Recombinant Haemophilus ducreyi 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (fabA)

What is the role of FabA in Haemophilus ducreyi fatty acid biosynthesis?

FabA (3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase) is a key enzyme in the bacterial type II fatty acid synthesis (FASII) pathway, particularly for unsaturated fatty acid biosynthesis. In bacteria like H. ducreyi, FabA catalyzes both the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP and isomerization to cis-3-acyl-ACP, which is essential for the production of unsaturated fatty acids. Similar to other dehydratases such as FabZ that catalyze dehydration reactions in the FASII pathway, FabA plays a critical role in membrane lipid composition, which affects bacterial survival in different environmental conditions .

The FASII pathway is distinct from the mammalian fatty acid synthesis system, making FabA a potential antimicrobial target. While the specific contribution of FabA to H. ducreyi pathogenesis hasn't been fully characterized, its function is expected to be essential for bacterial membrane formation and adaptation to host environments.

How does recombinant H. ducreyi FabA compare structurally to FabZ?

Both FabA and FabZ are β-hydroxyacyl-ACP dehydratases that share functional similarities but have distinct structural characteristics. While they both catalyze dehydration reactions in fatty acid biosynthesis, they differ in substrate specificity and catalytic properties.

Based on crystallographic studies of similar enzymes, FabA typically forms dimers with a "hot dog" fold structure, whereas FabZ often forms hexamers (trimers of dimers) as observed in the Candidatus liberibacter FabZ . Each monomer contains a catalytic histidine residue essential for the dehydration reaction. FabA possesses an additional catalytic ability to isomerize trans-2-acyl-ACP to cis-3-acyl-ACP, which is crucial for unsaturated fatty acid synthesis and is not typically performed by FabZ.

A more detailed structural comparison would require specific crystallographic data of H. ducreyi FabA, which could be obtained through methods similar to those used for C. liberibacter FabZ crystallization (2% v/v Tacsimate pH 5.0, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, 16% w/v polyethylene glycol 3350) .

What expression systems are most effective for producing recombinant H. ducreyi FabA?

For recombinant expression of H. ducreyi FabA, E. coli-based expression systems are typically most effective, particularly BL21(DE3) or derivatives optimized for membrane-associated protein expression. The process generally involves:

• Cloning strategy: The fabA gene from H. ducreyi genomic DNA can be amplified using PCR with specific primers and cloned into expression vectors containing appropriate promoters (T7, tac, or arabinose-inducible).

• Expression tags: Adding an N-terminal or C-terminal His-tag facilitates purification via nickel affinity chromatography. For crystallography studies, tags may need to be removed using specific proteases.

• Growth conditions: Expression is typically optimized at lower temperatures (16-25°C) after induction to enhance proper folding of the recombinant protein.

• Solubilization: As FabA interacts with membrane components, optimization of lysis buffers may be required, potentially including mild detergents or specialized solubilization methods.

Following an approach similar to that used for C. liberibacter FabZ, the protein can be purified to homogeneity using a combination of affinity chromatography and size exclusion chromatography, with protein concentrations of approximately 7 mg/mL for crystallization studies .

How might FabA contribute to H. ducreyi virulence and host adaptation?

H. ducreyi is an obligate human pathogen causing both cutaneous and genital ulcers, requiring adaptation to multiple host environments . While direct evidence for FabA's role in virulence is not explicitly described in the provided search results, several hypotheses can be formulated based on its function:

• Membrane adaptation: FabA-mediated unsaturated fatty acid synthesis likely contributes to membrane fluidity adjustments required for survival in different host microenvironments, including temperature adaptation and resistance to host antimicrobial peptides.

• Stress response integration: The Cpx two-component signal transduction system regulates numerous virulence determinants in H. ducreyi . Fatty acid composition, potentially influenced by FabA activity, could be regulated in response to environmental stressors through integration with the Cpx pathway.

• Metabolic adaptation: Similar to the significance of formate metabolism in H. ducreyi , fatty acid biosynthesis represents a central metabolic pathway that likely undergoes regulation during infection. This metabolism may be particularly important in the anaerobic or microaerobic environment of abscesses and ulcers.

A comprehensive transcriptomic analysis similar to that performed for the Cpx regulon would be valuable to determine if fabA expression changes during infection or in response to host factors .

What are the methodological approaches for assessing FabA inhibition in H. ducreyi?

Developing inhibition assays for H. ducreyi FabA requires multiple complementary approaches:

• In vitro enzyme assays:

  • Spectrophotometric assays measuring the formation of trans-2-decenoyl-ACP at 260 nm

  • Coupling assays with FabI to monitor NADH consumption at 340 nm

  • Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to directly monitor substrate conversion

• Whole-cell assays:

  • Minimum inhibitory concentration (MIC) determination against H. ducreyi strains

  • Growth curve analysis under various conditions

  • Fatty acid profiling using gas chromatography-mass spectrometry (GC-MS)

• Target validation approaches:

  • Construction of conditional fabA mutants (as complete knockouts may be lethal)

  • Complementation studies with wild-type and mutant variants

  • Overexpression studies to confirm mechanism of action for putative inhibitors

• Structural studies for rational design:

  • Co-crystallization with inhibitors

  • Molecular docking and virtual screening

  • Structure-activity relationship (SAR) studies with synthesized analogs

The combination of these approaches would provide comprehensive validation of FabA as an antimicrobial target and facilitate inhibitor development.

How does H. ducreyi FabA activity differ under various microenvironmental conditions relevant to infection?

H. ducreyi encounters varied microenvironments during infection, from initial attachment to progression within suppurative granulomas . The activity of FabA likely adapts to these conditions:

Experimental approaches to investigate these variations should include:

• Enzyme kinetics studies under varying pH, temperature, and oxygen conditions

• Membrane fatty acid profiling of H. ducreyi isolated from different infection stages

• Transcriptomic analysis comparing fabA expression across infection microenvironments

These studies would provide insight into how FabA activity is regulated during infection and potentially identify conditions where inhibitors would be most effective.

What are the optimal conditions for crystallizing recombinant H. ducreyi FabA?

Based on the successful crystallization of related β-hydroxyacyl-ACP dehydratases, the following conditions would serve as starting points for H. ducreyi FabA crystallization:

• Protein preparation:

  • Highly purified protein (>95% purity by SDS-PAGE)

  • Concentration range of 5-10 mg/mL

  • Buffer conditions: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol

• Crystallization screening:

  • Initial broad screening using commercial sparse matrix screens

  • Targeted optimization based on conditions successful for C. liberibacter FabZ: 2% v/v Tacsimate pH 5.0, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 16% w/v polyethylene glycol 3350 at 20°C

  • Vapor diffusion methods (hanging drop and sitting drop)

• Co-crystallization approaches:

  • With ACP substrate analogs

  • With potential inhibitors

  • With cofactors or stabilizing agents

• Cryo-protection:

  • Testing various cryoprotectants (glycerol, ethylene glycol, PEG 400)

  • Flash-cooling in liquid nitrogen

For phasing, selenomethionine substitution would be appropriate, similar to the approach used for C. liberibacter FabZ . X-ray diffraction data collection at synchrotron sources would allow for high-resolution structural determination.

What methods are most effective for quantifying H. ducreyi FabA enzymatic activity?

Several complementary methods can be employed to quantify H. ducreyi FabA enzymatic activity:

• Direct spectrophotometric assay:

  • Monitors increase in absorbance at 260 nm due to formation of the trans-2-enoyl-ACP product

  • Advantage: Real-time continuous measurement

  • Limitation: Relatively low sensitivity requiring higher enzyme concentrations

• Coupled enzyme assay:

  • Links FabA activity to NADH oxidation via FabI (enoyl-ACP reductase)

  • Monitors decrease in absorbance at 340 nm

  • Advantage: Higher sensitivity than direct assay

  • Limitation: Potential for false positives from inhibitors affecting coupling enzyme

• Chromatographic analysis:

  • HPLC or LC-MS methods to separate and quantify substrate and product

  • Advantage: Direct quantification of actual reaction products

  • Limitation: Discontinuous assay requiring sample processing

• Radiochemical assay:

  • Using radiolabeled substrates and measuring product formation

  • Advantage: Highest sensitivity for kinetic measurements

  • Limitation: Requires specialized facilities for radioactive materials

A typical reaction buffer would contain 100 mM sodium phosphate (pH 7.0), 50 mM NaCl, and 0.1 mM DTT, with substrate concentrations ranging from 10-200 μM for kinetic studies. Temperature optimization would likely show maximum activity between 30-37°C, reflecting the pathogen's adaptation to the human host.

How can RNA-Seq approaches be applied to understand the regulation of fabA expression in H. ducreyi?

RNA-Seq provides powerful insights into gene expression and regulation. For studying fabA regulation in H. ducreyi, the following approach would be effective:

• Experimental design:

  • Compare transcriptomes across growth phases (similar to the approach used for CpxRA studies)

  • Include wild-type H. ducreyi and relevant regulatory mutants (e.g., cpxR, cpxA)

  • Examine expression under various environmental conditions (pH, temperature, oxygen)

  • Analyze expression during infection using human infection models or relevant in vitro systems

• Technical considerations:

  • RNA extraction must be optimized for H. ducreyi, which can be challenging due to its fastidious nature

  • rRNA depletion rather than poly(A) selection for prokaryotic samples

  • Strand-specific library preparation for detecting antisense transcription

  • Minimum sequencing depth of 10-20 million reads per sample for adequate coverage

• Bioinformatic analysis pipeline:

  • Quality control and trimming of raw reads

  • Alignment to the H. ducreyi genome (GenBank accession no. AE017143)

  • Transcript quantification and normalization

  • Differential expression analysis

  • Regulatory motif identification similar to the CpxR binding motif analysis

• Validation approaches:

  • Quantitative RT-PCR for key findings

  • Promoter reporter fusion assays

  • Electrophoretic mobility shift assays (EMSA) to identify regulatory proteins

  • ChIP-Seq for direct identification of transcription factor binding sites

This comprehensive approach would reveal how fabA expression is regulated in response to environmental stimuli and potentially identify novel regulatory mechanisms that could be targeted for antimicrobial development.

How does the regulation of fabA intersect with known virulence regulatory systems in H. ducreyi?

The integration of fatty acid biosynthesis with virulence regulation in H. ducreyi likely occurs through multiple mechanisms:

• CpxRA two-component system: The CpxRA system is the only obvious intact two-component signal transduction system in the H. ducreyi genome and functions primarily as a repressor of virulence determinants . While fabA has not been specifically identified as part of the CpxR regulon, the regulation of membrane composition would be a logical connection point between stress responses and metabolic adaptation.

• Metabolic adaptation during infection: Similar to the upregulation of formate metabolism genes (focA, pflB) observed in pustules during human infection , fatty acid biosynthesis genes likely undergo expression changes in response to the host environment. These adaptations would be crucial for bacterial survival in the suppurative granuloma-like niche characteristic of H. ducreyi infections .

• Integration with phagocytosis resistance mechanisms: H. ducreyi has multiple mechanisms to avoid phagocytosis, including LspA1, LspA2, DsrA, and FgbA . Membrane composition, influenced by FabA activity, could affect the presentation and function of these surface-exposed virulence factors.

To experimentally investigate these intersections, researchers should consider:

• Comparative transcriptomics of wild-type and regulatory mutants under infection-relevant conditions

• Protein-protein interaction studies to identify potential direct interactions

• Membrane composition analysis in regulatory mutants

What is the potential of H. ducreyi FabA as a drug target compared to other enzymes in fatty acid biosynthesis?

The potential of H. ducreyi FabA as a drug target can be evaluated against several criteria:

FabA offers several advantages as a drug target:

• Its dual dehydratase/isomerase activity is unique and essential for unsaturated fatty acid biosynthesis

• The "hot dog" fold structure is amenable to structure-based drug design

• Its absence in humans reduces the likelihood of off-target effects

• Potential functional redundancy with FabZ for dehydration (but not isomerization)

• The hydrophobic active site may require specialized medicinal chemistry approaches

• Limited existing chemical scaffolds known to target FabA

A comprehensive target validation approach would include:

• Construction of conditional mutants to confirm essentiality

• Whole-cell activity assays with potential inhibitors

• In vivo efficacy testing in appropriate infection models

How can isotope labeling be used to track fatty acid biosynthesis in H. ducreyi during infection?

Isotope labeling provides powerful tools to track metabolic pathways during infection:

• Ex vivo stable isotope probing:

  • Culture H. ducreyi with 13C-labeled substrates (glucose, acetate)

  • Extract bacteria from infection models

  • Analyze 13C incorporation into fatty acids using GC-MS or LC-MS

  • Determine flux through FabA by examining labeled unsaturated fatty acids

• In vivo deuterium labeling:

  • Administer D2O (heavy water) to experimental models

  • Extract bacteria from infection sites

  • Analyze deuterium incorporation into newly synthesized fatty acids

  • Calculate synthesis rates based on isotope enrichment

• Pulse-chase experiments:

  • Pulse with labeled precursors before infection

  • Chase during infection process

  • Track label dilution to determine synthesis and turnover rates

• Multi-omics integration:

  • Combine isotope labeling with transcriptomics and proteomics

  • Create comprehensive models of metabolic adaptation

  • Identify regulatory nodes controlling fatty acid biosynthesis during infection

This approach would provide unprecedented insights into how H. ducreyi adapts its membrane composition during infection and could identify specific stages where FabA activity is critical for pathogen survival.

What novel techniques could advance our understanding of FabA structure-function relationships?

Several cutting-edge techniques could significantly advance our understanding of H. ducreyi FabA:

• Cryo-electron microscopy (Cryo-EM):

  • Visualization of FabA in complex with ACP without crystallization

  • Potential to capture different conformational states during catalysis

  • Resolution now approaching X-ray crystallography

• Time-resolved X-ray crystallography:

  • Capturing catalytic intermediates using rapid mixing and X-ray free electron lasers

  • Providing dynamic insights into the isomerization mechanism

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Mapping protein dynamics and conformational changes upon substrate binding

  • Identifying allosteric sites not evident in static structures

• Single-molecule FRET studies:

  • Real-time observation of conformational changes during catalysis

  • Determining the sequence of binding events with ACP and substrate

• AlphaFold2 and molecular dynamics simulations:

  • Predicting structures of FabA in complex with various substrates or inhibitors

  • Simulating the complete catalytic cycle

  • Identifying potential allosteric regulatory sites

These techniques would provide unprecedented insights into the unique dual functionality of FabA and guide rational drug design efforts targeting this enzyme.

How might CRISPR-based approaches be applied to study fabA function in H. ducreyi?

While CRISPR-Cas systems have revolutionized genetic manipulation in many organisms, their application to H. ducreyi requires careful adaptation:

• CRISPR interference (CRISPRi) for conditional knockdown:

  • Express catalytically inactive Cas9 (dCas9) with guide RNAs targeting fabA

  • Create tunable repression of fabA expression

  • Particularly valuable if fabA is essential and cannot be completely deleted

  • Requires optimization of dCas9 expression in H. ducreyi

• CRISPR-Cas9 recombineering:

  • Generate precise mutations in fabA to disrupt specific functions

  • Separate dehydratase from isomerase activity through targeted mutations

  • Create reporter fusions at the native locus

• Base editing approaches:

  • Introduce specific point mutations without double-strand breaks

  • Particularly useful for studying catalytic residues

  • Reduced cellular toxicity compared to standard CRISPR-Cas9

• Multiplex gene regulation:

  • Simultaneously modulate fabA with other fatty acid biosynthesis genes

  • Identify synthetic lethal interactions

  • Map genetic networks integrated with fatty acid biosynthesis

Implementation challenges include:

• Developing efficient transformation methods for H. ducreyi

• Optimizing promoters for Cas9/dCas9 expression

• Designing effective guide RNAs with minimal off-target effects

What transcriptional regulatory elements control fabA expression in H. ducreyi?

Understanding the transcriptional regulation of fabA in H. ducreyi would provide insights into how fatty acid biosynthesis is coordinated with other cellular processes:

• Promoter architecture analysis:

  • Identification of -10 and -35 elements

  • Mapping transcription start sites using 5' RACE or RNA-Seq

  • Identifying potential regulatory protein binding sites

  • Comparison with known CpxR binding motifs

• Transcription factor identification:

  • DNA affinity chromatography using the fabA promoter region

  • Electrophoretic mobility shift assays (EMSA) with candidate regulators

  • Bacterial one-hybrid screens to identify novel regulators

  • ChIP-Seq to map genome-wide binding of identified regulators

• Environmental responsiveness:

  • Promoter reporter fusions to monitor expression under various conditions

  • Correlating expression patterns with membrane composition changes

  • Identifying specific stimuli that modulate fabA transcription

• Small RNA regulation:

  • Identification of potential sRNA regulators using computational prediction

  • Validation through overexpression and deletion studies

  • Direct binding assays to confirm interactions

This comprehensive analysis would reveal how H. ducreyi coordinates fatty acid biosynthesis with other cellular processes during infection and adaptation to environmental stresses.