Recombinant Vibrio vulnificus 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (fabA)

What is the biological function of 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (fabA) in Vibrio vulnificus?

The fabA enzyme in V. vulnificus catalyzes a key step in unsaturated fatty acid biosynthesis, specifically the dehydration of 3-hydroxydecanoyl-ACP to trans-2-decenoyl-ACP and the isomerization to cis-3-decenoyl-ACP. This dual dehydratase/isomerase activity is essential for membrane phospholipid synthesis, enabling V. vulnificus to maintain appropriate membrane fluidity in response to environmental conditions. Given that V. vulnificus is a pathogen that inhabits brackish water environments with varying salinity and temperature levels, fabA likely plays a critical role in adapting to these changing conditions, similar to how other virulence factors in V. vulnificus undergo genetic variations to adapt to different environments .

How does fabA expression correlate with Vibrio vulnificus pathogenicity?

While direct correlations between fabA and pathogenicity haven't been extensively characterized in V. vulnificus, fatty acid biosynthesis enzymes like fabA are essential for bacterial survival and can influence virulence indirectly. V. vulnificus is known for its high pathogenicity, with approximately 1 in 5 infections resulting in death according to CDC data . The bacterium produces multiple virulence factors, including the MARTX toxin encoded by the rtxA1 gene, which exhibits genetic variation among strains . The relationship between fabA activity and expression of virulence factors represents an important area for investigation, particularly as V. vulnificus demonstrates genetic recombination and variation in virulence-associated genes over time .

What are the optimal expression systems for recombinant Vibrio vulnificus fabA production?

For recombinant V. vulnificus fabA expression, E. coli-based systems typically provide the highest yields with minimal toxicity. The BL21(DE3) strain is preferred due to its deficiency in lon and ompT proteases, reducing degradation of the target protein. Expression optimization should consider:

• Vector selection: pET series vectors with T7 promoter systems offer strong, inducible expression

• Codon optimization: Adjusting codons for E. coli preference may increase expression levels

• Growth conditions: Lower temperatures (16-25°C) after induction often improve soluble protein yield

• Induction parameters: IPTG concentration (0.1-1.0 mM) and induction time (3-18 hours) require optimization

Including a purification tag (His6, GST, or MBP) facilitates downstream purification while potentially enhancing solubility. For membrane-associated proteins like fabA, addition of detergents (0.1-1% Triton X-100 or n-dodecyl-β-D-maltoside) during cell lysis may improve extraction efficiency.

What purification strategies yield the highest purity recombinant fabA protein?

A multi-step purification protocol for recombinant V. vulnificus fabA typically yields >95% purity:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged fabA

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-300 mM imidazole gradient

  • Flow rate: 1-2 ml/min for optimal binding

• Intermediate purification: Ion exchange chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 0-500 mM NaCl gradient

  • Column selection based on fabA theoretical pI

• Polishing step: Size exclusion chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl

  • Column: Superdex 75 or 200, depending on protein size

Throughout purification, protein stability should be monitored using dynamic light scattering and thermal shift assays. Addition of glycerol (5-10%) and reducing agents (1-5 mM DTT or β-mercaptoethanol) often enhances stability. Similar multi-step purification approaches have been successfully applied to purify other recombinant proteins from V. vulnificus for structural and functional studies.

How can researchers troubleshoot low solubility issues with recombinant fabA?

Low solubility is a common challenge with recombinant fabA expression. Research-based solutions include:

• Fusion partners: MBP or SUMO tags can significantly enhance solubility

  • Cleavage sites (TEV or SUMO protease) should be incorporated for tag removal

  • Post-cleavage stability should be assessed by thermal shift assays

• Chaperone co-expression: GroEL/ES, DnaK/J, or trigger factor

  • Commercial chaperone plasmid sets are available with compatible selection markers

  • Induce chaperones prior to target protein expression

• Solubilizing additives during purification:

  • Detergents: 0.05-0.1% n-dodecyl-β-D-maltoside or CHAPS

  • Osmolytes: 0.5-1 M arginine, 5-10% glycerol, or 0.5-2 M urea

• Refolding protocols (if inclusion bodies form):

  • Solubilization: 6-8 M urea or 6 M guanidine hydrochloride

  • Refolding: Step-wise dialysis or rapid dilution methods

  • Monitoring refolding by circular dichroism to confirm secondary structure formation

For particularly challenging cases, high-throughput screening of different buffer conditions using techniques similar to those employed in RAA-CRISPR/Cas12a optimization for V. vulnificus detection can help identify optimal solubility conditions .

What are the key structural features of V. vulnificus fabA compared to other bacterial fabA homologs?

While V. vulnificus fabA-specific structural data is limited, comparative analysis with other bacterial fabA homologs (particularly from E. coli and P. aeruginosa) reveals several conserved features:

Potential V. vulnificus-specific features may include:

• Surface residue variations affecting substrate specificity or environmental adaptation

• Altered dimer interface affecting stability under different salt conditions

• Regulatory sites responsive to environmental cues relevant to V. vulnificus lifestyle

Structural characterization techniques should include:

• X-ray crystallography at 1.5-2.5 Å resolution

• Circular dichroism for secondary structure analysis

• Differential scanning fluorimetry to assess thermal stability

• Small-angle X-ray scattering for solution structure determination

Comparison with fabA structures from non-pathogenic Vibrio species could reveal adaptations specific to the pathogenic lifestyle of V. vulnificus, similar to how genomic comparisons have identified virulence-associated variations in other V. vulnificus genes .

How can researchers accurately measure fabA enzymatic activity and inhibition?

Accurate measurement of V. vulnificus fabA dehydratase/isomerase activity requires multi-faceted approaches:

Direct Activity Assays:

• Spectrophotometric assay tracking absorbance at 263 nm (for trans-2-enoyl-ACP formation)

  • Reaction buffer: 100 mM sodium phosphate pH 7.0, 50 mM NaCl

  • Temperature: 30°C (standard) and 37°C (physiological)

  • Substrate range: C8-C12 3-hydroxyacyl-ACPs at 10-100 μM

• Coupled assay with fabI (enoyl-ACP reductase)

  • Monitor NADH oxidation at 340 nm

  • Allows distinction between dehydration and isomerization activities

• LC-MS analysis of reaction products

  • Provides direct measurement of substrate consumption and product formation

  • Can distinguish between cis- and trans-isomers

Inhibition Studies:

• IC50 determination for potential inhibitors

  • Concentration range: 0.1-100 μM inhibitor

  • Pre-incubation time: 10-30 minutes

• Determination of inhibition mechanisms

  • Vary both substrate and inhibitor concentrations

  • Lineweaver-Burk analysis to distinguish competitive, non-competitive, and uncompetitive inhibition

• Thermal shift assays to confirm direct binding

  • Measure changes in protein melting temperature upon inhibitor binding

These methodological approaches enable comprehensive characterization of fabA activity and could help identify inhibitors with potential therapeutic value against V. vulnificus infections, which remain a significant health concern with a 20% mortality rate .

What techniques are most effective for studying fabA protein-protein interactions in V. vulnificus?

To investigate fabA protein-protein interactions within the V. vulnificus fatty acid synthesis pathway and potential regulatory networks, researchers should employ complementary techniques:

In vitro methods:

• Surface Plasmon Resonance (SPR)

  • Immobilize purified fabA on sensor chip

  • Flow potential interaction partners at 0.1-100 μM

  • Determine KD, kon, and koff values

• Isothermal Titration Calorimetry (ITC)

  • Directly measure binding thermodynamics (ΔH, ΔS, ΔG)

  • Typical concentrations: 10-20 μM fabA, 100-200 μM partner protein

  • Buffer matching critical for accurate measurements

• Analytical Ultracentrifugation (AUC)

  • Characterize complex formation in solution

  • Determine stoichiometry and assembly mechanisms

In vivo approaches:

• Bacterial Two-Hybrid (B2H) system

  • Adapt for use in V. vulnificus or heterologous host

  • Screen for interactions with fatty acid synthesis enzymes and potential regulators

• Co-immunoprecipitation with V. vulnificus cell lysates

  • Use anti-fabA antibodies or epitope tags

  • Validate interactions by mass spectrometry

• Proximity labeling methods (BioID or APEX2)

  • Express fabA fusion in V. vulnificus

  • Identify proximal proteins by streptavidin pulldown and mass spectrometry

Comparative analysis with interaction networks in other Vibrio species could reveal pathogen-specific adaptations, similar to how genomic analysis has identified unique genetic elements in V. vulnificus strains .

What CRISPR-based methodologies are most effective for fabA gene editing in Vibrio vulnificus?

CRISPR-Cas systems can be effectively adapted for V. vulnificus fabA genetic manipulation. Based on recent advances in V. vulnificus molecular techniques:

• CRISPR-Cas9 system optimization:

  • Expression vector: Broad-host-range plasmid with appropriate origin of replication

  • Promoter selection: Constitutive promoters like Plac or arabinose-inducible PBAD

  • gRNA design: Target unique regions of fabA with minimal off-target potential

  • PAM site selection: NGG for Cas9 from Streptococcus pyogenes

  • Transformation method: Electroporation (1.8-2.5 kV, 200 Ω, 25 μF)

• Homology-directed repair templates:

  • Homology arm length: 500-1000 bp flanking the target site

  • Selection markers: Chloramphenicol or kanamycin resistance cassettes

  • Scarless editing: Include counterselection markers (sacB) for marker removal

• Verification methods:

  • PCR screening of transformants

  • Sanger sequencing of the modified region

  • Whole-genome sequencing to confirm single integration site

This approach builds on the rapid and sensitive detection capabilities developed for V. vulnificus using CRISPR/Cas12a systems , adapting similar principles for genetic modification rather than detection.

How does environmental stress affect fabA expression in Vibrio vulnificus?

Environmental stress factors significantly influence fabA expression in V. vulnificus, reflecting its adaptation to diverse aquatic environments:

Temperature effects:

• Cold shock (15°C): Typically increases fabA expression to maintain membrane fluidity

• Heat shock (42°C): May decrease expression as part of stress response

• Seasonal variations: Expression likely correlates with seasonal temperature changes in marine environments

Salinity response:

• Low salinity: May increase fabA expression to modify membrane characteristics

• High salinity: Could alter expression patterns as part of osmotic stress response

• Brackish water adaptation: Expression levels likely optimized for typical V. vulnificus habitats

Nutrient availability:

• Carbon limitation: Potentially downregulates fabA to conserve resources

• Host-associated conditions: Expression may change during pathogenesis

Experimental approaches to measure these effects:

• qRT-PCR for transcript quantification

  • Reference genes: rpoA, recA, or gyrA for normalization

  • Sampling time course: 0, 15, 30, 60, 120 minutes post-stress

• Transcriptomics (RNA-seq)

  • Compare fabA expression across environmental conditions

  • Identify co-regulated genes in fatty acid biosynthesis pathways

• Promoter-reporter fusions

  • fabA promoter fused to fluorescent proteins or luciferase

  • Real-time monitoring of expression in different conditions

These approaches could reveal how fabA regulation contributes to V. vulnificus environmental adaptation and potentially its virulence, which is known to vary based on genetic factors and environmental conditions .

What methods should be used to analyze fabA expression in clinical versus environmental isolates of V. vulnificus?

Comparative analysis of fabA expression between clinical and environmental V. vulnificus isolates requires robust methodologies that account for strain diversity:

Sample collection and preparation:

• Clinical isolates: Blood culture specimens, wound samples, or oyster-related infection sources

• Environmental isolates: Brackish water, sediment, shellfish, particularly from areas with reported V. vulnificus presence

• Growth standardization: Identical media (LB with 2% NaCl) and growth phase (mid-log, OD600 = 0.4-0.6)

Expression analysis techniques:

• RT-qPCR protocol:

  • RNA extraction using RNeasy kits with on-column DNase treatment

  • cDNA synthesis with random hexamers or specific primers

  • fabA-specific primers with 90-110% efficiency

  • Reference genes validated across strain collection

• Proteomics approach:

  • Whole-cell proteome analysis by LC-MS/MS

  • Targeted fabA quantification using multiple reaction monitoring

  • Normalization to multiple housekeeping proteins

• Immunological detection:

  • Development of fabA-specific antibodies

  • Western blot or ELISA quantification

  • Standardization using recombinant fabA standard curve

Data analysis and interpretation:

• Statistical comparison between clinical and environmental groups

• Correlation with:

  • Genetic lineage (biotype, sequence type)

  • Virulence gene profiles (particularly rtxA1 variants)

  • Geographic origin and isolation source

This methodological approach parallels the genome-wide comparative analyses conducted on clinical versus environmental V. vulnificus isolates, which have revealed significant genomic diversity and potentially reduced virulence in some environmental strains .

How can fabA be targeted for development of novel antimicrobials against Vibrio vulnificus?

The essential role of fabA in V. vulnificus fatty acid biosynthesis makes it a promising antimicrobial target. Research strategies include:

Rational inhibitor design:

• Structure-based approach:

  • Use crystal structure of V. vulnificus fabA or homology models

  • Virtual screening of compound libraries targeting the active site

  • Fragment-based drug design focusing on catalytic residues

• Substrate analog development:

  • Synthesize 3-hydroxydecanoyl-ACP mimics with modifications at the hydration site

  • Incorporate reactive groups to form covalent bonds with active site residues

  • Design transition state analogs based on reaction mechanism

High-throughput screening approaches:

• Enzyme activity assays:

  • Miniaturized spectrophotometric assays in 384-well format

  • Screen chemical libraries of 10,000-100,000 compounds

  • Z-factor >0.7 for robust hit identification

• Whole-cell screening:

  • Growth inhibition assays using V. vulnificus clinical isolates

  • Secondary screens for fabA-specific mechanism of action

  • Counter-screens against human cell lines for toxicity assessment

Lead optimization process:

• Structure-activity relationship studies

• Pharmacokinetic property enhancement

• In vivo efficacy testing in mouse models of V. vulnificus infection

This targeted approach could lead to new treatment options for V. vulnificus infections, which have a high mortality rate of approximately 20% and often require rapid intervention with appropriate antibiotics.

What are the challenges and solutions for crystallizing recombinant V. vulnificus fabA for structural studies?

Crystallizing recombinant V. vulnificus fabA presents several challenges requiring systematic approaches:

Common challenges:

• Protein heterogeneity:

  • Post-translational modifications

  • Flexible regions causing conformational variability

  • Oligomerization state inconsistency

• Solubility limitations:

  • Aggregation at high concentrations needed for crystallization

  • Buffer incompatibility with crystallization conditions

• Crystal nucleation and growth issues:

  • Formation of microcrystals or thin needles

  • Poor diffraction quality

Methodological solutions:

• Protein engineering strategies:

  • Surface entropy reduction (replace clusters of Lys/Glu/Gln with Ala)

  • Truncation of disordered termini based on limited proteolysis

  • Introduction of crystal contacts through site-directed mutagenesis

• Crystallization optimization:

  • High-throughput screening of 500-1000 conditions

  • Microseeding from initial crystal hits

  • Additive screening (detergents, polyols, divalent cations)

  • Crystallization with substrates, products, or inhibitors

• Alternative crystallization methods:

  • Lipidic cubic phase for membrane-associated regions

  • Counter-diffusion in capillaries

  • Batch crystallization under oil

• Crystal handling and data collection:

  • Optimization of cryoprotection protocols

  • Annealing techniques for ice-damaged crystals

  • Utilization of microfocus synchrotron beamlines for small crystals

These approaches have been successful for crystallizing challenging proteins from various bacterial species and could be applied to V. vulnificus fabA to reveal structural features that might explain its adaptation to the unique environmental niches of this pathogen.

How can researchers integrate fabA function into systems-level models of V. vulnificus metabolism and virulence?

Integrating fabA function into systems-level models requires multi-omics approaches and computational modeling:

Data generation for model construction:

• Transcriptomics:

  • RNA-seq under various conditions (temperature, salinity, nutrient availability)

  • Identification of co-regulated gene clusters with fabA

• Proteomics:

  • Quantitative proteomics to measure fabA abundance

  • Protein-protein interaction mapping using proximity labeling

• Metabolomics:

  • Targeted fatty acid profiling using GC-MS

  • Untargeted metabolomics to identify broader metabolic changes

• Fluxomics:

  • 13C-labeled substrate incorporation into fatty acids

  • Measurement of flux through fatty acid biosynthesis pathways

Computational modeling approaches:

• Genome-scale metabolic model construction:

  • Include all reactions in fatty acid biosynthesis pathway

  • Connect to central carbon metabolism and virulence factor production

• Regulatory network modeling:

  • Integrate transcription factor binding data

  • Include environmental sensing systems

• Dynamic modeling of fatty acid biosynthesis:

  • Ordinary differential equations for key reactions

  • Parameter estimation from experimental data

Integration with virulence models:

• Correlate membrane composition with virulence factor expression

• Link environmental adaptation to pathogenicity

• Identify metabolic vulnerabilities as potential therapeutic targets

This systems biology approach would provide comprehensive insights into how fabA contributes to V. vulnificus adaptation and virulence, potentially explaining the genetic variation observed in virulence factors like MARTX toxins and helping predict how environmental changes might affect V. vulnificus population dynamics and pathogenicity.

What are the most pressing unanswered questions regarding V. vulnificus fabA function?

Several critical knowledge gaps remain in our understanding of V. vulnificus fabA:

• Regulatory mechanisms governing fabA expression in response to environmental cues specific to V. vulnificus habitats

• Structural adaptations of V. vulnificus fabA compared to homologs from non-pathogenic bacteria

• Potential moonlighting functions beyond fatty acid biosynthesis, particularly in virulence

• Impact of fabA activity on membrane composition during host infection

• Evolution of fabA in different V. vulnificus lineages and potential horizontal gene transfer events

Future research should prioritize these questions to enhance our understanding of V. vulnificus biology and pathogenesis, potentially leading to novel intervention strategies against this highly lethal pathogen with a mortality rate of approximately 20% .

How might climate change affect fabA function and expression in emerging V. vulnificus strains?

Climate change could significantly impact V. vulnificus fabA function and expression through multiple mechanisms:

• Rising water temperatures:

  • May select for fabA variants optimized for higher temperature environments

  • Could alter regulation of fabA expression to maintain membrane fluidity

  • Might extend geographical range of V. vulnificus into previously cooler waters

• Changed salinity profiles:

  • Increased rainfall and freshwater runoff may reduce coastal salinity

  • Could drive adaptation in fabA function for lower salinity environments

  • May select for strains with altered fatty acid composition

• Ecological niche expansion:

  • New environmental stresses may drive genetic recombination events

  • Could lead to novel fabA variants with altered catalytic properties

  • May result in strains with enhanced virulence potential

Research approaches to address these questions should include:

• Long-term monitoring of fabA sequence evolution in environmental isolates

• Experimental evolution studies under simulated climate change conditions

• Modeling the impact of temperature and salinity changes on enzyme kinetics

This research direction is particularly relevant given the evidence that V. vulnificus virulence factors already undergo significant genetic rearrangement and may be subject to environmental selection .

What interdisciplinary approaches could accelerate translation of fabA research into clinical applications?

Advancing V. vulnificus fabA research toward clinical applications requires interdisciplinary collaboration:

Computational biology and structural bioinformatics:

• Virtual screening of compound libraries against fabA models

• Development of machine learning algorithms to predict enzyme-inhibitor interactions

• In silico prediction of resistance mechanisms

Medicinal chemistry:

• Design and synthesis of novel fabA inhibitors

• Development of structure-activity relationships

• Optimization of pharmacokinetic properties

Microbiology and molecular biology:

• Validation of fabA as an essential gene in V. vulnificus

• Assessment of inhibitor efficacy in various strains

• Investigation of resistance development mechanisms

Immunology:

• Exploration of fabA as a potential vaccine antigen

• Understanding host immune responses to V. vulnificus lipids

• Development of adjuvant strategies

Clinical microbiology:

• Rapid detection methods for V. vulnificus based on fabA signatures

• Point-of-care testing for antibiotic susceptibility

• Development of fabA-targeted diagnostics

Public health and epidemiology:

• Surveillance of V. vulnificus strains in high-risk areas

• Modeling the impact of prevention strategies

• Education initiatives for at-risk populations