Recombinant Salmonella agona ATP synthase subunit b (atpF)

How does recombinant atpF protein differ from native protein in structural and functional studies?

When studying recombinant atpF protein versus native protein, researchers should consider:

Methodological approach:

• Expression system effects: Recombinant atpF is typically produced in E. coli expression systems, which may introduce post-translational modifications different from those in native Salmonella.

• Tag interference: Most recombinant proteins contain affinity tags that can affect protein folding, oligomerization, or interactions.

Experimental considerations:

• For structural studies, evaluate whether the tag position (N- or C-terminal) affects conformation

• For functional assays, compare ATP synthesis/hydrolysis rates between tagged and tag-cleaved proteins

• Include proper controls with native protein when assessing binding interactions

For accurate results, researchers should verify that recombinant atpF retains the ability to incorporate into the ATP synthase complex and maintain proper membrane interactions.

What experimental approaches are recommended for purifying recombinant Salmonella atpF?

Recommended purification protocol:

• Expression optimization:

  • Use pET expression systems with BL21(DE3) E. coli strain

  • Induce at OD600 0.6-0.8 with 0.5-1.0 mM IPTG

  • Lower induction temperature to 18-25°C to enhance solubility

• Membrane fraction isolation:

  • Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

  • Resuspend in buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl)

  • Disrupt cells by sonication or French press

  • Separate membrane fraction by ultracentrifugation (100,000 × g, 1 h, 4°C)

• Protein extraction:

  • Solubilize membranes with 1-2% detergent (DDM, LDAO, or Triton X-100)

  • Incubate with gentle agitation for 1-2 h at 4°C

  • Remove insoluble material by centrifugation (20,000 × g, 30 min, 4°C)

• Affinity chromatography:

  • Apply solubilized protein to appropriate affinity resin

  • Wash extensively to remove non-specific binding

  • Elute with imidazole (His-tag) or desthiobiotin (Strep-tag)

• Size exclusion chromatography:

  • Further purify by gel filtration to obtain homogeneous protein

  • Analyze fractions by SDS-PAGE for purity assessment

This protocol has been adapted from methods used for purifying membrane proteins from bacterial ATP synthase complexes .

How does ATP synthase subunit b contribute to Salmonella agona virulence and persistence?

ATP synthase subunit b plays an indirect but critical role in Salmonella virulence through its essential function in energy metabolism. Research findings indicate:

• Interaction with virulence factors:

  • The bacterial virulence protein MgtC targets the F1Fo ATP synthase complex, interacting primarily with the a subunit, but the stability provided by the b subunit is crucial for this interaction .

  • MgtC inhibits ATP-driven proton translocation and NADH-driven ATP synthesis, which is vital for Salmonella's survival within macrophages .

• Role in persistent infections:

  • S. agona has been increasingly recognized as a cause of persistent infections, with its strong biofilm formation capabilities contributing to this persistence .

  • ATP synthase function is modulated during the transition from acute to persistent infection, as evidenced by changes in ATP levels and cytoplasmic pH .

• Contribution to stress adaptation:

  • During macrophage invasion, Salmonella must adapt to the acidic phagosomal environment.

  • The ATP synthase complex helps maintain physiological ATP levels and cytoplasmic pH, which is essential for survival under these stress conditions .

The b subunit's role in maintaining ATP synthase structure is particularly important during persistent infections, where S. agona undergoes genomic rearrangements and metabolic adaptations to evade host immune responses .

What are the key experimental considerations when designing assays to study atpF interactions with Salmonella virulence factors?

When designing experiments to study interactions between atpF and virulence factors such as MgtC, researchers should consider the following:

Experimental design framework:

Critical methodological considerations:

• Membrane protein context:

  • atpF is a membrane protein, requiring special handling to maintain native conformation

  • Use mild detergents (DDM, LDAO) at minimal concentrations during extraction

  • Consider reconstitution in liposomes or nanodiscs for functional studies

• Intact complex requirements:

  • Some interactions may require the intact F1Fo complex rather than isolated subunits

  • Compare results from isolated recombinant atpF versus membrane preparations containing the complete ATP synthase

• Physiological relevance:

  • Include assays under conditions mimicking the macrophage environment (pH ~5.5)

  • Assess interaction effects on ATP synthesis/hydrolysis rates and proton translocation

The experimental design should include proper controls to validate specific interactions, particularly using genetic approaches with defined mutants in the virulence factors being studied .

How can researchers accurately measure the effects of atpF mutations on ATP synthesis and bacterial fitness?

Methodological approach for comprehensive assessment:

• ATP synthesis measurement in inverted membrane vesicles:

  • Prepare inside-out vesicles from wild-type and atpF mutant strains

  • Measure NADH-driven ATP synthesis using the luciferase reaction

  • Monitor ATP formation over time in the presence of ADP and Pi

  • Compare synthesis rates between wild-type and mutant vesicles

• ATP hydrolysis assays:

  • Quantify phosphate release using colorimetric methods (malachite green assay)

  • Test ATPase activity under different pH conditions (pH 5.5-7.5)

  • Include specific inhibitors to confirm ATP synthase-specific activity

• Proton translocation measurement:

  • Monitor fluorescence quenching of acridine orange or ACMA dye

  • Induce proton gradient with ATP or NADH addition

  • Quantify initial rates and extent of quenching

  • Calibrate with ionophores (nigericin, FCCP)

• In vivo bacterial fitness assays:

  • Growth curves in various media conditions (including low Mg2+ and low pH)

  • Competition assays between wild-type and mutant strains

  • Macrophage survival assays to assess intracellular fitness

  • Mouse infection models for in vivo virulence assessment

Data analysis should include statistical comparison of multiple independent experiments, with careful attention to the physiological relevance of ATP levels and pH changes in the context of Salmonella pathogenesis.

What genomic variations in the atpF gene have been observed during Salmonella agona persistent infections?

Recent studies examining S. agona isolates from persistent infections have revealed:

Genomic variation patterns:

• SNP accumulation during infection transition:

  • Increased SNP variation has been observed during early, convalescent carriage (3 weeks–3 months)

  • This variation likely reflects population expansion after acute infection

  • These genetic changes potentially represent immune evasion mechanisms enabling persistent infection

• Genomic structure variations:

  • Analysis of 207 isolates revealed conserved arrangement GS1.0 in most isolates (195)

  • Eight additional genomic structures were identified in 12 isolates

  • Rearranged isolates were typically associated with early convalescent carriage

• Phenotypic correlations:

  • Isolates from convalescent and temporary carriage showed significantly reduced biofilm formation ability compared to those from acute illness

  • Some persistent isolates displayed metabolic adaptations, including loss of 1,2-propanediol utilization

How does the ATP sensing mechanism in Salmonella regulate virulence gene expression?

Salmonella employs sophisticated ATP sensing mechanisms to regulate virulence gene expression:

Key regulatory pathways:

• mgtCBR operon regulation:

  • The mgtCBR leader mRNA senses cellular ATP levels

  • This mechanism upregulates mgtC transcription when Salmonella experiences mild acidification

  • Two short ORFs in the leader region (mgtM and mgtP) are involved in this ATP-sensing mechanism

• Experimental evidence:

  • Fluorescence was six-fold higher when a purine auxotroph was grown in media with high adenine compared to low adenine

  • This difference reflects the higher ATP levels present in bacteria grown at higher adenine concentration

  • When wild-type Salmonella was grown in glucose, fluorescence was twice as high as when grown in glycerol, reflecting larger ATP amounts generated with glucose as carbon source

• Structural basis:

  • The mgtCBR leader mRNA can adopt alternative structures (stem-loops A and B)

  • Mutations at positions 44-46 in the mgtCBR leader eliminated the response to ATP

  • These structural changes control the translation of MgtC, which in turn targets the F1Fo ATP synthase

• Physiological consequences:

  • MgtC inhibits F1Fo ATP synthase activity, reducing ATP levels

  • This adaptation is crucial for survival in the acidic environment of macrophage phagosomes

  • Dysregulated expression of MgtC and MgtB can reduce ATP to abnormally low levels, impairing bacterial growth

This elaborate sensing mechanism allows Salmonella to modulate its energy metabolism in response to environmental conditions, particularly during host infection.

What are the optimal conditions for expressing recombinant Salmonella agona atpF in heterologous systems?

Optimized expression protocol:

• Expression system selection:

  • Recommended: pET vector systems in E. coli BL21(DE3) or C43(DE3) strains

  • C43(DE3) strain is specifically advantageous for membrane proteins

  • Consider codon optimization for high-level expression

• Expression conditions optimization:

• Troubleshooting expression issues:

  • If inclusion bodies form: Lower temperature further (16°C) and IPTG concentration (0.05 mM)

  • If toxic to host: Use auto-induction media or tighter promoter control

  • If low expression: Check codon usage, consider fusion partners (MBP, SUMO)

• Verification methods:

  • Western blot using anti-His/anti-Strep antibodies or custom atpF antibodies

  • Membrane fractionation to confirm proper localization

  • Blue-native PAGE to assess incorporation into ATP synthase complex

The optimal expression approach should be determined empirically for each specific construct, as membrane proteins like atpF can vary in their expression characteristics.

How can researchers accurately assess the incorporation of recombinant atpF into functional ATP synthase complexes?

Methodological approach for functional assessment:

• Membrane isolation and ATP synthase complex extraction:

  • Isolate bacterial membranes by differential centrifugation

  • Solubilize using mild detergents (0.5-1% DDM, digitonin, or LDAO)

  • Perform blue native PAGE to visualize intact ATP synthase complexes

• Activity assays to confirm functionality:

  • ATP synthesis: Measure luciferin/luciferase-based ATP production in inverted membrane vesicles

  • ATP hydrolysis: Quantify inorganic phosphate release using colorimetric assays

  • Proton pumping: Monitor fluorescence quenching of pH-sensitive dyes (ACMA or acridine orange)

• Structural verification approaches:

  • Co-immunoprecipitation: Use antibodies against other ATP synthase subunits to pull down complexes

  • Size exclusion chromatography: Analyze complex formation by molecular weight

  • Cryo-EM or negative stain EM: Visualize complex architecture

• Quantitative assessment of incorporation:

  • Ratio determination: Compare stoichiometry of atpF to other subunits using quantitative Western blotting

  • Mass spectrometry: Use labeled reference peptides for absolute quantification

  • Functional complementation: Assess ability to rescue growth defects in atpF deletion strains

When substituting native atpF with recombinant variants, researchers should include controls to ensure that observed phenotypes are due to specific properties of the variant rather than expression level differences or incomplete incorporation.

What strategies can address common challenges in purifying and stabilizing recombinant atpF protein?

Challenge-solution framework for atpF purification:

Advanced stabilization techniques:

• Lipid supplementation:

  • Add cardiolipin or phosphatidylglycerol (0.1-0.5 mg/ml) to extraction and purification buffers

  • These lipids are natural components of bacterial membranes that stabilize ATP synthase

• Nanodisc reconstitution:

  • Incorporate purified atpF into MSP nanodiscs with defined lipid composition

  • Provides a more native-like membrane environment than detergent micelles

• Construct optimization:

  • Remove flexible termini that may cause aggregation

  • Create fusion constructs with well-folding partners

  • Introduce strategic disulfide bonds to enhance stability

These approaches must be empirically tested for each specific experimental goal, as the optimal conditions may vary depending on the downstream application (structural studies, activity assays, or interaction analyses).

How can recombinant atpF be used to study Salmonella agona persistence mechanisms?

Recombinant atpF provides valuable tools for understanding S. agona persistence:

Research application approaches:

• Structure-function studies of virulence factor interactions:

  • Generate site-directed mutants of atpF to map interaction interfaces with MgtC

  • Create chimeric proteins with atpF domains from non-persistent species

  • Assess effects on ATP synthesis inhibition and virulence factor binding

• Biofilm formation assessment:

  • Recent studies show that S. agona isolates from persistent infections display reduced biofilm formation capability

  • Investigate whether mutations in ATP synthase components correlate with changes in biofilm formation

  • Use recombinant atpF variants to complement deletion strains and assess biofilm restoration

• Experimental methodology for persistence models:

  • Develop fluorescently-tagged atpF constructs to track ATP synthase localization during infection

  • Create reporter systems linking atpF expression/interaction to measurable signals

  • Employ these tools in cellular infection models to monitor real-time changes during persistence establishment

• Comparative analysis between acute and persistent infections:

  • Analyze atpF sequence variations in isolates from different stages of infection

  • Express recombinant proteins representing these variants to assess functional differences

  • Correlate with genomic structure variations observed during persistent infections

This research can provide insights into the metabolic adaptations that enable S. agona to transition from acute to persistent infection, potentially revealing new therapeutic targets.

What techniques can detect conformational changes in atpF during ATP synthesis inhibition by virulence factors?

Advanced biophysical approaches:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Principle: Measures solvent accessibility of protein regions

  • Application: Detect regions of atpF that become protected or exposed upon interaction with virulence factors like MgtC

  • Advantage: Can work with membrane proteins in detergent micelles or nanodiscs

  • Protocol outline:

    • Incubate atpF alone or with MgtC in D2O buffer

    • Quench reaction at various timepoints

    • Digest with pepsin and analyze by LC-MS/MS

    • Identify peptides with altered deuterium incorporation

• Site-directed spin labeling EPR:

  • Principle: Measures distances between specific residues and their mobility

  • Application: Map conformational changes in atpF upon interaction with MgtC

  • Protocol:

    • Generate single-cysteine variants at strategic positions

    • Label with spin probes (MTSL)

    • Measure EPR spectra in presence/absence of interaction partners

    • Calculate distance constraints and mobility parameters

• Single-molecule FRET:

  • Principle: Measures distances between fluorophores at nanometer scale

  • Application: Real-time monitoring of conformational changes during ATP synthesis inhibition

  • Implementation:

    • Label specific residues in atpF with donor/acceptor fluorophores

    • Reconstitute into liposomes or immobilize on surfaces

    • Monitor FRET efficiency changes upon addition of MgtC or other virulence factors

• Cryo-electron microscopy:

  • Principle: Direct visualization of protein structure at near-atomic resolution

  • Application: Compare ATP synthase structures with/without virulence factors

  • Approach:

    • Purify intact ATP synthase complexes

    • Image in presence/absence of MgtC

    • Perform 3D reconstruction to identify conformational differences

These sophisticated biophysical techniques can reveal the molecular mechanisms by which virulence factors like MgtC inhibit ATP synthase function, potentially identifying new targets for antimicrobial development.

How do mutations in atpF affect Salmonella agona's response to environmental stresses during infection?

Comprehensive stress response analysis framework:

These studies can provide insights into how S. agona adapts its energy metabolism during the transition from acute to persistent infection, potentially revealing targets for disrupting this process .

How do genetic variations in atpF impact bacterial antibiotic susceptibility?

Mechanisms linking ATP synthase function to antibiotic resistance:

• Energy-dependent drug efflux:

  • Many efflux pumps require ATP or proton motive force

  • atpF mutations affecting ATP production may alter efflux efficiency

  • Experimental approach:

    • Compare accumulation of fluorescent substrates (ethidium bromide, Nile red) in wild-type vs. atpF mutants

    • Measure MICs of antibiotics with/without efflux pump inhibitors

    • Correlate ATP production capacity with efflux efficiency

• Membrane potential and antibiotic uptake:

  • ATP synthase function affects membrane potential (Δψ)

  • Many antibiotics require Δψ for cellular entry

  • Research method:

    • Measure membrane potential using voltage-sensitive dyes (DiSC3(5), JC-1)

    • Assess uptake of radiolabeled antibiotics in different atpF variants

    • Determine correlation between Δψ alterations and antibiotic susceptibility

• Persister cell formation:

  • Low ATP states are associated with antibiotic tolerance

  • atpF mutations may affect transition to persister state

  • Analytical approach:

    • Quantify persister formation under antibiotic stress

    • Measure ATP levels in persister populations

    • Evaluate effects of atpF complementation on persister formation

• Clinical relevance in persistent infections:

  • S. agona isolates from persistent infections show genomic variations

  • These variations may include changes affecting ATP synthase components

  • Research findings:

    • Persistent isolates display altered metabolic profiles

    • Biofilm formation capability is reduced in persistent isolates

    • These adaptations may contribute to antibiotic tolerance in vivo

Understanding the relationship between ATP synthase function and antibiotic susceptibility could lead to new therapeutic strategies targeting energy metabolism to overcome antimicrobial resistance in persistent Salmonella infections.

How can CRISPR-Cas9 genome editing be optimized for studying atpF function in Salmonella agona?

CRISPR-Cas9 optimization strategy for atpF manipulation:

• sgRNA design considerations:

  • Target regions with minimal off-target potential

  • Avoid essential domains that may prevent viable mutant recovery

  • Design multiple sgRNAs targeting different regions of atpF

  • Recommended tools: CHOPCHOP, E-CRISP with S. agona genome as reference

• Delivery methods optimization:

  • Plasmid-based: pCas9 and sgRNA on separate compatible plasmids

  • Temperature-sensitive plasmids for transient expression

  • Electroporation parameters: 2.5 kV, 25 μF, 200 Ω for highest efficiency

• Homology-directed repair template design:

  • For point mutations: 500-1000 bp homology arms

  • For deletions/insertions: 40-60 bp homology arms

  • Include silent mutations in PAM site to prevent re-cutting

  • Consider codon optimization while maintaining protein sequence

• Screening and verification protocol:

  • Primary screening: PCR with primers flanking the target region

  • Secondary confirmation: Sanger sequencing of PCR products

  • Functional verification: ATP synthesis/hydrolysis assays

  • Whole-genome sequencing to detect off-target effects

• Conditional mutagenesis approaches:

  • Since atpF is potentially essential, employ:

    • Inducible CRISPRi for knockdown rather than knockout

    • Tunable expression systems (tetO/tetR) for complementation

    • Temperature-sensitive alleles for conditional function

These optimized CRISPR-Cas9 approaches enable precise genetic manipulation of atpF to study its role in S. agona pathogenesis and persistence mechanisms.

What does current research reveal about the evolution of ATP synthase components in Salmonella pathogenicity?

Evolutionary insights from comparative genomics:

• Conservation patterns across Salmonella serovars:

  • The core structure of ATP synthase is highly conserved across Salmonella species

  • The a and b subunits show higher sequence conservation than peripheral components

  • Key residues involved in proton translocation and ATP synthesis are under purifying selection

• Adaptation signatures in persistent pathogens:

  • Typhoid-causing serovars show specific adaptations in energy metabolism genes

  • S. agona isolates from persistent infections exhibit increased genomic diversity

  • This diversity includes SNPs and structural rearrangements that may affect energy metabolism

• Host-specific adaptations:

  • Host-restricted serovars (e.g., S. Typhi, S. Gallinarum) show distinct metabolic adaptations

  • These include loss of function in certain metabolic pathways (1,2-propanediol utilization)

  • Similar metabolic adaptations have been observed in some persistent S. agona isolates

• Virulence factor interactions:

  • The MgtC virulence factor specifically targets the F1Fo ATP synthase a subunit

  • This interaction is conserved across multiple intracellular pathogens including S. enterica, M. tuberculosis, and others

  • The asparagine residue at position 92 in MgtC is conserved across these species and is critical for interaction with ATP synthase

These evolutionary patterns suggest that ATP synthase components play important roles in Salmonella adaptation to different hosts and persistence mechanisms, with specific adaptations occurring during the transition from acute to chronic infection.

How might recombinant atpF be exploited for developing novel antimicrobial strategies?

Innovative therapeutic approaches targeting ATP synthase:

• Structure-based inhibitor design:

  • Recombinant atpF enables structural studies revealing potential binding pockets

  • Virtual screening against these pockets can identify potential inhibitors

  • High-throughput screening using ATP synthesis assays can validate candidates

  • Rational design approach:

    • Target interface between b and a subunits

    • Design peptidomimetics based on MgtC-interaction domains

    • Focus on compounds that disrupt essential subunit interactions

• Immunotherapeutic strategies:

  • ATP synthase components are surface-exposed in some bacteria

  • Recombinant atpF can be used to develop:

    • Subunit vaccines targeting conserved epitopes

    • Monoclonal antibodies for passive immunization

    • Antibody-drug conjugates for targeted delivery

• Anti-virulence approaches:

  • Target virulence factor interactions with ATP synthase

  • Recombinant atpF enables:

    • Screening for compounds that block MgtC-ATP synthase interaction

    • Development of competitive inhibitors that prevent MgtC binding

    • Creation of decoy molecules that sequester virulence factors

• Combination therapy rationale:

  • ATP synthase inhibition may sensitize bacteria to existing antibiotics

  • Preliminary data suggests targeting energy metabolism can:

    • Reduce persister formation

    • Increase uptake of certain antibiotics

    • Prevent development of resistance mechanisms

These approaches offer promising alternatives to conventional antibiotics, particularly for addressing persistent infections where current therapies often fail.

What role does atpF play in the metabolic adaptation of Salmonella during transition to persistent infection?

Metabolic reprogramming during persistence establishment:

• Energy homeostasis regulation:

  • ATP synthase function is modulated during infection progression

  • MgtC-mediated inhibition fine-tunes ATP levels in response to environmental cues

  • This regulation is crucial for adaptation to the phagosomal environment

• Evidence from recent studies:

  • Persistent S. agona isolates show metabolic adaptations including:

    • Loss of 1,2-propanediol utilization in some carriage isolates

    • Reduced biofilm formation capacity

    • Changes in carbon source utilization profiles

  • These adaptations may reflect a shift from an acute infection metabolic state to a persistent state

• ATP synthase regulation mechanisms:

  • Transcriptional control: mgtCBR leader RNA senses ATP levels to regulate MgtC expression

  • Post-translational modification: Virulence factors like MgtC interact with ATP synthase components

  • These mechanisms allow dynamic adjustment of energy metabolism during infection stages

• Proposed model for atpF role in persistence: