Recombinant Salmonella enteritidis PT4 ATP synthase subunit beta (atpD)

What is the structure and function of ATP synthase subunit beta (atpD) in Salmonella enteritidis?

ATP synthase subunit beta (atpD) is a crucial component of the F1 portion of F0F1-ATP synthase, the primary enzyme responsible for ATP synthesis in Salmonella enteritidis. This protein contains the catalytic sites necessary for ATP synthesis and hydrolysis. The F0F1-ATP synthase complex consists of two major functional domains: the membrane-embedded F0 portion and the soluble F1 portion that contains the atpD subunit.

In Salmonella, ATP synthase levels have been observed to correlate inversely with osmotic stress, with F0F1-ATP synthase subunits β (AtpD) and γ (AtpG) showing reduced presence in filamented cells compared to control cells . This suggests that atpD plays a role in the bacterial response to environmental stressors, which is particularly relevant during host infection.

How does atpD expression change under different environmental conditions?

Under osmotic stress conditions, Salmonella enteritidis demonstrates reduced levels of ATP synthase subunits, including atpD. Research has shown that when S. enterica strains are cultured under high osmotic conditions (such as media supplemented with 7% NaCl), the levels of F0F1-ATP synthase subunits β (AtpD) decrease compared to cells grown in standard conditions . This adaptation appears to be part of the bacterial response to environmental stress, suggesting that energy metabolism regulation via atpD may be an important survival mechanism during infection or environmental persistence.

What is the relationship between atpD and bacterial filamentation during stress conditions?

Experimental data indicates an inverse relationship between ATP synthase levels and osmotic stress-induced filamentation in Salmonella. When S. enterica strains form filaments under osmotic stress (TSA-7NaCl), the levels of ATP synthase subunit β (AtpD) decrease compared to control cells . This suggests that atpD downregulation may be associated with the bacterial stress response that leads to filamentation. The morphological changes in Salmonella during stress conditions appear to coincide with metabolic adjustments, including altered ATP synthesis capacity through reduced expression of key components like atpD.

What are the most effective methods for expressing and purifying recombinant Salmonella enteritidis PT4 ATP synthase subunit beta (atpD)?

For expressing recombinant Salmonella enteritidis PT4 atpD, E. coli expression systems are typically employed using vectors that incorporate N-terminal or C-terminal His-tags for purification. Based on protocols used for similar ATP synthase subunits, the following methodology is recommended:

• Clone the full-length atpD gene into an expression vector with an appropriate tag (His-tag is commonly used)

• Transform into an E. coli expression strain (BL21(DE3) or similar)

• Induce expression using IPTG at optimal conditions (typically 0.5-1mM IPTG at 16-30°C for 4-16 hours)

• Lyse cells using appropriate buffer systems containing protease inhibitors

• Purify using immobilized metal affinity chromatography (IMAC)

• Perform size exclusion chromatography for higher purity

• Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0 to maintain stability

For long-term storage, lyophilization or storage at -20°C/-80°C with glycerol (20-50%) is recommended to prevent protein degradation .

What experimental approaches can be used to study the activity of atpD in the context of bacterial stress responses?

To investigate atpD activity during bacterial stress responses, researchers can employ several complementary approaches:

• Comparative proteomics: Using techniques like 2D-gel electrophoresis and mass spectrometry to compare atpD levels in stressed versus non-stressed conditions, similar to the approach that revealed decreased levels of ATP synthase subunits β (AtpD) in filamented cells versus control cells .

• ATP synthesis activity assays: Measuring ATP production capacity in membrane vesicles isolated from bacteria grown under various stress conditions.

• Gene expression analysis: RT-qPCR or RNA-seq to quantify atpD transcript levels under different conditions.

• Mutant analysis: Creating atpD knockdown or conditional mutants to assess the impact on bacterial survival during stress.

• Protein-protein interaction studies: Co-immunoprecipitation or bacterial two-hybrid systems to identify stress-specific interaction partners of atpD.

• Microscopy techniques: Immunofluorescence microscopy using anti-atpD antibodies to visualize localization changes during stress responses.

How can researchers accurately quantify changes in atpD levels during osmotic stress experiments?

For accurate quantification of atpD levels during osmotic stress experiments, researchers should consider multiple complementary approaches:

• Western blotting: Using specific antibodies against atpD, researchers can detect and quantify protein levels in membrane fractions isolated from bacteria grown under different osmotic conditions. For meaningful comparisons, normalization to total protein or to a stable reference protein is essential.

• Mass spectrometry-based proteomics: Label-free quantification or isotope-labeled approaches (SILAC, iTRAQ) can provide precise measurement of atpD abundance changes. This approach has previously revealed that F0F1-ATP synthase subunits β (AtpD) levels decrease in filamented cells compared to control cells .

• Activity-based protein profiling: For functional analysis, ATP binding or hydrolysis activity can be measured using membrane preparations from cells exposed to different osmotic conditions.

The experimental data should be analyzed using appropriate statistical methods, with significance typically determined at P < 0.05, as applied in studies examining protein level changes in Salmonella under osmotic stress .

How does atpD interact with other bacterial proteins during stress response and pathogenesis?

The interaction network of atpD during stress responses and pathogenesis likely involves both direct protein-protein interactions within the ATP synthase complex and indirect functional interactions with stress response pathways. Current understanding suggests:

• Interactions within the ATP synthase complex: atpD (subunit β) functionally interacts with other subunits, particularly atpA (subunit α) and atpG (subunit γ), to form the catalytic core of F1-ATP synthase. Under stress conditions, coordinated regulation of these subunits has been observed, with both atpD and atpG showing reduced levels in filamented cells experiencing osmotic stress .

• Potential interactions with stress response regulators: While direct interaction data is limited, the downregulation of atpD during stress suggests potential regulatory connections with stress response systems, possibly involving alternative sigma factors or two-component regulatory systems that sense environmental changes.

• Metabolic pathway integration: As a key enzyme in energy metabolism, ATP synthase activity influences multiple downstream metabolic pathways. The decreased levels of atpD observed during osmotic stress may represent a metabolic adaptation strategy that redirects energy utilization during stress conditions.

Advanced techniques such as bacterial two-hybrid screening, cross-linking mass spectrometry, or proximity labeling approaches would be valuable for comprehensively mapping the atpD interaction network under different conditions.

What are the implications of atpD regulation for bacterial adaptation to different host environments?

ATP synthase subunit beta (atpD) regulation appears to be an important component of Salmonella's adaptive response to changing environments during infection. The implications include:

• Metabolic flexibility: The observed inverse correlation between ATP synthase levels and osmotic stress suggests that Salmonella modulates its energy production machinery to adapt to different host microenvironments. This regulation may help the bacterium balance ATP production with other metabolic needs during adaptation to specific host niches.

• Morphological adaptations: Research demonstrates that ATP synthase subunits including atpD show decreased levels in filamented cells . This connection between energy metabolism and cell morphology may represent an integrated stress response that helps Salmonella survive hostile host conditions.

• Potential impact on antibiotic tolerance: Changes in energy metabolism and growth state, including filamentation, have been associated with altered antibiotic susceptibility in various bacteria. The regulation of atpD during stress responses may therefore have implications for antibiotic tolerance during infection.

Understanding how atpD regulation contributes to bacterial adaptation could potentially reveal new therapeutic targets that disrupt Salmonella's ability to adjust to host environments.

What controls should be included when studying the effects of environmental stress on atpD expression?

When investigating atpD expression under environmental stress conditions, researchers should implement the following controls:

• Growth phase-matched controls: Since protein expression can vary with growth phase, control and experimental cultures should be carefully matched for growth phase. Studies examining ATP synthase levels typically compare cells at similar optical densities (OD600) .

• Multiple reference genes/proteins: For expression studies, multiple reference genes that remain stable under the tested conditions should be used for normalization. Similarly, for protein quantification, several reference proteins should be monitored.

• Concentration gradients: Rather than testing a single stress condition, include a gradient of stress levels (e.g., different NaCl concentrations from 0% to 10%) to establish dose-response relationships, as demonstrated in studies of osmotic stress effects .

• Time course sampling: Environmental adaptation often involves temporal changes in gene expression. Include samples taken at multiple time points after stress induction.

• Genetic complementation controls: When using mutant strains, include complemented strains expressing the wild-type gene to confirm that observed phenotypes are specifically due to the targeted mutation.

These controls help distinguish specific effects on atpD from general stress responses and ensure the reproducibility and reliability of findings.

How can researchers differentiate between direct and indirect effects on atpD during stress responses?

Distinguishing direct from indirect effects on atpD during stress responses requires multiple complementary approaches:

• Promoter activity assays: Using reporter gene fusions (e.g., atpD promoter-GFP) to monitor transcriptional regulation in real-time during stress exposure can help identify direct transcriptional effects.

• Chromatin immunoprecipitation (ChIP): This technique can identify transcription factors that directly bind to the atpD promoter region under stress conditions.

• Post-translational modification analysis: Mass spectrometry-based approaches can reveal stress-induced modifications of atpD protein that may affect its stability or function independently of transcriptional changes.

• Targeted mutagenesis: Introducing mutations in potential regulatory elements of the atpD gene can help determine which regulatory mechanisms are essential for stress-responsive expression.

• Time-resolved experiments: Comparing the kinetics of atpD changes with other cellular responses can help establish cause-effect relationships. Primary responses typically occur earlier than secondary effects.

• Systems biology approaches: Network analysis integrating transcriptomic, proteomic, and metabolomic data can help position atpD changes within the broader stress response network.

By integrating these approaches, researchers can build a more complete understanding of how atpD regulation fits into the complex cellular response to environmental stressors.

What are the key methodological challenges in studying atpD function in the context of Salmonella infection models?

Studying atpD function during Salmonella infection presents several methodological challenges:

• Essential gene considerations: As a component of ATP synthase, atpD is likely essential for bacterial viability, making conventional knockout approaches problematic. Researchers should consider conditional expression systems, partial knockdowns, or point mutations that affect function without completely eliminating it.

• Isolation from infection settings: Extracting sufficient bacterial material from infected host cells or tissues for protein analysis is challenging. Techniques may include selective lysis of host cells, fluorescence-activated cell sorting (FACS) of bacteria expressing fluorescent proteins, or immunomagnetic separation.

• Distinguishing bacterial from host proteins: Host cells contain homologous ATP synthase components that may cross-react with antibodies or complicate mass spectrometry analysis. Using species-specific antibodies or peptide identification is essential.

• Temporal dynamics: atpD expression may change throughout the infection process. Time-course experiments with multiple sampling points are necessary but technically challenging in infection models.

• In vivo relevance of in vitro findings: Observations about atpD regulation under laboratory stress conditions (like high NaCl) need validation in relevant infection models to establish physiological significance.

• Functional redundancy: Energy metabolism often involves redundant pathways. Researchers should consider broader metabolic analysis to understand compensatory mechanisms when atpD function is altered.

Addressing these challenges requires integrating molecular techniques with appropriate infection models and sophisticated analytical approaches.

How should researchers interpret conflicting data regarding atpD expression levels in different experimental systems?

When faced with conflicting data about atpD expression across different experimental systems, researchers should consider several factors:

• Strain-specific variations: Different Salmonella enteritidis strains may show varying atpD expression patterns. For example, strain LT2 demonstrates different filamentation responses to osmotic stress compared to strains 4539H, E40, and M-09 , which could influence associated protein expression patterns.

• Growth phase effects: ATP synthase expression can vary with growth phase. The table below illustrates how protein measurements should be normalized and compared across conditions:

Data adapted from research on protein and DNA content during osmotic stress

• Methodological differences: Different protein quantification techniques (western blot, mass spectrometry, activity assays) may yield varying results. Cross-validation using multiple methods is essential.

• Environmental context: atpD expression may respond differently to various stressors. The inverse correlation with osmotic stress may not apply to other stress conditions.

• Statistical analysis: Ensure appropriate statistical tests are applied, with significance typically determined at P < 0.05 as used in comparable studies .

To resolve conflicts, researchers should explicitly state experimental conditions, use multiple analytical approaches, and consider performing meta-analyses when sufficient data is available across studies.

What statistical approaches are most appropriate for analyzing changes in atpD levels across different experimental conditions?

For robust statistical analysis of atpD level changes across experimental conditions, researchers should consider:

• Parametric vs. non-parametric tests: If data follows normal distribution, parametric tests like t-tests (for two conditions) or ANOVA (for multiple conditions) are appropriate. Studies examining protein levels in Salmonella under osmotic stress have typically used two-tailed t-tests to determine significance . If normality cannot be assumed, non-parametric alternatives like Mann-Whitney or Kruskal-Wallis tests should be used.

• Multiple testing correction: When comparing atpD levels across numerous conditions, correction for multiple testing (e.g., Bonferroni, Benjamini-Hochberg) is essential to control false discovery rates.

• Paired analyses: For experiments comparing the same bacterial strain before and after stress exposure, paired statistical tests can increase statistical power.

• Regression models for dose-response: When examining atpD response across different stress intensities (e.g., multiple NaCl concentrations), regression models can characterize the relationship between stress level and protein abundance.

• Mixed-effects models: For complex experimental designs with multiple factors (strain, stress type, duration), mixed-effects models can account for interactions while handling repeated measurements.

• Power analysis: A priori power calculations should guide experimental design to ensure sufficient sample size for detecting biologically relevant changes in atpD levels.

Researchers should clearly report not only p-values but also effect sizes and confidence intervals to provide a complete picture of atpD regulation across conditions.

How can researchers integrate atpD expression data with broader bacterial physiological responses?

Integrating atpD expression data with broader physiological responses requires a systems biology approach:

By integrating these approaches, researchers can move beyond isolated observations of atpD expression changes to understand their functional significance in the context of bacterial physiology and pathogenesis.

What are promising research avenues for understanding the role of atpD in Salmonella pathogenesis?

Several promising research directions could advance our understanding of atpD's role in Salmonella pathogenesis:

• Conditional expression systems: Developing strains with inducible atpD expression would allow researchers to modulate ATP synthase activity during different stages of infection, helping elucidate stage-specific requirements.

• Structure-function analysis: Creating point mutations in catalytic or regulatory domains of atpD could help identify which specific functions are critical for pathogenesis.

• Host compartment-specific regulation: Investigating how atpD expression changes in different host microenvironments (intestinal lumen, epithelial cells, macrophages) would provide insights into metabolic adaptation during infection progression.

• Intersection with virulence regulators: Exploring how virulence regulators interact with atpD expression could reveal integration between metabolic and virulence networks. Research has already demonstrated complex regulatory systems in Salmonella, such as DNA adenine methylase (Dam) modulation of inflammasome activation .

• Role in antibiotic tolerance: Investigating whether stress-induced changes in atpD expression contribute to antibiotic tolerance could have therapeutic implications.

• Vaccine development: Exploring atpD as a potential target for attenuated live vaccines by creating strains with altered ATP synthase function that maintain immunogenicity while reducing pathogenicity.

These directions would build upon observations linking ATP synthase regulation to stress responses and expand our understanding of how energy metabolism contributes to Salmonella's success as a pathogen.

What new technologies or methodological approaches could advance the study of atpD function?

Emerging technologies with significant potential for advancing atpD research include:

• CRISPR interference (CRISPRi): This technology allows titratable repression of gene expression without complete knockout, making it ideal for studying essential genes like atpD. Researchers could create strains with varying levels of atpD expression to determine threshold requirements under different conditions.

• Single-cell proteomics: New mass spectrometry approaches with increased sensitivity may enable protein quantification at the single-cell level, allowing researchers to characterize atpD expression heterogeneity within bacterial populations during infection.

• Protein-specific degradation systems: Systems like the auxin-inducible degron could enable rapid, conditional depletion of atpD protein in vivo, allowing temporal control over ATP synthase function during infection.

• Genetically encoded ATP sensors: Deploying fluorescent ATP biosensors in Salmonella could allow real-time monitoring of how atpD regulation affects intracellular ATP levels during host interaction.

• Cryo-electron tomography: This technique could visualize structural changes in the ATP synthase complex under different conditions, providing insights into how stress affects not just atpD levels but also the organization of the entire complex.

• Microfluidic infection models: Combining microfluidic devices with live-cell imaging would enable dynamic tracking of bacterial responses, including potential reporters of atpD expression, during host cell infection with precise control over microenvironmental conditions.

These technological advances would complement existing approaches that have revealed connections between ATP synthase regulation and stress responses in Salmonella .

How might understanding atpD regulation contribute to new antimicrobial strategies against Salmonella infections?

Understanding atpD regulation could inform novel antimicrobial strategies through several approaches:

• ATP synthase inhibitors: While ATP synthase inhibitors exist, deeper knowledge of atpD regulation during infection could reveal condition-specific vulnerabilities that allow more targeted inhibition with reduced side effects. The observed changes in ATP synthase levels during stress suggest that bacteria may be particularly susceptible to such inhibitors under specific conditions.

• Stress sensitization: If decreased atpD expression during stress represents an adaptive response, preventing this downregulation might sensitize bacteria to host-induced stresses. Compounds that interfere with regulatory mechanisms controlling atpD expression could potentially synergize with host defense mechanisms.

• Metabolic vulnerability exploitation: Understanding how atpD regulation integrates with broader metabolic networks could reveal collateral vulnerabilities that emerge when bacteria adjust their energy metabolism during infection. These vulnerabilities could be targeted by combination therapies.

• Anti-virulence approach: If atpD regulation is linked to virulence factor expression or function, modulating ATP synthase activity could potentially attenuate virulence without directly killing bacteria, potentially reducing selective pressure for resistance development.

• Host-directed therapies: Knowledge of how host conditions influence bacterial atpD expression could guide development of host-directed therapies that create an unfavorable environment for bacterial ATP synthase function.

• Diagnostic applications: Patterns of atpD expression could potentially serve as biomarkers for specific infection stages or antibiotic responses, informing more precise diagnostic approaches.

By targeting essential metabolic processes like those involving atpD, new therapeutic strategies may overcome existing antibiotic resistance mechanisms in Salmonella.