Recombinant Actinobacillus pleuropneumoniae serotype 3 N-acetylneuraminate lyase (nanA)

Basic Research Questions

• What is Actinobacillus pleuropneumoniae and what role does N-acetylneuraminate lyase (nanA) play in its pathobiology?

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a severe respiratory tract infection responsible for major economic losses in the swine industry worldwide. It is a Gram-negative, facultative anaerobic bacterium belonging to the Pasteurellaceae family with 15 recognized serotypes based on capsular and lipopolysaccharide antigens, with serotype 3 being prevalent in certain regions including China . A. pleuropneumoniae primarily infects the respiratory tract of pigs, causing acute fibrinohemorrhagic pneumonia and pleuritis, often leading to high mortality rates in acute cases or chronic production losses in surviving animals.

N-acetylneuraminate lyase (nanA) in A. pleuropneumoniae catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (sialic acid) to form N-acetylmannosamine and pyruvate. In bacterial pathogens, nanA typically serves multiple functions:

The enzyme is particularly relevant in respiratory pathogens like A. pleuropneumoniae, which must adapt to the sialic acid-rich environment of the respiratory tract to establish infection.

• How is the genomic context of nanA characterized in A. pleuropneumoniae serotype 3?

The genomic context of nanA in A. pleuropneumoniae serotype 3 is best understood within the framework of the complete genome of strain JL03, a Chinese field isolate of serotype 3. JL03 possesses a single circular chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences, six ribosomal rRNA operons, and 63 tRNA genes . Within this genomic landscape, nanA is typically part of a sialic acid utilization operon.

In many bacterial pathogens, including members of the Pasteurellaceae family, nanA is often clustered with other genes involved in sialic acid metabolism, such as:

• nanE (N-acetylmannosamine-6-phosphate epimerase)

• nanK (N-acetylmannosamine kinase)

• nanT (sialic acid transporter)

This genomic organization facilitates coordinated expression of enzymes required for the complete catabolism of sialic acid from uptake to entry into central metabolism.

When examining the nanA gene from A. pleuropneumoniae serotype 3, researchers should consider:

• Promoter elements and transcriptional regulators that control expression

• Presence of simple sequence repeats (SSRs) that might indicate phase variation

• Conservation across different serotypes of A. pleuropneumoniae

• Homology with nanA sequences from other bacterial species

Notably, A. pleuropneumoniae contains multiple phase-variable methyltransferases that can regulate gene expression through epigenetic mechanisms , making it important to investigate whether nanA expression might be influenced by such regulatory systems.

• What are the recommended protocols for cloning and expressing recombinant A. pleuropneumoniae serotype 3 nanA?

The successful cloning and expression of recombinant A. pleuropneumoniae serotype 3 nanA requires a systematic approach to ensure optimal yield and activity. The following protocol provides a comprehensive methodology:

Gene Amplification and Cloning:

• Design primers based on the nanA sequence from A. pleuropneumoniae serotype 3 (JL03 strain reference genome)

  • Forward primer: Include restriction site compatible with expression vector

  • Reverse primer: Include restriction site and consider whether to include or exclude the stop codon depending on desired tag orientation

  • Consider codon optimization if expressing in a heterologous host

• PCR amplification

  • Use high-fidelity DNA polymerase (e.g., Phusion or Q5)

  • Optimize annealing temperature (typically 58-62°C)

  • Include positive control with A. pleuropneumoniae genomic DNA

• Vector selection and preparation

  • pET-based vectors (e.g., pET-28a) for E. coli expression

  • Consider fusion tags: His-tag for purification, GST or MBP for solubility enhancement

  • Prepare vector with appropriate restriction enzymes

• Ligation and transformation

  • Ligate purified PCR product with prepared vector

  • Transform into cloning strain (E. coli DH5α)

  • Screen transformants by colony PCR and confirm by sequencing

Expression Optimization:

• Transform expression construct into E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

• Small-scale expression tests

  • Test multiple conditions in parallel:

    • Temperature: 16°C, 25°C, 37°C

    • IPTG concentration: 0.1mM, 0.5mM, 1.0mM

    • Induction time: 4h, 8h, overnight

• Solubility assessment

  • Separate soluble and insoluble fractions

  • Analyze by SDS-PAGE to determine optimal conditions for soluble expression

Purification Strategy:

• Cell lysis

  • Sonication or French press in buffer containing 50mM Tris-HCl pH 8.0, 300mM NaCl, 10% glycerol, 1mM DTT

• Affinity chromatography

  • Ni-NTA for His-tagged protein

  • Load clarified lysate and wash with increasing imidazole concentrations

  • Elute with 250-300mM imidazole

• Secondary purification

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Consider ion exchange chromatography if further purification is needed

• Buffer exchange and storage

  • Final buffer: 25mM Tris-HCl pH 7.5, 150mM NaCl, 10% glycerol, 1mM DTT

  • Flash-freeze aliquots in liquid nitrogen and store at -80°C

This comprehensive approach typically yields 5-10mg of purified recombinant nanA per liter of bacterial culture, suitable for subsequent biochemical and structural studies.

• What basic biochemical characterization should be performed on purified recombinant nanA?

A thorough biochemical characterization of purified recombinant A. pleuropneumoniae serotype 3 nanA provides essential baseline information before proceeding to more complex studies. The following characterization procedures are recommended:

Protein Quality Assessment:

• Purity analysis

  • SDS-PAGE with Coomassie staining (>95% purity recommended)

  • Western blot using anti-His antibody or nanA-specific antibodies

  • Mass spectrometry to confirm intact mass and peptide coverage

• Structural integrity evaluation

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assay to determine melting temperature and stability

  • Dynamic light scattering to check for aggregation state

Enzymatic Activity Characterization:

• Standard activity assay

  • Thiobarbituric acid (TBA) assay: Measures release of N-acetylmannosamine

  • Lactate dehydrogenase (LDH)-coupled assay: Measures pyruvate formation through NADH oxidation

  • Optimized conditions: 50mM Tris-HCl pH 7.5, 37°C

• Kinetic parameter determination

  • Substrate range: 0.1-10mM N-acetylneuraminic acid

  • Measure initial velocities and determine Km, Vmax, kcat, and kcat/Km

  • Plot data using Michaelis-Menten and Lineweaver-Burk analyses

• Optimal condition determination

  • pH profile: Test range 5.0-9.0 using appropriate buffers

  • Temperature profile: Test range 20-55°C

  • Metal ion requirements or inhibition

Table 1: Buffer Systems for nanA Activity Assays

Substrate Specificity Profile:

• Test activity on various sialic acid derivatives

  • N-acetylneuraminic acid (Neu5Ac, natural substrate)

  • N-glycolylneuraminic acid (Neu5Gc)

  • Various methylated or acetylated derivatives

• Compare relative activities

  • Express as percentage of activity relative to Neu5Ac

  • Determine specific kinetic parameters for each substrate

This comprehensive biochemical characterization provides the foundation for understanding the fundamental properties of A. pleuropneumoniae nanA and enables comparison with similar enzymes from other bacterial species.

Intermediate Research Questions

• How does the enzymatic activity of A. pleuropneumoniae nanA compare with N-acetylneuraminate lyases from other bacterial pathogens?

Comparative analysis of N-acetylneuraminate lyases from different bacterial species provides valuable insights into their evolutionary relationships and functional adaptations. When comparing A. pleuropneumoniae nanA with homologs from other pathogens, researchers should consider the following methodological approach:

Comparative Enzymatic Analysis Protocol:

• Express and purify nanA enzymes from multiple species under identical conditions

  • A. pleuropneumoniae (serotype 3)

  • Haemophilus influenzae (close relative in Pasteurellaceae)

  • Escherichia coli (well-characterized reference)

  • Clostridium perfringens (distinct phylogenetic lineage)

• Perform standardized activity assays under identical conditions

  • Buffer: 50mM Tris-HCl pH 7.5, 150mM NaCl

  • Temperature: 37°C

  • Substrate range: 0.1-10mM N-acetylneuraminic acid

• Determine and compare key kinetic parameters

  • Calculate Km, Vmax, kcat, and catalytic efficiency (kcat/Km)

  • Evaluate pH optima and pH-activity profiles

  • Assess temperature stability and thermodynamic parameters

Table 2: Comparative Kinetic Properties of Bacterial N-acetylneuraminate Lyases

*Values for A. pleuropneumoniae nanA must be experimentally determined

Substrate Specificity Comparison:
Evaluate relative activity on different sialic acid derivatives such as:

• N-acetylneuraminic acid (standard substrate)

• N-glycolylneuraminic acid

• O-acetylated derivatives

• Synthetic analogs

Structural Basis for Functional Differences:

• Analyze amino acid sequence alignments to identify conserved and variable regions

• Generate homology models if crystal structures are unavailable

• Perform molecular docking of substrates to compare binding modes

• Identify species-specific active site residues that might explain kinetic differences

This comparative analysis can reveal adaptations that might reflect the distinct ecological niches these bacteria occupy, potentially correlating with host specificity or tissue tropism. For A. pleuropneumoniae, which specifically infects the porcine respiratory tract, such adaptations might provide insights into host-pathogen coevolution and potential targets for intervention strategies.

• What mechanisms regulate nanA expression in A. pleuropneumoniae during infection?

Understanding the regulatory mechanisms controlling nanA expression in A. pleuropneumoniae during infection provides insights into the pathogen's adaptation to the host environment. Based on the available information about A. pleuropneumoniae gene regulation, several potential regulatory mechanisms warrant investigation:

Transcriptional Regulation:

• Catabolite repression

  • CRP (cAMP receptor protein) dependent regulation

  • Glucose availability affecting nanA expression

  • Experimental approach: Compare expression in glucose vs. sialic acid as carbon source

• Sialic acid-responsive regulators

  • NanR-type repressors common in other bacteria

  • Potential derepression in presence of substrate

  • Experimental approach: Identify putative regulator binding sites in nanA promoter

• Phase variation mechanisms

  • A. pleuropneumoniae contains multiple phase-variable DNA methyltransferases

  • These methyltransferases create phasevarions (phase-variable regulons)

  • Experimental approach: Examine nanA expression in strains with ON/OFF states of methyltransferases

Table 3: Potential Regulatory Elements for nanA Expression

Environmental Regulation:

• Temperature-dependent expression

  • Compare expression at 25°C vs. 37°C (host temperature)

  • Use qRT-PCR and reporter gene fusions

• pH-dependent regulation

  • Assess expression under different pH conditions (pH 6.0-8.0)

  • Relevant for adaptation to different microenvironments in respiratory tract

• Iron-dependent regulation

  • Iron restriction is a host defense mechanism

  • Examine nanA expression under iron-limited conditions

Host-Induced Regulation:

• Contact with host cells

  • Co-culture with porcine respiratory epithelial cells

  • RNA-seq to identify differential expression upon host contact

• Exposure to host factors

  • Mucin, pulmonary surfactant, antimicrobial peptides

  • Analyze expression changes using transcriptomics

• Biofilm-specific regulation

  • Compare planktonic vs. biofilm growth conditions

  • Use fluorescent reporters to visualize expression patterns

This multifaceted approach to understanding nanA regulation would reveal how A. pleuropneumoniae modulates sialic acid metabolism during different stages of infection. Given that A. pleuropneumoniae has phase-variable DNA methyltransferases that create epigenetic regulatory systems , it would be particularly interesting to determine if nanA is part of these phasevarions, as this would have implications for vaccine development and pathogen evolution.

• How does nanA contribute to A. pleuropneumoniae serotype 3 metabolism and adaptation to the host environment?

Metabolic Integration of nanA Activity:
A. pleuropneumoniae possesses complete glycolysis and gluconeogenesis pathways but has an incomplete TCA cycle, lacking citrate synthase, aconitase, and isocitrate dehydrogenase . In this metabolic context, nanA plays a pivotal role by:

Table 4: Predicted Growth Capabilities Based on nanA Status

Host Adaptation Functions:

• Colonization and persistence

  • Removal of sialic acids from host glycoproteins exposes underlying receptors

  • May enhance adhesion to porcine respiratory epithelium

  • Experimental approach: Compare wild-type and ΔnanA adhesion to porcine tracheal cells

• Immune evasion

  • Modification of bacterial surface sialylation patterns

  • Potentially reducing complement activation or neutrophil recognition

  • Experimental approach: Serum resistance assays comparing wild-type and ΔnanA mutants

• Biofilm formation

  • Sialic acid metabolism may influence extracellular matrix composition

  • Experimental approach: Crystal violet biofilm assays under different conditions

• Interspecies competition

  • Scavenging host sialic acids may deprive competing microbes

  • Experimental approach: Co-culture experiments with respiratory commensals

Experimental Approaches to Study nanA's Metabolic Role:

• Gene deletion studies

  • Create clean ΔnanA knockout using allelic exchange

  • Compare growth on different carbon sources

  • Assess in vivo fitness using competitive index experiments

• Metabolomics analysis

  • Compare metabolite profiles between wild-type and ΔnanA mutant

  • Use LC-MS/MS to identify metabolic bottlenecks

  • Trace isotope-labeled sialic acid metabolism

• Transcriptomics

  • RNA-seq to identify changes in gene expression in ΔnanA mutant

  • Identify compensatory pathways activated in absence of nanA

Understanding nanA's role in metabolism and host adaptation may reveal potential targets for therapeutic intervention or attenuation strategies for vaccine development against A. pleuropneumoniae.

• What are the structural features of A. pleuropneumoniae nanA and how do they relate to enzyme function?

The structural features of A. pleuropneumoniae nanA are crucial for understanding its catalytic mechanism, substrate specificity, and potential for inhibitor design. While specific structural data for A. pleuropneumoniae nanA is not directly available in the search results, a comprehensive structural analysis can be approached using comparative methods and experimental techniques:

Predicted Structural Organization:

• Primary structure analysis

  • Sequence alignment with structurally characterized nanA enzymes

  • Identification of catalytic residues (typically K165, Y137, D191 based on E. coli numbering)

  • Conservation analysis of substrate binding pocket residues

• Secondary and tertiary structure

  • Predicted to adopt a TIM barrel fold (β/α)₈ common to aldolases

  • N-terminal catalytic domain containing active site

  • C-terminal domain involved in oligomerization

• Quaternary structure

  • Likely forms homotetrameric assembly like other bacterial nanA enzymes

  • Tetramer stabilized by hydrophobic interactions and salt bridges

  • Experimental approach: Size exclusion chromatography and analytical ultracentrifugation

Table 5: Key Structural Elements Predicted in A. pleuropneumoniae nanA

*Residue numbers based on homology to E. coli nanA; actual positions in A. pleuropneumoniae nanA would need confirmation

Structure Determination Approaches:

• X-ray crystallography

  • Crystallization screening of purified recombinant protein

  • Co-crystallization with substrate or inhibitors

  • Resolution target: <2.0Å for detailed mechanistic insights

• Homology modeling

  • Template selection: E. coli nanA (PDB: 1NAL) or H. influenzae nanA

  • Model validation: Ramachandran plot, QMEAN score

  • Molecular dynamics refinement

• Cryo-electron microscopy

  • Particularly useful for examining quaternary structure

  • Single-particle analysis to resolve domain organization

Structure-Function Relationships:

• Catalytic mechanism

  • Schiff base formation between conserved lysine and substrate

  • Aldol cleavage facilitated by tyrosine-aspartate dyad

  • Water-mediated proton transfer network

• Substrate specificity determinants

  • Binding pocket architecture constrains substrate orientation

  • Species-specific residues may alter substrate preference

  • Experimental approach: Site-directed mutagenesis of binding pocket residues

• Allosteric regulation

  • Potential communication between subunits

  • Conformational changes upon substrate binding

  • Experimental approach: Hydrogen-deuterium exchange mass spectrometry

Understanding the structural features of A. pleuropneumoniae nanA would provide a foundation for rational inhibitor design and may reveal species-specific adaptations that could explain host tropism or virulence characteristics.

Advanced Research Questions

• How can nanA be targeted for the development of novel therapeutics against A. pleuropneumoniae infections?

The development of nanA-targeted therapeutics represents a promising approach for combating A. pleuropneumoniae infections, particularly given the increasing concerns about antimicrobial resistance. The following comprehensive strategy outlines the steps for developing nanA inhibitors as potential therapeutics:

Inhibitor Design Strategies:

• Structure-based design

  • Virtual screening against homology model or crystal structure

  • Fragment-based approach targeting catalytic site

  • Focus on transition-state analogs mimicking the oxocarbenium ion intermediate

• Substrate-based inhibitors

  • Modified sialic acid derivatives with enhanced binding but resistant to cleavage

  • Incorporation of fluorine or other electronegative atoms at C3 position

  • Phosphonate analogs replacing the carboxyl group

• Allosteric inhibitors

  • Target protein-protein interfaces in the tetrameric assembly

  • Disrupt conformational changes required for catalysis

  • May offer higher selectivity than active site inhibitors

Table 6: Chemical Classes for nanA Inhibitor Development

Screening and Evaluation Pipeline:

• Initial screening

  • High-throughput enzymatic assays using purified recombinant nanA

  • Fluorescence-based assays measuring inhibition of activity

  • Counter-screening against mammalian sialidases for selectivity

• Hit validation

  • Dose-response curves and IC₅₀ determination

  • Binding mechanism characterization (competitive, non-competitive)

  • Thermal shift assays to confirm target engagement

• Structure-activity relationship studies

  • Synthesize analogs of promising hits

  • Optimize potency, selectivity, and drug-like properties

  • X-ray crystallography of enzyme-inhibitor complexes

• Cellular and ex vivo evaluation

  • A. pleuropneumoniae growth inhibition assays

  • Biofilm formation inhibition

  • Porcine respiratory tissue infection models

• In vivo efficacy studies

  • Pharmacokinetic profiling in animal models

  • Efficacy in porcine respiratory infection model

  • Combination studies with existing antibiotics

Delivery Strategies for Respiratory Infections:

• Inhaled formulations

  • Dry powder inhalers for improved lung deposition

  • Nebulized solutions for acute treatment

  • Particle engineering for optimal distribution

• Sustained release approaches

  • Liposomal encapsulation for prolonged activity

  • Polymer-based microparticles

  • Mucoadhesive formulations for enhanced residence time

The development of nanA inhibitors offers a targeted approach that could potentially reduce the use of broad-spectrum antibiotics, addressing concerns about antimicrobial resistance while providing effective control of A. pleuropneumoniae infections in swine populations.

• What role does nanA play in A. pleuropneumoniae biofilm formation and persistence?

Biofilms represent a critical aspect of A. pleuropneumoniae pathogenesis, contributing to persistence and antibiotic tolerance. The role of nanA in biofilm formation and maintenance involves complex interactions between sialic acid metabolism, extracellular matrix production, and bacterial communication systems.

Biofilm Development and nanA's Role:

• Initial attachment phase

  • nanA may modify bacterial surface sialylation patterns

  • Altered surface charge affecting adhesion to host tissues

  • Experimental approach: Atomic force microscopy to measure adhesion forces

• Microcolony formation

  • Sialic acid metabolism potentially contributing to extracellular polymeric substances (EPS)

  • Production of precursors for polysaccharide synthesis

  • Experimental approach: Comparison of wild-type and ΔnanA biofilm architecture using confocal microscopy

• Mature biofilm maintenance

  • Recycling of sialic acids from biofilm matrix

  • Contribution to nutrient availability within biofilm

  • Experimental approach: Metabolomics analysis of biofilm matrix components

• Dispersal phase

  • Potential regulation of dispersal through sialic acid sensing

  • Modification of matrix composition affecting structural integrity

  • Experimental approach: Dynamic biofilm dispersal assays

Table 7: Biofilm Phenotypes and Analysis Methods

Molecular Mechanisms Linking nanA to Biofilm Biology:

• Second messenger signaling

  • c-di-GMP levels potentially affected by sialic acid metabolism

  • Altered regulation of biofilm-associated genes

  • Experimental approach: Quantification of c-di-GMP in wild-type vs. ΔnanA strains

• Quorum sensing interactions

  • Sialic acid derivatives as potential signaling molecules

  • Cross-talk with established quorum sensing systems

  • Experimental approach: Reporter strains to monitor quorum sensing activation

• Stress response coordination

  • Role in adaptation to oxygen limitation in biofilms

  • Contribution to pH homeostasis within biofilm microenvironments

  • Experimental approach: Gene expression analysis of stress response genes

Experimental Approaches to Study nanA in Biofilms:

• Static biofilm assays

  • Microtiter plate-based assays for quantification

  • Varying growth conditions to mimic host environments

  • Inclusion of host factors (mucin, respiratory secretions)

• Flow cell systems

  • Dynamic biofilm formation under controlled shear forces

  • Real-time imaging of biofilm development

  • Testing of inhibitor compounds under flow conditions

• Ex vivo models

  • Porcine respiratory tissue explants

  • Precision-cut lung slices

  • Primary porcine respiratory epithelial cells at air-liquid interface

Understanding nanA's role in biofilm biology would provide insights into A. pleuropneumoniae persistence and potentially identify novel approaches to disrupt biofilms, addressing a key challenge in treating established infections.

• How does nanA contribute to A. pleuropneumoniae virulence in experimental infection models?

Investigating the contribution of nanA to A. pleuropneumoniae virulence requires carefully designed experimental infection models that capture the complex host-pathogen interactions occurring during natural infection. The following approaches provide a comprehensive framework for assessing nanA's role in virulence:

Genetic Manipulation Strategies:

• Gene deletion mutants

  • Clean deletion of nanA using allelic exchange

  • Complementation with wild-type nanA on plasmid or chromosomally integrated

  • Catalytic mutants (K165A) maintaining protein structure but eliminating activity

• Controlled expression systems

  • Inducible promoters to modulate nanA expression levels

  • Reporter gene fusions to monitor expression in vivo

  • Tagged variants for localization studies

In Vitro Virulence Assays:

• Adhesion to porcine respiratory epithelial cells

  • Primary cell cultures or cell lines (NPTE, NPTr)

  • Quantification of adherent bacteria by CFU counts or immunofluorescence

  • Expected result: ΔnanA may show reduced adherence to sialylated surfaces

• Cytotoxicity assessment

  • LDH release assays from infected cell cultures

  • Apoptosis markers (caspase activation, annexin V staining)

  • Evaluation of cellular morphology changes

• Resistance to innate immune factors

  • Serum resistance assays

  • Survival in presence of antimicrobial peptides

  • Neutrophil phagocytosis and killing assays

Ex Vivo Models:

• Precision-cut lung slices (PCLS)

  • Maintains tissue architecture and cell diversity

  • Allows visualization of bacterial-tissue interactions

  • Measurement of inflammatory responses (cytokine production)

• Tracheal explant cultures

  • Assessment of ciliary clearance mechanisms

  • Biofilm formation on authentic respiratory surface

  • Histopathological evaluation of tissue damage

In Vivo Experimental Models:

• Intranasal/intratracheal challenge in pigs

  • Natural host provides most relevant model

  • Competitive index experiments (wild-type vs. ΔnanA co-infection)

  • Clinical scoring, bacterial loads in tissues, histopathology

• Signature-tagged mutagenesis (STM) approach

  • Similar to technique described in search result

  • Identification of attenuation in complex pools of mutants

  • Recovery and enumeration of bacteria from different tissue sites

Table 8: Expected Virulence Phenotypes in Different Models

Molecular Analysis of Host Responses:

• Transcriptomics

  • RNA-seq of infected tissues comparing wild-type and ΔnanA infection

  • Pathway analysis to identify differentially affected processes

  • Validation of key findings by qRT-PCR

• Immunological assessment

  • Cytokine/chemokine profiling in serum and BALF

  • Flow cytometric analysis of immune cell recruitment

  • Histopathological scoring of lesion severity

This comprehensive approach to studying nanA's contribution to virulence would provide insights into its role in A. pleuropneumoniae pathogenesis and help evaluate its potential as a therapeutic target or vaccine component.

• What are the challenges and approaches for developing nanA-based vaccines against A. pleuropneumoniae?

The development of nanA-based vaccines against A. pleuropneumoniae presents both unique opportunities and significant challenges. Based on the information in search result regarding phasevarions and vaccine development, a systematic approach to nanA-based vaccine development would include:

Antigen Characterization and Validation:

• Conservation analysis

  • Sequence comparison across all 15 serotypes

  • Identification of conserved epitopes

  • Assessment of potential phase variation affecting expression

• Expression profiling

  • Verification of in vivo expression during infection

  • Evaluation of expression levels across growth phases

  • Determination if nanA is regulated by phase-variable DNA methyltransferases

• Immunogenicity assessment

  • Animal immunization with purified recombinant nanA

  • Characterization of antibody responses (titer, isotype, neutralization)

  • T cell response evaluation (proliferation, cytokine production)

Table 9: Vaccine Formulation Strategies for nanA-Based Vaccines

Vaccine Delivery Considerations:

• Route of administration

  • Intramuscular: Traditional, reliable

  • Intranasal: Mucosal immunity at site of infection

  • Aerosol: Direct delivery to respiratory tract

• Adjuvant selection

  • Must balance immunopotentiation with safety

  • Consideration of pig-specific immune responses

  • Compatibility with antigen and formulation

• Formulation stability

  • Cold chain requirements

  • Shelf-life under field conditions

  • Compatibility with existing vaccination programs

Efficacy Evaluation Pipeline:

• In vitro assessment

  • Antibody binding to whole bacteria

  • Opsonophagocytic activity

  • Neutralization of enzymatic activity

• Challenge models

  • Homologous challenge (same serotype)

  • Heterologous challenge (different serotypes)

  • Natural exposure studies in field conditions

• Correlates of protection

  • Antibody titers in serum and mucosal secretions

  • Cellular immunity markers

  • Protective efficacy parameters (clinical scores, lung lesions)

Key Challenges in nanA-Based Vaccine Development:

• Phase variation considerations

  • Search result indicates A. pleuropneumoniae encodes phase-variable methyltransferases

  • These create phasevarions affecting gene expression

  • Important to determine if nanA expression is stable or phase-variable

• Cross-serotype protection

  • 15 serotypes with varying virulence and geographical distribution

  • Need to ensure nanA-based vaccine provides broad protection

  • May require combination with other conserved antigens

• Differentiation of infected from vaccinated animals (DIVA)

  • Important for surveillance and control programs

  • May require companion diagnostic tests

  • Consider marker vaccines or unique epitope tags

From search result , we know that "characterisation of phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen." This highlights the importance of assessing whether nanA is subject to phasevarion regulation before proceeding with vaccine development.