Recombinant Vibrio vulnificus 3-oxoacyl-[acyl-carrier-protein] synthase 3 protein 1 (fabH1)

What is the role of fabH1 in Vibrio vulnificus pathogenesis?

The 3-oxoacyl-[acyl-carrier-protein] synthase 3 protein 1 (fabH1) plays a crucial role in fatty acid biosynthesis in Vibrio vulnificus. It catalyzes the first condensation reaction that initiates fatty acid synthesis, potentially governing the total rate of fatty acid production. This enzyme possesses both acetoacetyl-ACP synthase and acetyl transacylase activities .
While not directly involved in toxin production, fabH1 contributes to bacterial survival and adaptation through its role in membrane lipid synthesis. Studies indicate that fatty acid biosynthesis enzymes in pathogenic Vibrio species can influence virulence by affecting membrane integrity, biofilm formation, and stress responses. Research on similar enzymes in other pathogenic bacteria suggests that fabH1 may indirectly influence virulence by impacting the bacterium's ability to survive hostile host environments .

What are the optimal expression systems for producing recombinant V. vulnificus fabH1?

Several expression systems have been successfully employed for recombinant fabH1 production:

• Temperature optimization (typically 16-25°C for induction)

• IPTG concentration (0.1-1 mM)

• Induction time (4-24 hours)
  For improved solubility, co-expression with chaperones or fusion with solubility-enhancing tags may be necessary. The choice between His-tag and other affinity tags will depend on downstream applications and whether native protein activity is required .

How can I design an enzymatic activity assay for recombinant fabH1?

Two complementary approaches have been validated for measuring fabH1 activity:

Radiochemical filter disc assay

This highly sensitive method uses [14C]acetyl-CoA to detect the β-[14C]ketobutyryl-ACP product. The assay involves:

• Incubating purified fabH1 with radioactive acetyl-CoA substrate

• Trapping the β-ketobutyryl-ACP product on filter discs

• Measuring radioactivity with scintillation counting
  This method is reliable for determining activity with labeled primers but requires specialized radioisotope facilities .

Spectrophotometric assay

For non-radioactive alternatives, researchers can:

• Monitor the decrease in absorbance at 340 nm as NADPH is consumed

• Utilize coupled enzyme assays where fabH1 activity is linked to a detectable product
  For substrate specificity analysis, compare reaction rates with different acyl-CoA primers such as acetyl-CoA, propionyl-CoA, and branched chain acyl-CoAs. Bacterial FabH homologs show variability in substrate preference, with bFabH1 and bFabH2 utilizing both straight-chain and branched-chain primers, while E. coli FabH (eFabH) preferentially uses straight-chain substrates .

What structural analyses have been performed on fabH1 and how do they inform function?

Structural analysis of fabH1 reveals several key features that inform its function:

• The protein contains a conserved catalytic triad (Cys-His-Asn) essential for activity

• An N-terminal domain with a thiolase fold that binds acetyl-CoA

• A C-terminal domain that interacts with malonyl-ACP
  Homology modeling based on related FabH structures indicates that fabH1 from V. vulnificus forms a homodimer or homotetramer, with each monomer having a molecular weight of approximately 33.9 kDa .
  Specific structural features that determine substrate specificity include:

• The size and shape of the binding pocket, which influences preference for straight or branched-chain substrates

• Residues lining the acyl binding channel
  Studies comparing FabH enzymes from different bacteria reveal that substrate specificity is determined by specific amino acid substitutions in the binding pocket. For example, comparative analysis with B. subtilis FabH homologs (bFabH1 and bFabH2) showed only 33.2% amino acid sequence homology, yet they share significant similarity in the NAD(P)-binding domain and the conserved region of the active site .

How can recombinant fabH1 be used for screening potential antimicrobial compounds?

Recombinant fabH1 provides an excellent target for antimicrobial drug screening due to its essential role in bacterial fatty acid synthesis and absence in mammalian systems. A comprehensive screening approach includes:

Primary screening assays:

• Enzymatic inhibition assays: Measure fabH1 activity in the presence of test compounds using spectrophotometric or radiochemical methods

• Thermal shift assays: Detect compounds that bind to fabH1 by altering its thermal stability

• Surface plasmon resonance: Quantify binding kinetics between potential inhibitors and fabH1

Secondary validation approaches:

• Bacterial growth inhibition: Test compounds that inhibit fabH1 activity for their effect on V. vulnificus growth

• Fatty acid profiling: Analyze changes in bacterial fatty acid composition using GC-MS

• Synergy testing: Evaluate potential synergistic effects with existing antibiotics
  When designing screening libraries, focus on compounds that mimic substrates or transition states of the fabH1-catalyzed reaction. Research indicates that small molecules targeting the active site Cys112 residue show promise as inhibitors. Platensimycin derivatives and thiolactomycin analogs have shown efficacy against related FabH enzymes in other pathogens .

How does fabH1 differ from other FabH isoforms in Vibrio vulnificus, and what are the implications for bacterial metabolism?

V. vulnificus, like several other bacteria, contains multiple FabH isoforms that differ in substrate specificity and metabolic roles:

• V. vulnificus fabH1 can utilize both straight-chain and branched-chain acyl-CoA primers, though with different efficiencies

• This versatility contrasts with E. coli FabH, which predominantly accepts straight-chain substrates

• The ability to use branched-chain substrates correlates with specific amino acid substitutions in the binding pocket
  The presence of multiple FabH isoforms allows bacteria to maintain membrane fluidity under various environmental conditions. For V. vulnificus, which encounters changing temperatures and salt concentrations in marine environments, this adaptability is particularly important .

What experimental approaches can detect interactions between fabH1 and other proteins in the fatty acid synthesis pathway?

Several complementary methods can be used to investigate protein-protein interactions involving fabH1:

In vitro approaches:

• Pull-down assays: Use purified His-tagged fabH1 as bait to identify interacting partners from bacterial lysates

• Surface plasmon resonance: Measure binding kinetics between fabH1 and potential interacting proteins

• Isothermal titration calorimetry: Determine thermodynamic parameters of specific protein-protein interactions

In vivo approaches:

• Bacterial two-hybrid systems: Screen for protein interactions within a bacterial host

• Crosslinking followed by mass spectrometry: Identify proteins that are in close proximity to fabH1 in the native environment

• Co-immunoprecipitation: Isolate protein complexes containing fabH1 from bacterial lysates
  Research on related systems suggests that fabH1 likely interacts with:

• Acyl carrier protein (ACP)

• FabD (malonyl-CoA:ACP transacylase)

• FabF/B (3-oxoacyl-ACP synthases)
  Understanding these interactions can provide insights into the organization of the fatty acid synthesis machinery and potential points for intervention .

How does the recombinant expression of fabH1 affect protein folding and activity compared to the native enzyme?

The recombinant expression of fabH1 introduces several challenges that can affect protein folding and activity:

Factors affecting recombinant fabH1 quality:

• Expression host: E. coli-expressed fabH1 may lack post-translational modifications present in the native enzyme

• Affinity tags: N-terminal or C-terminal tags can interfere with dimerization or substrate binding

• Folding environment: Cytoplasmic conditions in expression hosts differ from native V. vulnificus

What role does fabH1 play in the quorum sensing mechanisms of Vibrio vulnificus?

Recent research has revealed unexpected connections between fatty acid biosynthesis and quorum sensing (QS) in Vibrio species:
FabH1 influences QS through several potential mechanisms:

• AHL precursor synthesis: Acyl-ACPs generated through the fabH1-initiated pathway serve as precursors for N-acylhomoserine lactone (AHL) synthesis, which are key QS signaling molecules

• Regulation of QS gene expression: Studies in related bacteria show that deletion mutations in fabH1 significantly reduce AHL production

• Crosstalk with regulatory systems: The fatty acid biosynthesis pathway interacts with global regulators that also control QS circuits
  Experimental evidence demonstrates that fabH1 deletion or inhibition leads to:

• Reduced AHL synthesis

• Altered biofilm formation

• Changes in virulence factor expression
  In P. aeruginosa, the fabH1 gene showed a 33.2-fold decrease in expression in a vqsM mutant (a global regulator of QS), suggesting that fabH1 is part of the QS regulon . This relationship indicates a regulatory feedback loop where QS controls fatty acid synthesis, which in turn affects the availability of AHL precursors .

How can site-directed mutagenesis of fabH1 provide insights into substrate specificity and catalytic mechanisms?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in fabH1:

Key residues for targeted mutagenesis:

• Catalytic triad: Cys112, His244, and Asn274 (numbering based on related FabH enzymes)

• Substrate binding pocket: Residues that line the acyl-binding channel

• Dimer interface: Amino acids involved in subunit interactions

Experimental design for mutagenesis studies:

• Generate single point mutations using PCR-based techniques

• Express and purify mutant proteins using the same conditions as wild-type

• Compare enzymatic parameters (kcat, Km) with various substrates

• Assess structural changes using circular dichroism or thermal stability assays
  Research on related FabH enzymes has shown that:

• Mutations in the catalytic cysteine abolish activity

• Specific residues in the binding pocket determine preference for straight vs. branched-chain substrates

• The size of the binding pocket influences chain-length specificity
  For example, studies with B. subtilis FabH homologs demonstrated that specific residues in the binding pocket determine whether the enzyme can accept branched-chain substrates. Similar approaches could be applied to V. vulnificus fabH1 to understand its substrate range and potential for inhibitor development .

What are the methodological approaches for studying the role of fabH1 in Vibrio vulnificus virulence using animal models?

Investigating fabH1's role in V. vulnificus virulence requires carefully designed animal studies:

Mouse models for V. vulnificus infection:

• Oral infection model: Mimics food-borne route of human infection

  • Iron-overloaded mice are administered bacteria intragastrically

  • Bacterial loads in organs and blood are quantified at various timepoints

  • Survival rates are monitored for 7-14 days

• Wound infection model: Simulates wound infections

  • Subcutaneous injection at the dorsal site

  • Local tissue damage and systemic spread are assessed

Experimental approaches to study fabH1's role:

• Gene knockout studies:

  • Create fabH1 deletion mutants using homologous recombination

  • Compare virulence between wild-type and ΔfabH1 strains

  • Complement mutants to confirm phenotype is due to fabH1 deletion

• Inhibitor studies:

  • Treat bacteria with specific fabH1 inhibitors prior to infection

  • Administer inhibitors during infection to assess therapeutic potential

• Immunization approaches:

  • Use recombinant fabH1 as a potential vaccine candidate

  • Assess protective immunity against subsequent challenge
    Research has shown that disruption of fatty acid synthesis pathways often attenuates bacterial virulence. For V. vulnificus specifically, studies with MARTX toxin variants demonstrated that genetic variations in virulence factors can significantly impact pathogenicity in mouse models .

How do environmental conditions affect fabH1 expression and activity in Vibrio vulnificus?

V. vulnificus inhabits marine and estuarine environments with varying conditions that influence fabH1 expression and activity:

Environmental factors affecting fabH1:

Experimental approaches to study environmental effects:

• qRT-PCR analysis of fabH1 expression under various conditions

• Reporter gene constructs (fabH1 promoter fused to GFP/luciferase)

• Proteomic analysis to quantify fabH1 protein levels

• Enzymatic assays to measure activity under different conditions
  Research in related Vibrio species suggests that temperature is a particularly important factor influencing fatty acid synthesis enzyme expression. Cold adaptation often involves increased production of branched-chain fatty acids, which would require specific FabH activity .

What techniques are most effective for resolving contradictory data in fabH1 functional studies?

When facing contradictory results in fabH1 research, several approaches can help resolve discrepancies:

Sources of contradictions in fabH1 studies:

• Strain variations: Different V. vulnificus isolates may exhibit genetic diversity in fabH1

• Experimental conditions: Temperature, pH, and buffer composition affect enzyme activity

• Protein preparation methods: Tag position, purification strategy, and storage can impact function

• Assay limitations: Different activity assays measure different aspects of enzyme function

Resolution strategies:

• Standardized protocols:

  • Use consistent expression systems and purification methods

  • Standardize enzyme assay conditions across laboratories

  • Include positive controls (e.g., E. coli FabH) in comparative studies

• Multi-method validation:

  • Employ orthogonal techniques to confirm protein-protein interactions

  • Validate enzymatic activity using both direct and coupled assays

  • Confirm in vitro findings with in vivo experiments

• Comprehensive controls:

  • Test multiple V. vulnificus strains to account for genetic variation

  • Include catalytically inactive mutants as negative controls

  • Consider contextual factors like temperature and pH
    Research on V. vulnificus rtxA1 gene has demonstrated significant genetic variation among strains, suggesting similar diversity might exist for metabolic enzymes like fabH1. When evaluating contradictory literature, consider whether differences might reflect actual biological diversity rather than experimental artifacts .

How can systems biology approaches integrate fabH1 function into the broader metabolic network of Vibrio vulnificus?

Systems biology provides powerful frameworks for understanding fabH1's role within the broader metabolic context:

Integration approaches:

• Genome-scale metabolic modeling:

  • Incorporate fabH1 reactions into genome-scale metabolic reconstructions

  • Perform flux balance analysis to predict the impact of fabH1 perturbations

  • Identify metabolic bottlenecks and alternative pathways

• Multi-omics integration:

  • Combine transcriptomic, proteomic, and metabolomic data

  • Map fabH1 expression to metabolite pools and flux changes

  • Identify regulatory networks controlling fabH1 expression

• Protein-protein interaction networks:

  • Map fabH1 interactions with other proteins

  • Identify functional modules within which fabH1 operates

  • Discover unexpected connections to other cellular processes

What are the current challenges and future directions in fabH1 research for antimicrobial development?

The development of fabH1 inhibitors as potential antimicrobials faces several challenges:

Current challenges:

• Selectivity: Designing inhibitors that target V. vulnificus fabH1 without affecting human fatty acid metabolism

• Resistance development: Potential for bacteria to develop resistance through mutations or alternate pathways

• Delivery barriers: Getting inhibitors across the Gram-negative cell envelope

• Validation gaps: Limited in vivo validation of fabH1 as an antimicrobial target

Future research directions:

• Structure-guided drug design:

  • Obtain high-resolution crystal structures of V. vulnificus fabH1

  • Use computational approaches to identify novel binding sites

  • Design transition-state analogs as potential inhibitors

• Combination therapies:

  • Explore synergistic effects between fabH1 inhibitors and existing antibiotics

  • Target multiple steps in the fatty acid synthesis pathway simultaneously

  • Combine with membrane permeabilizers to enhance delivery

• Alternative applications:

  • Develop fabH1 inhibitors as antivirulence agents rather than growth inhibitors

  • Target fabH1's role in quorum sensing to disrupt biofilm formation

  • Use as adjuncts to enhance immune clearance of infection
    Research suggests that targeting bacterial fatty acid synthesis remains promising despite challenges. The contribution of fabH1 to both growth and virulence regulation makes it particularly attractive. Furthermore, the genetic variation observed in V. vulnificus virulence factors suggests that resistance monitoring would be essential for any fabH1-targeting therapeutics .