Recombinant Salmonella paratyphi A ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Salmonella paratyphi A?

ATP synthase subunit c (atpE) in Salmonella paratyphi A is a 79-amino acid protein that functions as a critical component of the F0 sector of ATP synthase. The protein spans the bacterial membrane and forms the c-ring structure that facilitates proton translocation during ATP synthesis. Its amino acid sequence (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) reveals a hydrophobic profile consistent with its transmembrane function . The protein contains conserved regions that participate in proton binding and interactions with other ATP synthase subunits, playing an essential role in the bacterium's energy metabolism.

What is the significance of studying recombinant Salmonella paratyphi A atpE in pathogenesis research?

Studying recombinant Salmonella paratyphi A atpE provides valuable insights into the pathogenesis mechanisms of paratyphoid fever, for which an effective vaccine remains elusive . The ATP synthase complex is essential for bacterial survival and plays a role in virulence. Research indicates that S. paratyphi A regulates MAPK and insulin pathways in host organisms, contributing to increased oxidative stress and altered immune responses . Understanding the structure-function relationship of atpE could reveal potential drug targets, as ATP synthase inhibition may disrupt the pathogen's energy metabolism. Additionally, recombinant atpE can be used to develop detection systems and evaluate immune responses in vaccine development pipelines.

How can researchers effectively express and purify recombinant Salmonella paratyphi A atpE while maintaining its native conformation?

Expression and purification of functional recombinant S. paratyphi A atpE requires specialized approaches due to its hydrophobic nature and membrane integration. A recommended methodology involves:

• Vector selection: Use pET expression systems with mild induction conditions, incorporating a cleavable fusion tag (His6 or GST) to facilitate detection and purification.

• Expression optimization: Transform into E. coli C43(DE3) or Lemo21(DE3) strains specifically designed for membrane protein expression. Culture at lower temperatures (18-25°C) with reduced IPTG concentrations (0.1-0.5 mM) to minimize inclusion body formation.

• Membrane fraction isolation: Employ differential centrifugation (40,000-100,000 × g) following cell disruption.

• Detergent screening: Test a panel of detergents including n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), and digitonin at concentrations just above their critical micelle concentration to solubilize the protein while preserving native structure.

• Purification strategy: Implement a two-step chromatography approach using immobilized metal affinity chromatography followed by size exclusion chromatography in detergent-containing buffers.

Validation of proper folding can be assessed through circular dichroism spectroscopy, which should reveal characteristic α-helical content expected for this transmembrane protein.

What experimental models best represent Salmonella paratyphi A atpE function in host-pathogen interactions?

Based on current research, several experimental models effectively represent S. paratyphi A atpE function in host-pathogen interactions:

• Caenorhabditis elegans model: C. elegans provides a valuable platform for studying S. paratyphi A pathogenesis. This model has revealed that infection activates immune responses mediated by tir-1, nsy-1, sek-1, pmk-1, mpk-1, skn-1, daf-2, and daf-16, suggesting regulation of MAPK and insulin pathways . Advantages include genetic tractability, transparent body, and conserved innate immune signaling.

• Cell culture systems: Human intestinal epithelial cell lines (Caco-2, HT-29) can be utilized to investigate ATP synthase involvement in adhesion, invasion, and intracellular survival.

• Reconstituted proteoliposome systems: For biophysical studies, purified atpE can be reconstituted into liposomes with defined lipid compositions to measure proton conductance and ATP synthesis activities.

• Humanized mouse models: These provide a more relevant immunological context for studying pathogen-host interactions while allowing controlled experimental conditions.

The choice of model should align with specific research questions, with C. elegans offering particular advantages for studying conserved defense mechanisms against pathogenic infection due to its simplified immune network .

How does oxidative stress influence the expression and function of Salmonella paratyphi A ATP synthase during infection?

Oxidative stress significantly impacts S. paratyphi A ATP synthase expression and function during infection. Research demonstrates that S. paratyphi A infection increases host oxidative stress markers . This relationship operates bidirectionally:

• Host-derived oxidative stress effects on ATP synthase:

  • Reactive oxygen species (ROS) can damage membrane proteins, including ATP synthase components

  • Oxidative modifications to atpE cysteine residues can alter c-ring assembly and proton conductance

  • Sustained oxidative stress triggers compensatory upregulation of ATP synthase genes to maintain energy homeostasis

• ATP synthase contribution to oxidative stress responses:

  • ATP synthase function affects intracellular redox balance through PMF maintenance

  • Bacterial ATP levels influence expression of oxidative stress response genes

  • ATP-dependent chaperones protect cellular proteins from oxidative damage

Experimental data from C. elegans models showed that S. paratyphi A infection increases hydrogen peroxide levels, accompanied by altered expression of immune response genes . This suggests that monitoring atpE expression and ATP synthase activity during oxidative stress could reveal mechanisms of pathogen adaptation and persistence during infection.

What are the optimal protocols for studying atpE gene expression in Salmonella paratyphi A under different environmental conditions?

Optimal protocols for studying atpE gene expression in S. paratyphi A should incorporate multiple complementary approaches:

Quantitative RT-PCR methodology:

• Culture S. paratyphi A under varying conditions (pH, temperature, nutrient limitation, oxidative stress)

• Extract total RNA using TRIzol reagent followed by DNase I treatment

• Synthesize cDNA using reverse transcriptase with random hexamers

• Perform qPCR using atpE-specific primers normalized to reference genes (16S rRNA, gyrB)

• Calculate expression changes using the 2^-ΔΔCT method

Primer design for atpE amplification:

Additional recommended methods:

• RNA-Seq for transcriptome-wide analysis

• Reporter gene fusions (atpE-lacZ/GFP) for real-time expression monitoring

• Western blotting using anti-atpE antibodies for protein-level verification

• ChIP-seq to identify transcription factors regulating atpE expression

Environmental conditions should simulate infection-relevant scenarios, including acidic pH (5.0-6.5), physiological temperature (37°C), oxygen limitation, and exposure to sublethal concentrations of antimicrobials. This comprehensive approach provides robust data on atpE expression regulation in response to environmental stressors.

What techniques can be used to evaluate the interaction between Salmonella paratyphi A atpE and host immune system components?

Several advanced techniques can effectively evaluate interactions between S. paratyphi A atpE and host immune components:

• Protein-protein interaction assays:

  • Co-immunoprecipitation with anti-atpE antibodies followed by mass spectrometry

  • Yeast two-hybrid screening using atpE as bait against host immune protein libraries

  • Surface plasmon resonance to measure binding kinetics with purified immune proteins

  • Biolayer interferometry for real-time interaction analysis

• Immunological detection methods:

  • ELISA using recombinant atpE to measure antibody responses in patient sera

  • Flow cytometry to assess binding of atpE to immune cells

  • Immunohistochemistry to visualize atpE localization during infection

• Functional immune assays:

  • Cytokine profiling (IL-6, TNF-α, IL-1β) after exposing macrophages to recombinant atpE

  • Neutrophil activation assays measuring respiratory burst following atpE stimulation

  • Complement activation testing in the presence of purified atpE

• Genetic approaches:

  • Generation of S. paratyphi A atpE mutants with altered immunogenic epitopes

  • Host cell transfection with atpE constructs to examine intracellular signaling

Studies in C. elegans have demonstrated that S. paratyphi A infection activates immune pathways including MAPK and insulin signaling . Applying these techniques to mammalian systems would provide translational insights into how atpE contributes to pathogenesis and immune evasion.

How can researchers effectively analyze the structural and functional alterations in atpE mutants?

Comprehensive analysis of structural and functional alterations in atpE mutants requires an integrated approach:

Structural analysis techniques:

• X-ray crystallography: Particularly challenging for membrane proteins like atpE, but provides atomic-level detail when successful

• Cryo-electron microscopy: Increasingly powerful for membrane protein complexes, revealing c-ring assembly in its native lipid environment

• NMR spectroscopy: Suitable for specific domains or the entire protein in detergent micelles

• Molecular dynamics simulations: Provide insights into conformational changes and proton translocation mechanisms

Functional characterization methods:

• ATP synthesis assays: Measure ATP production in inverted membrane vesicles using luciferase-based detection

• Proton transport measurements: Use pH-sensitive fluorescent dyes (ACMA, pyranine) to track proton movement

• Membrane potential analysis: Employ potentiometric dyes like DiSC3(5) to assess changes in membrane potential

• Growth phenotyping: Compare growth rates in different media and stress conditions

Data interpretation framework:

$\text{Normalized Activity} = \frac{(Activity_{mutant} - Activity_{background})}{(Activity_{wildtype} - Activity_{background})} \times 100\%$

Correlation between structural properties and functional outcomes can be established through systematic mutagenesis targeting:

• Proton-binding residues (typically acidic amino acids)

• Residues at subunit interfaces

• Conserved transmembrane helical regions

• Lipid-interacting domains

This multi-faceted approach enables researchers to connect specific structural alterations to functional consequences, providing mechanistic insights into atpE's role in ATP synthase operation.

What statistical approaches are most appropriate for analyzing atpE expression data in different experimental contexts?

Selecting appropriate statistical methods for analyzing atpE expression data depends on experimental design and data characteristics:

For qRT-PCR data analysis:

• Normalization strategies:

  • Multiple reference gene normalization using geNorm or NormFinder algorithms

  • Calculation of stability values (M) for reference genes under experimental conditions

  • Application of the 2^-ΔΔCT method with validated reference genes

• Statistical tests for differential expression:

  • Paired t-test for before/after comparisons in the same samples

  • One-way ANOVA with post-hoc tests (Tukey's HSD) for multiple condition comparisons

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) for non-normally distributed data

For RNA-Seq data analysis:

• Normalization methods:

  • TPM (Transcripts Per Million)

  • RPKM/FPKM (Reads/Fragments Per Kilobase Million)

  • DESeq2 or edgeR normalization approaches

• Differential expression analysis:

  • Negative binomial models (DESeq2, edgeR)

  • Significance cutoffs: adjusted p-value < 0.05 and log2 fold change > 1

For time-series experiments:

• Regression analysis for temporal trends

• Repeated measures ANOVA with appropriate post-hoc tests

• Mixed-effects models to account for both fixed and random effects

Data visualization recommendations:

When analyzing data from C. elegans infection models, researchers should consider potential confounding factors such as developmental stage, ensuring appropriate controls are included for accurate interpretation of atpE expression changes in response to infection .

How can researchers interpret contradictory results between different model systems studying Salmonella paratyphi A atpE function?

When faced with contradictory results between model systems studying S. paratyphi A atpE function, researchers should implement a systematic reconciliation approach:

• Source of variation analysis:

  • Model-specific differences (C. elegans vs. cell culture vs. mouse models)

  • Experimental conditions (temperature, media composition, bacterial growth phase)

  • Strain variations in S. paratyphi A isolates

  • Measurement techniques and their limitations

• Hierarchical evaluation framework:

  • Establish which aspects of the contradiction relate to mechanistic understanding vs. quantitative differences

  • Determine if differences reflect model-specific biology rather than technical artifacts

  • Consider evolutionary conservation of pathways across model organisms

• Integration strategies:

  • Meta-analysis techniques to synthesize findings across studies

  • Bayesian approaches to weight evidence based on methodological robustness

  • Pathway-focused analysis rather than single-gene interpretation

• Validation in multiple systems:

  • Cross-validation of key findings in alternative models

  • Complementary assays measuring the same parameter using different techniques

  • Genetic complementation studies to confirm gene-function relationships

Research with C. elegans has shown that S. paratyphi A infection activates specific immune pathways, including MAPK and insulin signaling . If these findings contradict observations in other models, researchers should examine whether the contradiction reflects genuine biological differences in host response mechanisms or methodological variations. The simplified immune network in C. elegans might highlight fundamental pathways obscured by the complexity of mammalian systems .

What bioinformatic tools provide the most reliable analysis of atpE sequence conservation and functional domains across Salmonella species?

For comprehensive bioinformatic analysis of atpE sequence conservation and functional domains across Salmonella species, researchers should employ the following tools and approaches:

Sequence alignment and conservation analysis:

• Multiple sequence alignment tools:

  • MUSCLE or MAFFT for accurate alignment of atpE sequences

  • T-Coffee for incorporating structural information into alignments

  • Clustal Omega for large-scale alignments across multiple Salmonella serovars

• Conservation visualization and analysis:

  • ConSurf for mapping conservation onto structural models

  • WebLogo for generating sequence logos highlighting conserved motifs

  • MEGA-X for phylogenetic analysis and evolutionary rate calculation

Functional domain prediction:

• Transmembrane topology tools:

  • TMHMM or HMMTOP for predicting transmembrane helices

  • TOPCONS for consensus prediction of membrane protein topology

  • PSIPRED for secondary structure prediction

• Functional site prediction:

  • ProFunc for identifying potential active sites and binding regions

  • 3DLigandSite for ligand binding site prediction

  • COACH for protein-protein interaction site prediction

Structural modeling resources:

• Homology modeling platforms:

  • SWISS-MODEL for automated model generation

  • I-TASSER for integrative threading-based modeling

  • AlphaFold2 for highly accurate structure prediction

Comparative genomics databases:

• Specialized resources:

  • Microscope for comparative analysis of microbial genomes

  • OMA Browser for orthology inference

  • STRING for functional protein association networks

Analysis of S. paratyphi A atpE should focus on conserved functional regions, particularly those involved in proton binding and c-ring assembly. The 79-amino acid sequence contains highly conserved regions across Salmonella serovars, with identical sequences observed between S. paratyphi A and S. paratyphi C for the atpE protein . Researchers should pay particular attention to transmembrane domains and residues involved in ion conductance when performing comparative analyses.

What are the emerging approaches for targeting Salmonella paratyphi A ATP synthase for vaccine development?

Emerging approaches for targeting S. paratyphi A ATP synthase in vaccine development represent promising directions for addressing the lack of effective vaccines against paratyphoid fever :

• Epitope-based vaccine strategies:

  • Identification of immunodominant B-cell and T-cell epitopes within conserved regions of atpE

  • Multi-epitope constructs combining atpE epitopes with other immunogenic Salmonella proteins

  • Computational epitope mapping followed by experimental validation in animal models

• Attenuated live vector systems:

  • Development of S. paratyphi A strains with modified ATP synthase expression for balanced attenuation

  • Creation of safe bacterial vectors expressing engineered atpE constructs

  • Prime-boost strategies combining DNA vaccines with protein boosters

• Structural vaccinology approach:

  • Structure-based design of atpE immunogens with enhanced stability and immunogenicity

  • Rational engineering of conformational epitopes to elicit neutralizing antibodies

  • Nanoparticle display of atpE epitopes in optimal orientation

• Innovative adjuvant combinations:

  • TLR agonists to enhance immune response against atpE antigens

  • Mucosal adjuvants for improved intestinal immunity

  • Cytokine-adjuvant combinations tailored to elicit balanced Th1/Th2/Th17 responses

Recent research using C. elegans as a model organism has provided insights into S. paratyphi A pathogenesis mechanisms, including the activation of specific immune pathways . These findings can inform rational vaccine design by targeting conserved mechanisms of host-pathogen interaction. Future vaccine candidates should capitalize on the conserved nature of ATP synthase components while addressing the challenges of generating protective immunity against this intracellular pathogen.

How might CRISPR-Cas9 technology be applied to study the role of atpE in Salmonella paratyphi A pathogenesis?

CRISPR-Cas9 technology offers powerful approaches for investigating atpE's role in S. paratyphi A pathogenesis:

• Precise genome editing strategies:

  • Generation of clean atpE deletion mutants without polar effects

  • Introduction of point mutations to target specific functional residues

  • Creation of domain swaps between different Salmonella serovars

  • Development of conditional knockdown systems for essential genes

• CRISPR interference (CRISPRi) applications:

  • Titratable repression of atpE expression using catalytically inactive Cas9 (dCas9)

  • Temporal control of atpE expression during different infection stages

  • Multiplexed targeting of ATP synthase genes to assess synthetic interactions

• CRISPR activation (CRISPRa) approaches:

  • Upregulation of atpE to assess consequences of overexpression

  • Modulation of regulatory elements controlling ATP synthase operons

  • Activation of compensatory pathways in atpE-deficient backgrounds

• High-throughput screening platforms:

  • Genome-wide CRISPR screens to identify genetic interactions with atpE

  • Pooled screening in infection models to discover atpE-dependent virulence factors

  • Base editing approaches for systematic assessment of residue function

Experimental design considerations:

• Guide RNA design should consider PAM availability and potential off-target effects

• Validation of edits using sequencing and functional assays

• Complementation studies to confirm phenotype specificity

Given that S. paratyphi A has been shown to regulate host immune pathways including MAPK and insulin signaling , CRISPR-engineered bacterial strains could be used in C. elegans and other models to precisely map which bacterial factors, including atpE variants, are responsible for specific host response patterns.

What interdisciplinary approaches could advance our understanding of the role of ATP synthase in Salmonella paratyphi A infection dynamics?

Advancing our understanding of ATP synthase's role in S. paratyphi A infection requires innovative interdisciplinary approaches that integrate multiple scientific disciplines:

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Flux balance analysis to model energy metabolism during infection

  • Network modeling of ATP synthase interactions with other cellular components

  • Agent-based modeling of host-pathogen dynamics at tissue level

• Biophysics-immunology interface:

  • Single-molecule techniques to study ATP synthase function during infection

  • Super-resolution microscopy to visualize ATP synthase distribution in bacterial cells

  • Microfluidics platforms to study bacterial energetics under changing conditions

  • Real-time monitoring of bacterial ATP production in infection models

• Synthetic biology approaches:

  • Engineering of ATP synthase variants with altered properties

  • Creation of biosensors for monitoring ATP levels during infection

  • Development of synthetic genetic circuits responsive to energy state

  • Design of minimal ATP synthase systems for fundamental studies

• Translational research connections:

  • High-throughput screening for ATP synthase inhibitors with therapeutic potential

  • Development of diagnostic tools based on ATP synthase detection

  • Collaborative clinical studies linking ATP synthase variation to disease outcomes

Research in C. elegans has demonstrated that S. paratyphi A infection leads to increased oxidative stress and activation of specific immune pathways . Interdisciplinary approaches could build on these findings by integrating real-time bioenergetic measurements with immunological readouts in more complex models, creating a comprehensive picture of how ATP synthase function influences infection dynamics and host response patterns.