Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni ATP synthase subunit alpha (atpA), partial

What are the unique structural features of ATP synthase in pathogenic Leptospira compared to other bacteria?

The ATP synthase in Leptospira interrogans has significant structural similarity to sodium ion-specific ATP synthases rather than the more common proton-specific ATP synthases found in many bacteria. Specifically, L. interrogans ATP synthase shows the highest similarity to the sodium ion-specific ATP synthase of Propionigenium modestum, suggesting a sodium-dependent energy production mechanism . This distinction is important for understanding the bioenergetics of Leptospira and its adaptation to various environmental conditions.

Experimental evidence for this structural uniqueness comes from comparative genomic analyses and proteomic studies. The sodium specificity is believed to be determined by specific amino acid residues in the membrane-spanning segments of the ATP synthase complex, particularly in the a-subunit and c-ring.

How does the function of atpA in Leptospira interrogans correlate with the organism's adaptability to different host environments?

ATP synthase plays a crucial role in the energy metabolism of Leptospira, allowing the pathogen to adapt to various environmental conditions. Research indicates that ATP production mechanisms may shift during transition from environmental to host conditions. Under host conditions (37°C, increased osmolarity, and serum presence), expression profiles of energy production proteins, including ATP synthase components, show notable changes .

Experimental data from iTRAQ and LC-ESI-tandem mass spectrometry analyses revealed that proteins involved in energy production are differentially regulated when L. interrogans transitions from in vitro to in vivo-like conditions (-Fe/FBS media) . This adaptability in energy metabolism may contribute to the pathogen's survival during infection and colonization of different host tissues.

What are the optimal expression systems for producing functional recombinant atpA from L. interrogans for structural studies?

For optimal expression of recombinant L. interrogans atpA, E. coli-based expression systems have been successfully employed with several important considerations:

• Vector selection: pRSET plasmids have been effective for expressing leptospiral proteins as His₆-tagged fusion proteins, facilitating purification by affinity chromatography .

• E. coli strain optimization: BL21(DE3) strains typically yield good expression levels for leptospiral proteins. For atpA, which is a large, complex subunit, co-expression with chaperones (such as GroEL/GroES) may improve correct folding.

• Induction conditions: Expression at lower temperatures (16-20°C) after induction with 0.1-0.5 mM IPTG for 16-20 hours often improves the solubility of large leptospiral proteins.

• Purification strategy: A two-step purification approach is recommended:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Size exclusion chromatography to obtain highly pure protein

These methodologies have been validated for several leptospiral proteins and can be adapted specifically for atpA expression .

What challenges are associated with maintaining the native conformational state of recombinant atpA, and how can they be addressed?

Maintaining the native conformation of recombinant atpA presents several challenges:

• Protein aggregation: atpA tends to form inclusion bodies when overexpressed in E. coli.

  • Solution: Use of solubility tags (MBP, SUMO) or co-expression with chaperones.

• Subunit interaction dependencies: In its native state, atpA functions as part of a multisubunit complex.

  • Solution: Co-expression with other ATP synthase subunits, particularly atpB and atpG, to facilitate proper folding.

• Post-translational modifications: Potential modifications in the native protein may be absent in recombinant systems.

  • Solution: Analysis of the native protein by mass spectrometry to identify modifications, followed by synthetic addition of these modifications if necessary.

• Stability issues: The isolated subunit may have reduced stability compared to the complete complex.

  • Solution: Optimization of buffer conditions (pH 7.2-7.5, 150-300 mM NaCl, 5-10% glycerol, 1-5 mM MgCl₂) to enhance stability.

Research groups have reported improved results by performing protein purification at 4°C and including ATP (1-2 mM) in buffers to stabilize the conformation of the nucleotide-binding domain of atpA .

How does the genetic sequence of atpA differ across Leptospira serovars, and what are the implications for cross-protection in vaccine development?

Comparative genomic analysis shows that atpA is highly conserved across pathogenic Leptospira species, with sequence identity typically exceeding 95%. This conservation makes it a potential target for cross-protective vaccine development.

Specific findings include:

What proteomic approaches have been most effective for identifying post-translational modifications in native atpA, and how do these modifications affect protein function?

Advanced proteomic approaches have revealed several post-translational modifications (PTMs) in native atpA that may affect its function:

• Mass Spectrometry-Based Approaches:

  • LC-MS/MS with electron transfer dissociation (ETD) has been effective in identifying phosphorylation sites

  • MALDI-TOF-TOF MS for identifying multiple PTMs simultaneously

• Identified Modifications in atpA:

  • Phosphorylation at Ser/Thr residues, particularly during environmental stress

  • Acetylation at specific lysine residues, potentially regulating enzymatic activity

  • Oxidative modifications under host-induced oxidative stress conditions

• Functional Implications:

  • Phosphorylation appears to regulate ATP synthase activity under varying environmental conditions

  • Acetylation may play a role in adapting to different energy states during infection

  • Oxidative modifications may represent host-induced stress effects

Global proteomic analyses comparing in vitro and in vivo-like conditions have shown differential regulation of ATP synthase components, suggesting that PTMs may play a role in adapting energy metabolism during infection .

How does the expression of atpA change during host infection, and what experimental models best capture these changes?

Expression of atpA and other ATP synthase components undergoes significant regulation during host infection. The following experimental models have provided insights into these changes:

• In vitro models mimicking host conditions:

  • Cultures in iron-limited media supplemented with serum (-Fe/FBS) show differential regulation of energy production proteins including ATP synthase components

  • Temperature shifts (from 28°C to 37°C) combined with increased osmolarity induce changes in ATP synthase expression

• Animal infection models:

  • Hamster models have revealed that during acute infection, L. interrogans modulates expression of metabolic proteins including ATP synthase

  • Guinea pig models show similar regulation patterns and are valuable for studying virulent strains

• Cell culture infection systems:

  • Macrophage interaction models demonstrate that L. interrogans alters expression of many genes involved in energy production and metabolism when exposed to host immune cells

Key findings include:

• atpA expression is typically downregulated during early infection stages, possibly as an energy conservation mechanism

• During persistent infection in reservoir hosts, expression returns to higher levels

• Expression patterns differ between reservoir hosts (where infection is chronic) and accidental hosts (where infection is acute and severe)

Is there evidence that atpA or other ATP synthase components interact with host immune systems, and could these interactions contribute to pathogenesis?

Recent research has uncovered evidence that ATP synthase components may interact with host immune systems:

• Interaction with Pattern Recognition Receptors (PRRs):

  • Some ATP synthase components, when exposed on the bacterial surface during stress conditions, can be recognized by TLR4 and TLR2

  • This recognition may contribute to inflammatory responses during leptospirosis

• Antibody Recognition:

  • Serum from leptospirosis patients shows reactivity to certain ATP synthase components, suggesting exposure of these typically internal proteins during infection

  • Reactivity patterns differ between acute and convalescent sera

• Potential Role in Immune Evasion:

  • There is preliminary evidence that ATP synthase components may bind to complement regulators, potentially contributing to immune evasion

  • Similar to findings with other bacterial pathogens where ATP synthase components have been shown to bind factor H

• Na⁺/K⁺-ATPase Interaction:

  • Research suggests that leptospiral components may interact with host Na⁺/K⁺-ATPase, potentially contributing to inflammatory and metabolic alterations

  • This interaction may represent a novel pathogenic mechanism

While these interactions require further validation, they suggest that ATP synthase components may have moonlighting functions beyond their classical role in energy metabolism, potentially contributing to the complex pathogenesis of leptospirosis .

What bioinformatic tools and approaches are most effective for analyzing the structural and functional domains of atpA across Leptospira species?

For comprehensive bioinformatic analysis of atpA across Leptospira species, researchers have successfully employed a combination of tools and approaches:

• Sequence Analysis:

  • Multiple sequence alignment using MUSCLE or MAFFT for identifying conserved and variable regions

  • BLAST and HMMer for identifying homologs in newly sequenced genomes

  • ConSurf for identifying functionally important residues based on evolutionary conservation

• Structural Analysis:

  • Homology modeling using SWISS-MODEL or I-TASSER with crystal structures of ATP synthase from other bacteria as templates

  • Molecular dynamics simulations using GROMACS to assess structural stability and dynamics

  • Docking simulations to analyze interactions with other ATP synthase subunits

• Functional Domain Prediction:

  • InterProScan for identifying functional domains and motifs

  • PROSITE for identifying catalytic sites and binding motifs

  • Pfam for classification of protein domains

• Evolutionary Analysis:

  • MEGA or PAML for phylogenetic analysis and detection of selection pressure

  • CodeML for identifying sites under positive selection

  • Gene synteny analysis to examine genomic context conservation

These approaches have revealed that atpA contains several highly conserved domains across Leptospira species, with the ATP-binding domain showing the highest conservation. The membrane-adjacent regions show more variability, potentially reflecting adaptation to different host environments .

What advanced mass spectrometry approaches can identify interactions between atpA and other proteins in the Leptospira proteome?

Advanced mass spectrometry (MS) techniques have revolutionized the study of protein-protein interactions in bacterial systems, including Leptospira:

• Cross-linking Mass Spectrometry (XL-MS):

  • Employs chemical cross-linkers (e.g., DSS, BS3) to capture transient protein interactions

  • LC-MS/MS analysis of cross-linked peptides reveals interaction sites

  • Has been used to map interactions within the ATP synthase complex in other bacteria

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Purification of atpA with its interacting partners using TAP-tagging or antibody-based pulldowns

  • MS identification of co-purified proteins

  • Quantitative approaches like SILAC or TMT labeling enhance specificity

• Hydrogen-Deuterium Exchange MS (HDX-MS):

  • Maps protein interaction surfaces through differential deuterium uptake

  • Provides dynamic information about conformational changes upon binding

  • Particularly useful for studying regulatory interactions

• Native MS:

  • Analysis of intact protein complexes preserving non-covalent interactions

  • Provides stoichiometric information and complex topology

  • Requires specialized instrumentation for high mass ranges

These techniques have identified interactions between atpA and other ATP synthase subunits (atpB, atpG, atpD), as well as potential interactions with regulatory proteins and metabolic enzymes. The ATP synthase complex appears to be part of larger "respirasomes" or metabolic assemblies in bacterial membranes .

How does the immunogenicity of recombinant atpA compare to established immunodominant antigens like LipL32 and LigA for diagnostic applications?

Comparative immunogenicity studies have yielded valuable insights into the potential of atpA as a diagnostic antigen:

These findings indicate that:

• atpA shows moderate immunogenicity compared to the highly immunodominant LipL32 and LigA

• atpA-based serological tests would be more suitable for convalescent rather than acute phase diagnosis

• atpA exhibits moderate cross-reactivity with other bacterial infections, particularly with other spirochetes

While atpA alone may not be an ideal diagnostic antigen, it could potentially be incorporated into multi-antigen chimeric constructs. Recent approaches using chimeric proteins containing multiple epitopes have shown promise for improved serodiagnosis .

What experimental evidence supports or refutes the potential of atpA as a vaccine candidate against leptospirosis?

The evaluation of atpA as a vaccine candidate has yielded mixed results:

Supporting Evidence:

• atpA is highly conserved across pathogenic Leptospira species, suggesting potential for broad protection

• As an essential protein for bacterial survival, immune targeting may be effective

• Certain epitopes of atpA are surface-exposed during infection, as evidenced by seroreactivity in patient samples

Refuting Evidence:

• Challenge studies in animal models have not demonstrated significant protection with atpA-based immunization

• Comparative immunization studies show that established vaccine candidates like LigA and LigB provide superior protection

• The primarily cytoplasmic localization of atpA may limit accessibility to antibodies

Experimental Data:
Hamster immunization studies with various leptospiral recombinant proteins have shown:

• rLigA immunization provided 100% survival against challenge

• rLipL32 showed variable protection (40-70%) in different studies

• ATP synthase components, when tested, showed minimal protection (<30%)

Recent approaches focusing on chimeric constructs or DNA vaccines may overcome some limitations, but current evidence suggests that ATP synthase components, including atpA, are not leading vaccine candidates compared to surface-exposed lipoproteins and adhesins .

What strategies overcome the challenges of generating neutralizing antibodies against functional domains of atpA for inhibition studies?

Generating neutralizing antibodies against functional domains of atpA presents unique challenges that can be addressed through specialized techniques:

• Structural Epitope Selection:

  • Use computational epitope prediction (BepiPred, DiscoTope) combined with structural analysis

  • Target surface-exposed loops within the catalytic domain

  • Focus on regions involved in nucleotide binding or subunit interactions

• Immunization Strategies:

  • Use of cyclized peptides that maintain native conformation of epitopes

  • Prime-boost approaches combining DNA and protein immunization

  • Carrier protein conjugation (KLH, TTox) for enhanced immunogenicity

• Antibody Screening Methods:

  • Development of ATP hydrolysis inhibition assays for functional screening

  • Surface plasmon resonance (SPR) to measure binding to specific domains

  • Bacterial growth inhibition assays in iron-limited conditions where ATP synthase function is critical

• Antibody Engineering:

  • Generation of recombinant antibody fragments (Fab, scFv) for improved access to conformational epitopes

  • Affinity maturation through directed evolution

  • Bispecific antibodies targeting multiple functional domains simultaneously

Researchers have successfully employed these approaches to generate antibodies against other bacterial ATP synthases, demonstrating the feasibility of targeting specific functional domains to inhibit enzymatic activity .

What are the most effective approaches for studying atpA function in live Leptospira given the challenges of genetic manipulation in this organism?

Studying atpA function in live Leptospira remains challenging due to difficulties in genetic manipulation, but several innovative approaches have proven effective:

• Conditional Gene Expression Systems:

  • Tetracycline-inducible promoters for controlled expression

  • Riboswitch-based systems for post-transcriptional regulation

  • These approaches allow modulation of atpA expression without complete knockout, which would likely be lethal

• CRISPR Interference (CRISPRi):

  • Use of catalytically inactive Cas9 (dCas9) for targeted gene repression

  • Allows titration of atpA expression levels

  • Has been successfully adapted for use in spirochetes

• Chemical Genetics:

  • Use of specific ATP synthase inhibitors (e.g., modified forms of oligomycin)

  • Allows temporal control of inhibition and dose-dependent effects

  • Can be combined with metabolomic analysis to assess impact

• Heterologous Expression Systems:

  • Expression of Leptospira atpA in genetically tractable surrogate hosts

  • Complementation studies in E. coli ATP synthase mutants

  • Chimeric ATP synthase complexes to study specific subunit functions

• Transposon Mutagenesis with Deep Sequencing (Tn-seq):

  • Genome-wide essentiality screening

  • Identification of synthetic lethal interactions with atpA

  • Characterization of the essential nature of different atpA domains

These approaches have revealed that atpA function is essential under standard culture conditions but may be partially dispensable under specific environmental conditions, suggesting metabolic flexibility in Leptospira .

How might single-molecule techniques advance our understanding of ATP synthase function in Leptospira compared to traditional biochemical approaches?

Single-molecule techniques offer unprecedented insights into ATP synthase function that traditional biochemical approaches cannot provide:

• Single-Molecule FRET (smFRET):

  • Enables real-time observation of conformational changes during catalytic cycles

  • Can resolve intermediate states not detectable in ensemble measurements

  • Would allow visualization of how the unique sodium specificity affects rotational dynamics

• Magnetic Tweezers and Optical Traps:

  • Direct measurement of mechanical forces and torque generated by ATP synthase

  • Quantification of the mechanochemical coupling efficiency

  • Would reveal how the Leptospira ATP synthase compares to other bacterial synthases in terms of mechanical output

• Nanodiscs and Lipid Bilayer Recordings:

  • Single-channel recordings of proton/sodium flux through the Fo portion

  • Determination of ion specificity and selectivity under various conditions

  • Would confirm the proposed sodium specificity of the Leptospira ATP synthase

• High-Speed AFM:

  • Visualization of ATP synthase dynamics in near-native membrane environments

  • Observation of subunit rearrangements during catalytic cycles

  • Would reveal potential unique structural features of the Leptospira complex

These techniques could resolve key questions about the Leptospira ATP synthase, such as:

• Whether its sodium specificity confers unique advantages in host environments

• How environmental factors like pH and ion concentrations affect its activity

• If its rotational mechanism differs from that of other bacterial ATP synthases

What research gaps remain in understanding the relationship between atpA function and Leptospira virulence in different host species?

Several critical research gaps remain in understanding the relationship between atpA function and Leptospira virulence:

• Host-Specific Adaptations:

  • Limited understanding of how ATP synthase function varies between reservoir hosts (where infection is chronic) versus accidental hosts (where infection is acute)

  • Unclear whether ATP synthase regulation differs in immunocompetent versus immunocompromised hosts

  • Unknown if sodium/proton utilization preference shifts in different host environments

• Connection to Virulence Regulation:

  • Poor understanding of how energy metabolism connects to virulence factor expression

  • Limited knowledge of whether ATP synthase inhibition affects expression of key virulence factors like LigA/B, LipL32, and Loa22

  • Unknown if the ATP synthase complex participates in signaling networks that regulate virulence

• Tissue-Specific Functions:

  • Lack of data on whether ATP synthase function differs during colonization of different tissues (kidney versus liver)

  • Limited understanding of tissue-specific metabolic adaptations

  • Unknown if different Leptospira serovars show variation in ATP synthase regulation during tissue tropism

• Therapeutic Targeting Potential:

  • Insufficient evaluation of ATP synthase as a drug target

  • Limited testing of ATP synthase inhibitors in animal models

  • Unknown whether ATP synthase inhibition could attenuate virulence without promoting resistance

Addressing these gaps requires integrating techniques from multiple disciplines, including:

• Tissue-specific transcriptomics and proteomics

• Metabolic flux analysis in different host environments

• Comparative studies across multiple Leptospira species and serovars in different host species