Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Pantothenate synthetase (panC)

What is Pantothenate Synthetase (PanC) in Leptospira interrogans and why is it significant for research?

Pantothenate synthetase (PanC; EC 6.3.2.1) is an enzyme encoded by the panC gene that catalyzes the essential adenosine triphosphate (ATP)-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, including Leptospira interrogans. Pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP) .

The significance of PanC in research stems from several critical factors:

• PanC is absent in mammals but essential for bacterial growth, making it an attractive target for antimicrobial development

• Both CoA and ACP are essential cofactors for bacterial metabolism

• L. interrogans, particularly serovar Copenhageni, is a major causative agent of leptospirosis, a global zoonotic disease with significant public health impact

• The enzyme represents a potential target for developing selective inhibitors that could be effective against leptospirosis

How is the panC gene organized in the Leptospira interrogans genome?

The organization of the panC gene in L. interrogans shows distinctive characteristics compared to other bacterial species:

• Unlike many bacterial genes that are organized into operons, L. interrogans genes, including panC, are typically scattered across chromosome I

• The L. interrogans serovar Copenhageni genome consists of two circular chromosomes (chromosome I and chromosome II)

• The genome contains various metabolic pathways distributed across both chromosomes with some functional links between them

• Genome analysis reveals that L. interrogans has complex regulatory systems and signal transduction mechanisms that likely control metabolic genes like panC in response to environmental stimuli

What are the biochemical properties and enzymatic mechanism of L. interrogans PanC?

The biochemical properties of L. interrogans PanC reflect its role in bacterial metabolism:

• Enzymatic Mechanism: PanC catalyzes a two-step reaction:

  • Activation of pantoate with ATP to form pantoyl-adenylate

  • Nucleophilic attack by beta-alanine to form pantothenate with release of AMP

• Optimal Conditions: The enzyme functions optimally under conditions that match L. interrogans physiological parameters:

  • pH range: 7.2-7.6 (optimal pH 7.4), consistent with L. interrogans' neutralophilic properties

  • Temperature range: 28-30°C, reflecting the mesophilic nature of L. interrogans

• Cofactor Requirements:

  • Requires divalent metal ions (usually Mg²⁺) for ATP coordination

  • May interact with components of L. interrogans' complex metabolic network

How does the metabolic context of PanC integrate with Leptospira interrogans physiology?

PanC functions within a broader metabolic network in L. interrogans:

The pantothenate synthesis pathway is critical because L. interrogans requires long-chain fatty acids for growth and metabolism, and these processes depend on CoA, which is derived from pantothenate .

What methodologies are most effective for expressing and purifying recombinant L. interrogans PanC?

Based on current research practices, the following methodology has proven effective:

Expression System:

• E. coli expression system using BL21(DE3) or similar strains

• Expression vector with N-terminal or C-terminal His-tag for purification

• Optimal induction conditions: 0.5-1 mM IPTG at 30°C for 4-6 hours or 18°C overnight

Purification Protocol:

• Cell lysis using sonication or pressure-based methods in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

• Initial purification by Ni-NTA affinity chromatography

• Secondary purification by size exclusion chromatography

• Final polishing step using ion-exchange chromatography if needed

Quality Assessment:

• SDS-PAGE analysis for purity (>90% purity is typically achievable)

• Enzymatic activity assay measuring ATP utilization via coupled enzyme reactions

• Thermal shift assay to assess protein stability

• Dynamic light scattering to confirm monodispersity

Storage:

• Optimal storage in buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, 6% trehalose at -80°C

• Addition of glycerol (final concentration 20-50%) for preventing freeze-thaw damage

• Avoiding repeated freeze-thaw cycles to maintain enzymatic activity

How can high-throughput screening be optimized to identify inhibitors of L. interrogans PanC?

Optimizing high-throughput screening for PanC inhibitors involves several key approaches:

Enzymatic Assay Development:

• A coupled enzymatic reaction can be used to monitor PanC activity by measuring the reduction in absorbance at 340 nm due to NADH oxidation

• This coupled assay involves:

  • PanC reaction producing AMP from ATP

  • Myokinase converting AMP to ADP

  • Pyruvate kinase converting ADP to ATP with phosphoenolpyruvate

  • Lactate dehydrogenase converting pyruvate to lactate with NADH oxidation

Assay Optimization Parameters:

• Signal-to-background ratio optimization: ≥3:1

• Z'-factor: ≥0.7 for robust screening

• DMSO tolerance: up to 2% final concentration

• Reaction components optimization:

  • Enzyme concentration: typically 10-50 nM

  • Substrate concentrations: at or slightly below Km values

  • Buffer composition: typically 50 mM HEPES pH 7.5, 10 mM MgCl₂, 50 mM KCl

Screening Strategy:

• Primary screen at single concentration (10-20 μM)

• Confirmation of hits in dose-response format

• Counter-screening against coupling enzymes to eliminate false positives

• Evaluation of potential inhibitors in whole-cell assays against L. interrogans

Hit Evaluation:

• IC₅₀ determination for promising compounds

• Mechanism of inhibition studies (competitive, noncompetitive, uncompetitive)

• Structure-activity relationship analysis

• Assessment of selectivity against mammalian enzymes

This approach has successfully identified inhibitor classes such as 3-biphenyl-4-cyanopyrrole-2-carboxylic acids for Mycobacterium tuberculosis PanC, which could inform similar work on L. interrogans PanC .

How does PanC contribute to the pathogenesis of L. interrogans infection?

While PanC is not a classical virulence factor, its contribution to L. interrogans pathogenesis is multifaceted:

Metabolic Support for Pathogenesis:

• PanC enables CoA synthesis, which is essential for fatty acid metabolism - the primary energy source for L. interrogans during infection

• L. interrogans' ability to survive in host tissues depends on essential metabolic pathways supported by PanC

Relationship to Virulence Mechanisms:

• L. interrogans has a complex array of virulence factors including LigA and LigB proteins, LipL32, and Loa22

• While PanC doesn't directly interact with these virulence factors, it provides the metabolic foundation for their expression and function

• The energy derived from CoA-dependent pathways supports motility via the periplasmic flagella, which is crucial for bacterial invasion and dissemination

Host-Pathogen Interaction Context:

• L. interrogans infection triggers host immune responses including TLR2 and TLR4 activation

• The metabolic activity supported by PanC enables the bacteria to persist despite these immune responses

• L. interrogans can cause biphasic illness with both anicteric and icteric phases, requiring sustained metabolic activity throughout infection

Research indicates that metabolic enzymes like PanC represent potential vulnerability points in bacterial pathogens, as they are essential for survival during infection but are absent in mammalian hosts .

How does PanC from L. interrogans serovar Copenhageni compare with PanC from other Leptospira species and serovars?

Comparative analysis of PanC across Leptospira species reveals important distinctions:

Interspecies Comparison:

Serovar-Level Variations:

Evolutionary Considerations:

• Pathogenic Leptospira species form a monophyletic group distinct from non-pathogenic species

• L. interrogans serovar Copenhageni is closely related to L. interrogans serovar Lai and L. kirschneri

• Unlike L. borgpetersenii, which shows genome reduction and increased host dependence, L. interrogans maintains more metabolic flexibility

What are the implications of targeting PanC for therapeutic development against leptospirosis?

Targeting PanC for therapeutic development presents both opportunities and challenges:

Advantages:

• PanC is absent in mammals, minimizing potential off-target effects

• It is essential for bacterial growth and survival of L. interrogans

• The enzyme is part of a conserved metabolic pathway, allowing for potential broad-spectrum activity

• High-throughput screening methods have been successfully developed for PanC inhibitor discovery

Challenges:

• L. interrogans has a complex double membrane structure that may limit inhibitor access

• The bacterium possesses numerous efflux mechanisms as part of its transport system

• Achieving appropriate tissue distribution to target L. interrogans in infected kidneys and other tissues

• Developing inhibitors that can function in the physiological conditions where L. interrogans thrives

Potential Approaches:

• Structure-based drug design using recombinant L. interrogans PanC

• Development of prodrugs that can penetrate bacterial membranes

• Combination therapy with agents that increase membrane permeability

• Exploration of natural products with anti-leptospiral activity as starting points

Current Status:

• PanC inhibitors have been identified for other bacterial species such as Mycobacterium tuberculosis

• These include 3-biphenyl-4-cyanopyrrole-2-carboxylic acids that show activity against both the enzyme and whole bacteria

• Similar approaches could be adapted for L. interrogans PanC, though species-specific optimization would be necessary

• Current leptospirosis treatment relies on antibiotics like penicillin and doxycycline

How can recombinant L. interrogans PanC be utilized in diagnostic applications for leptospirosis?

Recombinant L. interrogans PanC has potential applications in leptospirosis diagnostics:

Current Diagnostic Landscape:

• The microscopic agglutination test (MAT) is the gold standard but has limitations including reduced sensitivity in early disease

• PCR methods are effective in early disease but require specialized equipment

• Current recombinant antigen-based diagnostics utilize proteins like LipL32, LigA/B, and GroEL

Potential Applications of Recombinant PanC:

• Serological Assays:

  • ELISA-based detection of anti-PanC antibodies in patient sera

  • Integration into multi-antigen panels to improve sensitivity and specificity

  • Development of rapid lateral flow assays for point-of-care testing

• Protein Microarray Technology:

  • Inclusion of PanC in protein microarray panels containing multiple leptospiral antigens

  • Similar approaches with other leptospiral proteins have identified antigens that can distinguish between acute and convalescent cases

• Chimeric Protein Constructs:

  • Development of chimeric multi-epitope proteins incorporating PanC epitopes along with other immunogenic regions

  • Similar approaches have shown promise, as with the rChi2 chimeric protein that demonstrated 75% and 82.5% responder rates for MAT-negative and MAT-positive samples, respectively

Implementation Considerations:

• Validation with serum panels from confirmed leptospirosis cases and appropriate controls

• Assessment of cross-reactivity with other infectious diseases (dengue, malaria, etc.)

• Determination of sensitivity in different phases of infection

• Evaluation of serovar cross-reactivity across different Leptospira interrogans serovars

Research on other leptospiral antigens has demonstrated that recombinant proteins can achieve high specificity (90-97%) in distinguishing leptospirosis from other conditions, suggesting similar potential for properly characterized recombinant PanC .

What experimental approaches can assess the immunogenicity of recombinant L. interrogans PanC?

Comprehensive assessment of recombinant PanC immunogenicity requires multiple complementary approaches:

In Vitro Immunological Assays:

• Antibody Recognition Studies:

  • Western blotting using sera from leptospirosis patients to detect recognition of recombinant PanC

  • ELISA to quantify antibody binding and determine titers

  • Epitope mapping to identify immunodominant regions

• Cell-Based Assays:

  • Peripheral blood mononuclear cell (PBMC) stimulation to assess T-cell responses

  • Cytokine profiling following stimulation with recombinant PanC

  • Antigen presentation assays with dendritic cells

Animal Immunization Studies:

• Protocol Design:

  • Immunization of hamsters or other appropriate animal models with purified recombinant PanC

  • Typical regimen: primary immunization followed by booster at 3 weeks

  • Adjuvant selection (Alhydrogel has been successful with other leptospiral proteins)

• Immunological Evaluation:

  • Measurement of total IgG responses at multiple timepoints

  • Assessment of antibody avidity maturation

  • Analysis of antibody recognition of native PanC in L. interrogans lysates

  • Evaluation of antibody cross-reactivity with different Leptospira species and serovars

• Protection Assessment:

  • Challenge studies with virulent L. interrogans

  • Monitoring survival, bacterial burden, and histopathological changes

  • Evaluation of sterilizing immunity using culture and PCR methods

This approach follows similar methodologies used successfully with other leptospiral proteins such as LigA, LigB, and chimeric constructs like rChi2 .

How should structure-function studies of L. interrogans PanC be designed?

Structure-function studies should combine multiple experimental approaches:

Structural Analysis:

• X-ray Crystallography:

  • Crystallization of purified recombinant PanC (apo-enzyme)

  • Co-crystallization with substrates, product, and potential inhibitors

  • Analysis of active site architecture and substrate binding determinants

• Alternative Structural Methods:

  • Nuclear Magnetic Resonance (NMR) for dynamic studies

  • Cryo-electron microscopy for larger complexes

  • Small-angle X-ray scattering (SAXS) for solution conformation

Functional Characterization:

• Enzyme Kinetics:

  • Determination of key kinetic parameters (Km, kcat, kcat/Km) for both substrates

  • Assessment of potential allosteric regulation

  • pH and temperature dependence profiles

  • Metal ion requirements and effects

• Mutagenesis Studies:

  • Site-directed mutagenesis of catalytic residues

  • Alanine-scanning of substrate binding pocket

  • Creation of chimeric enzymes with PanC from other species

Molecular Dynamics and Computational Approaches:

• Simulation Studies:

  • Molecular dynamics simulations to analyze protein flexibility

  • Substrate binding and product release pathways

  • Identification of potential allosteric sites

• Virtual Screening:

  • Structure-based virtual screening for potential inhibitors

  • Docking studies to predict binding modes

  • Quantum mechanical/molecular mechanical (QM/MM) studies of reaction mechanism

Correlation with Biological Function:

• Gene Knockout/Complementation:

  • Generation of L. interrogans panC mutants (if technically feasible)

  • Complementation studies with wild-type and mutant versions

  • Assessment of impact on bacterial physiology and virulence

• Expression Analysis:

  • Examination of panC expression under various environmental conditions

  • Effect of host factors on expression levels

  • Correlation with expression of related metabolic genes

What are the key considerations for developing selective inhibitors of L. interrogans PanC?

Development of selective PanC inhibitors requires a systematic approach:

Target Validation and Assay Development:

• Biochemical Validation:

  • Confirmation that L. interrogans PanC is essential for bacterial growth

  • Development of reliable enzymatic assays for inhibitor screening

  • Establishment of appropriate positive controls and assay parameters

• Assay Cascade Design:

  • Primary biochemical screen

  • Secondary whole-cell activity assessment

  • Selectivity against human enzymes

  • ADME and toxicity evaluation

Inhibitor Discovery Strategies:

• High-Throughput Screening:

  • Diverse compound libraries (>100,000 compounds)

  • Fragment-based screening approaches

  • Natural product libraries

• Structure-Based Design:

  • Utilization of X-ray crystal structures

  • Virtual screening of in silico libraries

  • Fragment growing/linking strategies

  • Structure-activity relationship development

Optimization Parameters:

• Potency Considerations:

  • IC₅₀ < 1 μM against purified enzyme

  • MIC < 10 μg/mL against L. interrogans

  • Selectivity index >50 (ratio of cytotoxicity to antibacterial activity)

• Physicochemical Properties:

  • Compliance with Lipinski's rules for oral bioavailability

  • Optimization for penetration of the leptospiral outer membrane

  • Stability in physiological conditions

• Pharmacokinetic Considerations:

  • Distribution to tissues where Leptospira localizes (particularly kidneys)

  • Appropriate half-life to maintain effective concentrations

  • Limited metabolism and excretion

This approach has been successful for M. tuberculosis PanC, where 3-biphenyl-4-cyanopyrrole-2-carboxylic acids were identified as potent inhibitors with whole-cell activity . A similar methodology could be adapted for L. interrogans PanC.

How does the genomic context of panC in L. interrogans inform research approaches?

The genomic context provides important insights for research design:

Genomic Organization:

• Unlike many bacteria, leptospiral genes including panC are not organized into operons but are scattered across chromosome I

• This dispersed organization may indicate unique regulatory mechanisms that should be considered in expression studies

• Comparative genomics between L. interrogans and other spirochetes like Borrelia and Treponema can highlight important differences in metabolic organization

Regulatory Considerations:

• Promoter Analysis:

  • Identification of potential regulatory elements in the panC promoter region

  • Analysis of binding sites for known transcription factors

  • Reporter gene assays to assess promoter activity under different conditions

• Expression Coordination:

  • Investigation of potential co-regulation with other metabolic genes

  • RNA-seq analysis under various environmental conditions

  • Correlation with expression of other genes in the CoA biosynthetic pathway

Evolutionary Context:

• Phylogenetic Analysis:

  • Comparison of panC sequences across Leptospira species and serovars

  • Assessment of selection pressure on different domains

  • Correlation with host range and pathogenicity

• Genome Reduction Analysis:

  • Unlike L. borgpetersenii, which shows genome reduction and increased pseudogenes , L. interrogans maintains metabolic flexibility

  • This suggests that metabolic genes like panC may be particularly important for L. interrogans' lifestyle

Functional Genomics Approaches:

• Transposon Mutagenesis:

  • Assessment of panC essentiality through transposon insertion site analysis

  • Identification of genetic interactions through synthetic lethality screens

  • Conditional expression systems to regulate panC levels

• CRISPR-Cas Systems:

  • L. interrogans possesses CRISPR-Cas systems that could potentially be adapted for genetic manipulation

  • Development of CRISPR-based methods for precise genomic editing

  • Use of CRISPRi for controlled downregulation of panC expression

The genomic context analysis helps inform experimental design by highlighting L. interrogans-specific features that distinguish it from model organisms where PanC has been studied previously.

What methodological approaches are best for evaluating interactions between PanC and the host immune system?

Evaluating PanC-host immune interactions requires multiple complementary approaches:

Antigen Presentation and Recognition:

• B-cell Epitope Mapping:

  • Peptide array analysis to identify immunodominant epitopes

  • Phage display libraries to define antibody binding sites

  • Structural analysis of antibody-antigen complexes

• T-cell Response Analysis:

  • Identification of MHC-binding epitopes using prediction algorithms and validation assays

  • T-cell proliferation assays with synthetic peptides

  • Cytokine profiling to characterize Th1/Th2/Th17 responses

Animal Model Studies:

• Immune Response Characterization:

  • Detailed analysis of antibody isotypes and subclasses following immunization

  • Assessment of cell-mediated immunity through adoptive transfer experiments

  • Cytokine responses in different tissues following challenge

• Protection Mechanisms:

  • Passive transfer of anti-PanC antibodies to evaluate protection

  • Depletion of specific immune cell populations to determine their contribution

  • Correlation of immune parameters with protection against challenge

Human Studies:

• Patient Sample Analysis:

  • Screening of sera from leptospirosis patients for anti-PanC antibodies

  • Comparison of acute and convalescent phases

  • Correlation with disease severity and outcomes

• Cross-reactivity Assessment:

  • Evaluation of potential cross-reactivity with human proteins

  • Assessment of autoimmune potential

  • Analysis of cross-reactivity with other bacterial PanC enzymes

Immunomodulatory Effects:

• Innate Immune Response:

  • Assessment of interaction with pattern recognition receptors

  • Analysis of dendritic cell maturation and antigen presentation

  • Evaluation of NK cell activation

• Adaptive Immune Programming:

  • Impact on T-cell differentiation pathways

  • Effects on B-cell affinity maturation

  • Long-term memory formation

Similar approaches with other leptospiral antigens have demonstrated their utility in understanding immune responses to proteins such as LigA/B, LipL32, and chimeric constructs .