Recombinant Francisella tularensis subsp. tularensis ATP synthase subunit alpha (atpA), partial

Basic Research Questions

• What is Francisella tularensis and why is atpA of research interest?

Francisella tularensis is a zoonotic intracellular pathogen that causes tularemia, a potentially debilitating febrile illness. It has a low infectious dose (fewer than 50 bacteria can cause disease), multiple transmission routes, and potential to cause life-threatening infections, leading to its classification as a Category A Select Agent with bioterrorism potential .

The ATP synthase subunit alpha (atpA) is a critical component of the F1F0-ATP synthase complex responsible for cellular energy production. Research interest in atpA stems from:

• Its essential role in bacterial energy metabolism and potential as a drug target

• Possible contribution to the bacterium's adaptation to intracellular life

• Potential involvement in pathogenesis and stress response

• Structural features that might be unique to F. tularensis

Understanding atpA function could provide insights into F. tularensis bioenergetics during infection and reveal therapeutic vulnerabilities.

• What genetic tools are available for manipulating and expressing atpA in F. tularensis?

Several genetic tools have been developed for F. tularensis that can be applied to atpA studies:

• Shuttle plasmid pFNLTP1: This hybrid plasmid transforms F. tularensis LVS, F. novicida U112, and E. coli at frequencies exceeding 1 × 10^7 CFU/μg of DNA. It maintains stable replication both in laboratory media and during macrophage infection without antibiotic selection .

• Allelic exchange systems: These allow creation of gene knockouts, tagged variants, or merodiploid strains. For essential genes like atpA, conditional expression systems or merodiploid approaches (where a second copy is introduced before modifying the native gene) are recommended .

• Temperature-sensitive vectors: Derivatives of pFNLTP1 containing temperature-sensitive mutations that prevent replication at nonpermissive temperatures, enabling conditional gene expression .

Methodological considerations:

• Include appropriate regulatory elements (like the rpsL promoter) to ensure proper expression levels

• Consider gene dosage effects, as high-level expression may not be well-tolerated

• For purification purposes, incorporate epitope tags such as a 5×His tag as demonstrated with other F. tularensis proteins

• What are the optimal conditions for purifying functional recombinant F. tularensis atpA?

Purification of functional recombinant F. tularensis atpA requires careful optimization:

Cell Lysis and Extraction:

• Buffer composition is critical - consider including:

  • Stabilizing agents: 5-10% glycerol, low concentrations of ATP

  • Protease inhibitors to prevent degradation

  • Reducing agents (DTT or β-mercaptoethanol) if the protein contains cysteines

• Gentle lysis methods to preserve protein structure:

  • Enzymatic lysis with lysozyme for initial cell wall disruption

  • Sonication or French press with optimized parameters

Purification Strategy:

Quality Control:

• SDS-PAGE for purity assessment

• Western blotting for identity confirmation

• Activity assays to verify function (ATP hydrolysis)

• Circular dichroism to confirm proper folding

Since atpA is part of a multi-subunit complex, consider co-expression strategies with other ATP synthase components to improve stability and functionality.

• How can atpA function be reliably measured in experimental settings?

Reliable measurement of atpA function requires multiple complementary approaches:

ATP Hydrolysis Assays:

• Enzyme-coupled spectrophotometric assays:

  • Link ADP production to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitor absorbance decrease at 340 nm in real-time

• Colorimetric phosphate detection:

  • Malachite green assay measures released inorganic phosphate

  • Provides endpoint measurements with high sensitivity

Binding Studies:

• Isothermal Titration Calorimetry (ITC):

  • Directly measures binding thermodynamics of ATP/ADP

  • Provides KD, ΔH, ΔS, and stoichiometry information

• Fluorescence-based approaches:

  • Intrinsic tryptophan fluorescence changes upon nucleotide binding

  • TNP-ATP displacement assays for binding site characterization

Structure-Function Analysis:

• Site-directed mutagenesis of catalytic residues with activity correlation

• Comparative analysis with well-characterized ATP synthase alpha subunits

• Complementation studies in F. tularensis or heterologous systems

Critical Experimental Controls:

• Heat-inactivated protein as negative control

• Commercial F1-ATPase preparations as positive control

• Testing multiple buffer conditions to determine optimal activity parameters

• What biosafety considerations are important when working with recombinant F. tularensis atpA?

Working with components from F. tularensis requires careful attention to biosafety:

Biosafety Level Requirements:

• Recombinant atpA work typically requires BSL-2 facilities when:

  • Using attenuated strains like LVS

  • Working with the isolated gene or protein in non-pathogenic expression hosts

• BSL-3 facilities are required when:

  • Using virulent F. tularensis subsp. tularensis (type A) strains

  • Working with live bacteria for infection studies

Risk Mitigation Strategies:

• Expression system selection:

  • Use of safer heterologous hosts like E. coli for protein production

  • Avoid creating potentially hazardous recombinants with enhanced virulence

• Proper decontamination procedures:

  • Effective chemical disinfectants (hypochlorite, phenolics)

  • Validated autoclave protocols for solid waste

Regulatory Compliance:

• Select Agent regulations apply to virulent F. tularensis strains

• Institutional Biosafety Committee approval requirements

• Proper documentation and inventory control

Training and Emergency Response:

• Specialized training for personnel

• Exposure response protocols

• Medical surveillance recommendations

Always consult current CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) guidelines for specific recommendations regarding work with F. tularensis and derived materials.

Advanced Research Questions

• How might atpA function contribute to F. tularensis intracellular survival and pathogenesis?

The ATP synthase alpha subunit likely plays critical roles in F. tularensis pathogenesis:

Energy Provision for Intracellular Lifecycle:
F. tularensis undergoes a complex intracellular lifecycle involving phagosomal escape and cytosolic replication . Each stage presents unique bioenergetic challenges:

• Phagosomal acidification resistance may require ATP synthase adaptation

• Cytosolic replication demands high energy production

• Metabolic flexibility during different infection phases likely involves ATP synthase regulation

Potential Coordination with Virulence Factors:

• ATP-dependent secretion systems require energy from ATP synthase

• Acid phosphatases that suppress oxidative burst may function in concert with ATP synthase

• Fatty acid biosynthesis, essential for F. tularensis virulence, requires ATP

Experimental Approaches to Assess Contribution:

• Conditional expression systems to modulate atpA levels during infection

• ATP synthase inhibitor studies during different infection stages

• Metabolic profiling under various infection conditions

• Correlating atpA expression with virulence factor production

Research Considerations:

• Different F. tularensis strains may show variation in atpA expression and regulation

• Host cell type affects intracellular pH and nutrient availability, potentially impacting ATP synthase function

• Opsonization conditions influence uptake mechanisms and subsequent intracellular trafficking

• How can genetic manipulation systems be optimized for functional studies of atpA in F. tularensis?

Advanced genetic manipulation of atpA in F. tularensis requires sophisticated approaches:

Conditional Expression Systems:

• Since atpA is likely essential, true knockouts may not be viable

• Approaches for regulated expression include:

  • Tetracycline-responsive promoters adapted for Francisella

  • Riboswitch-based systems for small molecule control

  • Degron-tagged constructs for protein-level regulation

Site-Specific Mutagenesis Strategies:

• Targeting catalytic residues while maintaining structural integrity

• Creating chromosomal point mutations using scarless techniques

• Allelic replacement using counterselectable markers like sacB

Validation of Genetic Manipulations:

Advanced Applications:

• CRISPR-Cas9 adaptation for F. tularensis

• Fluorescent protein fusions for localization studies

• Split-protein complementation for interaction mapping

• Dual-plasmid systems for complex genetic manipulations

When implementing these systems, consider:

• The restricted host range of certain plasmids

• The challenges of multi-copy vs. single-copy expression

• Potential polar effects on downstream genes

• The need for comprehensive controls to validate phenotypes

• What are the challenges in resolving contradictory data about F. tularensis ATP synthase function under different experimental conditions?

Resolving contradictory findings about F. tularensis ATP synthase function requires systematic approaches:

Sources of Experimental Variation:

• Strain differences: Significant genetic variation exists between F. tularensis subspecies and laboratory strains

• Growth conditions: Media composition and growth phase dramatically affect metabolism

• Host cell models: Different macrophage types show variable intracellular environments

• Opsonization conditions: Serum vs. non-opsonized bacteria follow different uptake pathways

Methodological Considerations:

• Standardization of experimental protocols:

  • Defined media compositions

  • Consistent growth phases for experiments

  • Standardized infection models and MOI

• Comprehensive controls:

  • Multiple F. tularensis strains tested in parallel

  • Careful selection of reference genes for expression studies

  • Both in vitro and ex vivo validation

Statistical and Analytical Approaches:

• Meta-analysis of published data

• Multivariate analysis to identify key variables affecting outcomes

• Bayesian modeling to incorporate prior knowledge

Examples from Related Research:
The literature shows contradictory findings about F. tularensis phagosomal acidification, with some studies showing acidification while others show resistance . Similar contradictions may arise with ATP synthase function and can be addressed by:

• Carefully controlling experimental conditions

• Directly comparing methods side-by-side

• Combining multiple complementary techniques

• Considering temporal dynamics and microenvironmental variations

• How can structural biology approaches enhance our understanding of F. tularensis atpA?

Advanced structural biology techniques offer powerful insights into atpA function:

Comparative Structural Analysis:

• Homology modeling based on solved ATP synthase structures

• Identification of F. tularensis-specific structural features

• Mapping of potential regulatory sites and interaction surfaces

Experimental Structure Determination:

• X-ray crystallography of the isolated alpha subunit

• Cryo-EM of the entire ATP synthase complex

• NMR studies of specific domains or interaction interfaces

Dynamics and Conformational Changes:

• Molecular dynamics simulations to model nucleotide binding effects

• Hydrogen-deuterium exchange mass spectrometry to map conformational changes

• FRET-based approaches to monitor intramolecular movements

Structure-Function Correlations:

Therapeutic Applications:

• Structure-based drug design targeting F. tularensis-specific features

• Identification of allosteric sites for selective inhibition

• Epitope mapping for diagnostic antibody development

Combining structural insights with functional studies will provide a comprehensive understanding of how F. tularensis ATP synthase has adapted to the intracellular lifestyle.

• How does atpA expression change during different stages of F. tularensis infection, and what are the implications?

Understanding the dynamic expression of atpA during infection provides critical insights:

Temporal Expression Analysis:

• Whole-genome transcriptional profiling has revealed that F. tularensis metabolic genes are actively transcribed during infection

• For atpA, expression likely changes throughout the infection cycle:

  • Initial uptake phase: Baseline expression

  • Phagosomal escape: Potential upregulation to counter stress

  • Cytosolic replication: High expression to support rapid division

  • Persistent infection: Regulated expression for long-term survival

Methodological Approaches:

• RNA-seq from infected tissues at multiple timepoints

• Promoter-reporter fusions to visualize expression dynamics

• Proteomics to correlate transcriptional and translational regulation

• Single-cell approaches to capture population heterogeneity

Regulatory Networks:

• Nutrient-responsive regulation

• Stress-responsive expression changes

• Coordination with virulence factor expression

• Host immune response effects

Therapeutic Implications:

• Identification of critical infection stages for ATP synthase targeting

• Development of stage-specific intervention strategies

• Understanding bacterial adaptation to host environments

Research Considerations:

• Different infection models may show variable expression patterns

• Host-specific adaptations may occur in different animal species

• In vitro culture conditions poorly mimic in vivo expression dynamics

Understanding these dynamic changes could reveal optimal timing for therapeutic interventions targeting the ATP synthase complex in F. tularensis.