Recombinant Listeria monocytogenes serotype 4b ATP synthase subunit alpha 1 (atpA1)

What is the function of ATP synthase subunit alpha 1 (atpA1) in Listeria monocytogenes?

The ATP synthase subunit alpha 1 (atpA1) is a critical component of the F1 domain of the F-type ATP synthase complex in Listeria monocytogenes. This complex catalyzes ATP synthesis from ADP and inorganic phosphate using the energy of an electrochemical proton gradient across the bacterial membrane. The alpha subunit contains nucleotide binding sites essential for the catalytic function of the enzyme.

In L. monocytogenes, the F-type ATP synthase has demonstrated importance for growth and survival under various conditions. Research has shown that 8 of the 9 genes encoding this enzyme were depleted after growth in gallbladders , indicating their significance in host environments. Beyond its canonical role in energy production, the F-type ATP synthase is hypothesized to be crucial during anaerobic growth for combating acid stress and generating proton motive force . This multifunctional role makes atpA1 an integral part of L. monocytogenes metabolism and stress response systems.

What methods are used to clone and express recombinant atpA1 from L. monocytogenes serotype 4b?

The cloning and expression of recombinant atpA1 from L. monocytogenes serotype 4b typically follows these methodological steps:

• Gene amplification: The atpA1 gene is PCR-amplified from L. monocytogenes serotype 4b genomic DNA using primers designed to include appropriate restriction sites for subsequent cloning .

• Vector construction: The amplified gene is digested with restriction enzymes and ligated into an expression vector. Common vectors include pGEM for initial cloning and pKSV7 or pPL2 for expression in Listeria .

• Transformation: The recombinant vector is first transformed into E. coli (such as XL1-Blue) and positive transformants are selected using antibiotic markers, typically on LB plates supplemented with appropriate antibiotics (100 μg/ml ampicillin), and sometimes with IPTG (0.5 mM) and X-Gal (80 μg/ml) for blue-white screening .

• Sequence verification: Plasmid DNA is isolated from positive transformants and sequenced to confirm the integrity of the insert .

• Expression in Listeria: For expression in L. monocytogenes, the construct is introduced via electroporation. For chromosomal integration, temperature-sensitive vectors like pKSV7 are used, with transformants cultured at 30°C and then shifted to 42°C (non-permissive temperature) to select for integration .

• Confirmation of expression: Protein expression is verified using techniques such as SDS-PAGE, Western blotting, or mass spectrometry-based proteomics approaches .

How can atpA1 gene deletions be created in Listeria monocytogenes?

Creating atpA1 gene deletions in Listeria monocytogenes requires careful genetic manipulation techniques:

• Allelic exchange approach: This widely used method involves:

  • Amplifying ~700 bp regions flanking the atpA1 gene using PCR

  • Cloning these fragments into a temperature-sensitive shuttle vector like pKSV7

  • Transforming the construct into L. monocytogenes via electroporation

  • Selecting integrants using antibiotic resistance (typically chloramphenicol) at 42°C, the non-permissive temperature for pKSV7 replication

  • Culturing the integrants without antibiotics at 30°C to allow plasmid excision

  • Screening for colonies that have lost the antibiotic resistance marker, indicating successful plasmid excision

  • Confirming deletion using PCR and sequencing

• Complementation: To verify that observed phenotypes are specifically due to atpA1 deletion, complementation is performed by:

  • Amplifying the wild-type atpA1 gene with its native promoter

  • Cloning into an integration vector like pPL2

  • Introducing into the deletion mutant via conjugation or electroporation

  • Confirming integration and expression

This methodology ensures the creation of clean, unmarked deletion mutants suitable for subsequent functional characterization studies.

What growth conditions affect atpA1 expression in L. monocytogenes?

The expression of atpA1 in L. monocytogenes is significantly influenced by various environmental and growth conditions:

• Oxygen availability: F-type ATP synthase expression, including atpA1, is particularly important during anaerobic growth. Studies with other ATP synthase components (such as atpB) have shown differential growth patterns under aerobic versus anaerobic conditions .

• Nutrient availability: Growth in nutrient-rich versus nutrient-limited media affects ATP synthase expression. For instance, an atpB deletion strain showed severe growth attenuation in rich BHI medium but could replicate in bile under anaerobic conditions .

• Host environments: During infection, L. monocytogenes undergoes significant proteome remodeling in response to host conditions. Studies using metabolic labeling techniques combined with TMT (tandem mass tag) approaches have demonstrated dynamic changes in protein synthesis and degradation rates during infection .

• Stress responses: The expression and stability of ATP synthase components are regulated as part of broader stress response mechanisms. For example, ClpC, which forms a protease complex with ClpP, influences protein degradation and turnover during adaptation to stress conditions and infection .

• Growth phase: ATP synthase expression levels typically vary between exponential and stationary growth phases, reflecting changing energy demands.

How does atpA1 contribute to L. monocytogenes virulence during infection?

The contribution of atpA1 to L. monocytogenes virulence involves multiple mechanisms that support bacterial survival and replication in host environments:

• Adaptation to intracellular conditions: Genome-wide screening has identified ATP synthase components as essential for growth and survival in host environments, including ex vivo gallbladder models . This suggests atpA1 plays a crucial role in adapting to the metabolic constraints of intracellular life.

• Energy provision during critical infection stages: The F-type ATP synthase supports energy needs during key steps of the infection cycle, including phagosomal escape, cytosolic replication, and cell-to-cell spread. ATP synthase mutants (such as ΔatpB) show attenuation in infection models .

• pH homeostasis: During infection, L. monocytogenes encounters varying pH environments, from the acidic phagosome to the neutral cytosol. The F-type ATP synthase contributes to maintaining internal pH homeostasis, with evidence suggesting it functions to combat acid stress and generate proton motive force during anaerobic growth .

• Integration with virulence regulation: Proteome remodeling during infection, which includes modulation of ATP synthase components, appears to be coordinated with virulence factor expression. Studies of the ClpC-dependent proteome have revealed extensive remodeling upon host interaction, supporting the hypothesis that protein degradation and synthesis are carefully regulated during infection .

• Metabolic adaptation: atpA1 function facilitates shifts in carbon metabolism during infection, ensuring energy production continues despite changing nutrient availability in different host compartments.

These multifaceted contributions make atpA1 an integral component of the virulence machinery of L. monocytogenes, despite not being a classical virulence factor.

What methods can be used to study atpA1 protein interactions within the ATP synthase complex?

Advanced methodologies to study atpA1 protein interactions within the ATP synthase complex include:

• Protein crosslinking coupled with mass spectrometry:

  • Chemical crosslinkers can capture transient protein interactions

  • Digestion and mass spectrometry analysis identify crosslinked peptides

  • This approach can map the interaction interfaces between atpA1 and other ATP synthase subunits

• Time-resolved proteomics approaches:

  • pSILAC (pulse Stable Isotope Labeling with Amino acids in Cell culture) combined with TMT (Tandem Mass Tag) labeling enables simultaneous determination of protein synthesis and degradation rates

  • This approach can track the assembly kinetics of the ATP synthase complex

  • The technique has been successfully applied to monitor L. monocytogenes proteome dynamics during infection

• Co-immunoprecipitation studies:

  • Using antibodies against atpA1 or epitope-tagged versions

  • Mass spectrometry identification of co-precipitated proteins

  • Quantitative analysis to distinguish specific from non-specific interactions

• Bacterial two-hybrid systems:

  • Modified for use in Gram-positive bacteria

  • Screening for protein-protein interactions involving atpA1

  • Validation of interactions identified through other methods

• Blue native PAGE:

  • Separation of intact protein complexes under native conditions

  • Western blotting or mass spectrometry for component identification

  • Useful for studying the integrity of the ATP synthase complex in various mutants

These methodologies can be complemented with structural approaches such as cryo-electron microscopy to obtain a comprehensive understanding of atpA1's position and interactions within the ATP synthase complex.

How does the proteome of L. monocytogenes change in atpA1 mutants during infection?

The proteome of L. monocytogenes undergoes significant remodeling in atpA1 mutants during infection, reflecting both direct effects of ATP synthase dysfunction and compensatory responses:

• Energy metabolism reconfiguration: Loss of functional atpA1 forces L. monocytogenes to adjust its energy production pathways. While specific data for atpA1 mutants is not provided in the search results, studies of the L. monocytogenes proteome during infection have revealed extensive remodeling of metabolic pathways .

• Stress response activation: ATP synthase deficiency triggers stress response systems, including upregulation of chaperones and proteases. The ClpC-dependent protein degradation system, which has been studied using time-resolved proteomics approaches , would likely show altered activity in atpA1 mutants.

• Virulence factor modulation: Proteome studies have demonstrated connections between metabolism and virulence. In atpA1 mutants, expression of virulence factors may be altered due to energy limitations or changes in regulatory circuits.

• Compensatory upregulation of alternative energy pathways: To compensate for ATP synthase dysfunction, alternative energy generation pathways may be upregulated. This could include fermentative pathways or other mechanisms to maintain redox balance.

• Membrane protein composition changes: The bacterial membrane proteome likely undergoes significant changes to maintain membrane potential and integrity in the absence of fully functional ATP synthase.

The methodology for such studies would mirror the approach described in result , which combined pSILAC with TMT labeling to simultaneously track protein synthesis and degradation. This technique allows for time-resolved quantitative proteome analysis across multiple time points during infection, providing insights into the dynamic changes occurring in atpA1 mutants compared to wild-type bacteria.

What is the relationship between atpA1 and other virulence factors in L. monocytogenes serotype 4b?

The relationship between atpA1 and other virulence factors in L. monocytogenes serotype 4b involves complex regulatory networks and functional interdependencies:

• Energy provision for virulence factor expression: ATP synthase function, including atpA1, provides the energy required for the synthesis of classical virulence factors. Genome-wide screening has demonstrated that ATP synthase components are crucial for growth in conditions that simulate infection .

• Coordinate regulation with LIPI-1 genes: The Listeria pathogenicity island 1 (LIPI-1) contains essential virulence genes. Although not directly mentioned in the search results in relation to atpA1, the coordinated expression of metabolic and virulence genes is well-established in bacterial pathogens. The actA gene from LIPI-1, responsible for actin polymerization and cell-to-cell spread, is variably present in different L. monocytogenes strains , suggesting complex relationships between core metabolism and virulence.

• Protein quality control systems: ClpC, which forms a protease complex with ClpP, has been shown to play an important role in virulence development and is involved in extensive proteome remodeling upon host interaction . This system likely influences both atpA1 stability and virulence factor expression, creating a regulatory link between them.

• Stress adaptation pathways: Both ATP synthase and virulence factors are regulated in response to environmental stresses encountered during infection. Shared regulatory networks likely coordinate these responses to optimize bacterial survival and virulence.

• Metabolic regulation: The PTS systems (sugar transport) have been identified alongside ATP synthase components in infection models , suggesting coordinated regulation between nutrient acquisition, energy production, and virulence factor expression.

Understanding these relationships requires integrated approaches combining genetics, proteomics, and functional assays to map the regulatory networks connecting primary metabolism and virulence in L. monocytogenes.

How can recombinant atpA1 be utilized in vaccine development against listeriosis?

Recombinant atpA1 presents several opportunities for listeriosis vaccine development, leveraging both the immunogenicity of this essential protein and the established capacity of Listeria as a vaccine vector:

• Direct antigen approach:

  • Recombinant atpA1 protein or peptides can be used as antigens in subunit vaccines

  • Being essential for bacterial metabolism, antibodies targeting atpA1 could effectively neutralize L. monocytogenes

  • Adjuvant systems would be required to enhance immunogenicity

• PEST sequence engineering:

  • PEST sequences (protein degradation signals) have been shown to enhance the immunogenicity of antigens when included in Listeria vaccines

  • Fusion of atpA1 epitopes with PEST sequences could improve their processing and presentation to the immune system

  • Research has demonstrated that "Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice" , suggesting a similar approach could work for atpA1

• Live attenuated vaccine vectors:

  • Attenuated L. monocytogenes strains with modified atpA1 could serve as live vaccine vectors

  • These would induce robust cellular immunity while maintaining sufficient attenuation for safety

  • The modified atpA1 could be engineered to express epitopes from other pathogens, creating multivalent vaccines

• DNA vaccine approach:

  • DNA vaccines encoding atpA1 could generate both humoral and cell-mediated immunity

  • Codon optimization and appropriate promoters would enhance expression in mammalian cells

• Combination with other antigens:

  • atpA1 could be combined with established Listeria antigens like listeriolysin O (LLO)

  • Previous research has shown that "fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy"

  • Similar fusion approaches with atpA1 could enhance vaccine efficacy

These strategies would need to be evaluated in appropriate animal models, with careful assessment of both safety and protective efficacy against challenge with virulent L. monocytogenes strains.

What is known about atpA1 structure-function relationships and potential inhibitor binding sites?

While the specific structure of L. monocytogenes serotype 4b atpA1 has not been fully determined, inferences can be made from homologous proteins and functional studies:

• Structural domains:

  • The alpha subunit of F1-ATP synthase typically contains three major domains:

  • N-terminal domain containing a β-barrel motif

  • Central nucleotide-binding domain with the Walker A motif (P-loop)

  • C-terminal domain involved in inter-subunit interactions

  • These domains work together during the conformational changes that drive ATP synthesis

• Catalytic mechanism:

  • atpA1 contains nucleotide-binding sites that participate in the binding-change mechanism

  • During catalysis, each alpha subunit cycles through different conformational states

  • These conformational changes are coupled to the rotation of the central stalk of the ATP synthase

• Inhibitor binding sites:

  • Potential inhibitor binding sites include:

  • The nucleotide-binding pocket

  • Interface regions between alpha and adjacent beta subunits

  • Allosteric sites that could disrupt conformational cycling

  • Small molecule inhibitors targeting these sites could disrupt ATP synthesis function

• Critical residues:

  • Conserved residues in the Walker A motif are essential for nucleotide binding

  • Residues at the alpha-beta interface are critical for inter-subunit communication

  • Mutations in these regions could generate attenuated strains potentially useful for vaccine development

• Species-specific features:

  • While the core structure is conserved, L. monocytogenes atpA1 may contain unique surface features

  • These features could be targeted for species-specific inhibitor development

  • Comparative analysis with human ATP synthase could identify bacterial-specific targets for antimicrobial development

Structural studies of the complete ATP synthase complex, rather than isolated subunits, would be most informative for understanding atpA1 function in its native context and for identifying potential sites for therapeutic intervention.

What methodologies can be used to study atpA1 gene regulation under different environmental conditions?

Understanding atpA1 gene regulation under different environmental conditions requires sophisticated methodological approaches:

• Transcriptional analysis:

  • RT-qPCR to quantify atpA1 mRNA levels under different conditions

  • RNA-seq for genome-wide transcriptional profiling to identify co-regulated genes

  • 5' RACE to map transcription start sites and identify promoter regions

  • ChIP-seq to identify transcription factors binding to the atpA1 promoter region

• Promoter analysis:

  • Reporter gene fusions (e.g., lacZ, gfp) to monitor promoter activity

  • Site-directed mutagenesis of putative regulatory elements

  • Electrophoretic mobility shift assays (EMSA) to detect protein-DNA interactions

  • DNase I footprinting to precisely map protein binding sites

• Translational regulation:

  • Ribosome profiling to measure translation efficiency

  • Analysis of 5' UTR secondary structures that might influence translation

  • Investigation of potential small RNA regulators

• Post-translational regulation:

  • Proteome analysis using pSILAC combined with TMT labeling to measure protein synthesis and degradation rates

  • Identification of post-translational modifications that affect activity

  • Analysis of protein-protein interactions that influence stability or activity

• Environmental simulation:

  • In vitro models simulating different host environments (pH, oxygen levels, nutrient availability)

  • Ex vivo models such as the gallbladder model described in result

  • Infection models to study regulation during different stages of pathogenesis

• Single-cell approaches:

  • Single-cell RNA-seq to capture heterogeneity in gene expression

  • Fluorescent reporters for real-time monitoring of gene expression in individual bacteria

  • Time-lapse microscopy to track dynamic changes in expression

These methodologies can be applied across a range of conditions relevant to L. monocytogenes lifecycle, including: varying oxygen availability, nutrient limitation, acid stress, bile exposure, intracellular environment, and biofilm formation.

How does atpA1 function compare between different serotypes of L. monocytogenes?

Comparative analysis of atpA1 function across different L. monocytogenes serotypes reveals both conservation and specialization:

• Sequence conservation:

  • The catalytic core of atpA1 is highly conserved across serotypes due to its essential function

  • Sequence variations primarily occur in non-catalytic regions that might influence regulatory interactions or complex assembly

  • Serotype 4b strains, which are frequently associated with invasive listeriosis, may contain specific adaptations in atpA1 that contribute to their enhanced virulence

• Expression patterns:

  • Different serotypes show varied expression patterns of ATP synthase components

  • These differences may reflect adaptation to specific ecological niches or host environments

  • For example, clinical isolates from serotype 4b might show expression patterns optimized for intracellular survival

• Functional integration:

  • atpA1 function integrates with serotype-specific metabolic networks

  • Different serotypes possess varying complements of virulence factors (e.g., LIPI-3 is variably present across lineages )

  • ATP synthase function must coordinate with these serotype-specific traits

• Stress resistance:

  • Serotypes differ in their resistance to various stresses encountered during infection

  • ATP synthase function, including atpA1, contributes to stress resistance, particularly acid tolerance

  • Serotype-specific variations in atpA1 may contribute to differential stress resistance profiles

• Host adaptation:

  • Lineage I strains (including many serotype 4b isolates) are more frequently isolated from human clinical cases

  • Lineage II strains are more commonly found in food and environmental samples

  • These ecological differences may be reflected in subtle adaptations in atpA1 function

Methodological approaches for comparing atpA1 function across serotypes include:

• Complementation studies exchanging atpA1 genes between serotypes

• Comparative biochemical characterization of purified atpA1 proteins

• Analysis of growth phenotypes under various stress conditions

• Infection models comparing wild-type and chimeric strains with exchanged atpA1 alleles