analysis of atpE sequences across serovars

  • Identification of host-adapted signatures in ATP synthase components

  • Detection of selection pressures on specific residues

  • Correlation with pathogenic potential and host range

• Functional divergence studies:

  • Cross-complementation experiments between serovars

  • Chimeric ATP synthase construction to identify functionally important variations

  • In vitro activity comparisons under various environmental conditions

  • Linking sequence differences to functional adaptations

• Host adaptation mechanisms:

  • Analysis of atpE modifications in host-restricted serovars (e.g., S. Typhi, S. Paratyphi)

  • Comparison with broad-host-range serovars (e.g., S. Typhimurium)

  • Investigation of atpE interactions with host-specific factors

  • Evidence: Typhoidal serovars like S. Typhi and S. Paratyphi show distinct adaptations for persistent human infection

• Co-evolutionary analyses:

  • Identification of coordinated changes between atpE and other bacterial proteins

  • Mapping of co-evolution with regulatory systems like MgtC-CigR

  • Investigation of compensatory mutations maintaining ATP synthase function

  • Application: Understanding how core metabolic machinery evolves alongside virulence traits