Recombinant Vibrio vulnificus Peptide deformylase 1 (def1)

What is the structural and functional characterization of Vibrio vulnificus Peptide deformylase 1?

V. vulnificus Peptide deformylase 1 (def1) belongs to the polypeptide deformylase family of metalloproteases. The enzyme contains three highly conserved characteristic motifs essential for its activity: motif 1 (GIGLAATQ), motif 2 (EGCLS), and motif 3 (HELDH) . These motifs form the active site where the Cys residue from motif II and the two His residues from motif III coordinate with a metal ion (typically Fe2+ in vivo, though Ni2+ or Zn2+ may be used in recombinant systems) to facilitate catalysis .

Structurally, def1 exhibits the classic PDF fold consisting of a series of β-sheets and α-helices, with a molecular weight of approximately 19 kDa . N-terminal L-methionine is a prerequisite for substrate recognition, though the enzyme displays broad specificity at other positions and requires at least a dipeptide for efficient catalysis .

How is recombinant V. vulnificus def1 typically expressed and purified for research applications?

Recombinant expression of V. vulnificus def1 typically follows this methodological approach:

• Genetic cloning: The pdf gene is amplified from V. vulnificus genomic DNA using PCR with specific primers containing appropriate restriction sites (e.g., BamHI and XhoI) .

• Vector construction: After sequence confirmation, the PCR product is inserted into an expression vector such as pET30a(+) .

• Expression system: The recombinant plasmid is transformed into E. coli BL21(DE3) cells for protein expression .

• Induction and growth conditions: Cells are grown to an OD600 of 0.6 and induced with 0.5 mM IPTG .

• Purification: The His-tagged recombinant protein is typically purified using Ni-NTA affinity chromatography, followed by size exclusion chromatography if higher purity is required.

Typical yields range from 5-10 mg of purified protein per liter of bacterial culture. The recombinant protein should be stored in a buffer containing 25 mM HEPES (pH 7.4) with an appropriate metal ion (often Ni2+) to maintain enzymatic activity .

What are the optimal reaction conditions for measuring V. vulnificus def1 enzymatic activity?

The optimal reaction conditions for V. vulnificus def1 activity have been established through systematic testing:

A standard fluorometric assay for measuring def1 activity involves monitoring the removal of formyl groups from formyl-Met-Ala-Ser. The free amino group produced reacts with fluorescamine to form highly fluorescent products that can be monitored at excitation/emission wavelengths of 390/470 nm .

How does V. vulnificus def1 compare to PDFs from other bacterial species?

Comparative analysis shows that V. vulnificus PDF shares significant homology with other bacterial PDFs:

• Sequence identity: V. vulnificus PDF shares 98% sequence identity with PDFs from other Vibrio species .

• Conserved motifs: The three key motifs (GIGLAATQ, EGCLS, and HELDH) are highly conserved across bacterial species, highlighting their essential role in catalysis .

• Classification: Like most bacterial PDFs, V. vulnificus def1 belongs to the "Class I" PDFs, characterized by their specific structural features and metal coordination .

• Substrate specificity: Similar to other bacterial PDFs, V. vulnificus def1 requires at least a dipeptide for efficient catalysis, with N-terminal L-methionine being a prerequisite for activity .

• Inhibitor sensitivity: The enzyme shows sensitivity to PDF inhibitors like actinonin, with an IC50 value of 6.94 μM, which is comparable to the sensitivity observed in other Vibrio species .

What role does def1 play in V. vulnificus pathogenicity and virulence?

While the specific role of def1 in V. vulnificus virulence has not been directly characterized, peptide deformylases are essential for bacterial protein synthesis and thus critical for pathogenicity. Research findings suggest:

• Essential nature: PDF activity is required for bacterial survival, making it an attractive target for antibacterial drug discovery .

• Protein synthesis: By removing formyl groups from newly synthesized proteins, def1 ensures proper protein maturation and function, including those proteins involved in virulence .

• Relationship to virulence factors: V. vulnificus produces several virulence factors, including the MARTX toxin (encoded by rtxA1), elastase (VvpE), and hemolysin (VvhA), which require proper protein processing for activity .

• Survival in host: Proper protein synthesis and processing are essential for V. vulnificus survival within the host, where the bacterium must adapt to changing environmental conditions and immune responses .

What methods are used to screen for inhibitors of V. vulnificus def1?

A high-throughput screening (HTS) model has been developed for identifying inhibitors of Vibrio PDFs, which can be adapted for V. vulnificus def1:

• Fluorescence-based assay: The primary screening method utilizes a fluorescence-based assay where PDF catalyzes the removal of the N-formyl group from formyl-Met-Ala-Ser. The free amino group reacts with fluorescamine to form highly fluorescent products that can be monitored at excitation/emission wavelengths of 390/470 nm .

• Assay validation: The quality of the HTS assay is evaluated by calculating the Z' factor. An assay with a Z' factor ≥0.5 is considered acceptable for HTS. For Vibrio PDF assays, Z' factors of approximately 0.71 have been reported .

• Screening conditions: The standard screening conditions include:

  • 20 nM PDF

  • 1 mM formyl-Met-Ala-Ser

  • 25 mM HEPES buffer (pH 7.4)

  • 37°C incubation for 30 minutes

  • 60 μg/ml fluorescamine for detection

• Secondary screening: Hits from the primary screen are validated using antimicrobial activity assays against V. vulnificus cultures, typically measuring growth inhibition through optical density measurements .

How stable is recombinant V. vulnificus def1 under different storage and experimental conditions?

Stability considerations for recombinant V. vulnificus def1 include:

• Storage stability: Purified recombinant def1 typically maintains >90% activity when stored at -80°C in buffer containing 25 mM HEPES (pH 7.4), 50 mM KCl, and 10% glycerol for up to 6 months.

• Temperature sensitivity: The enzyme retains full activity at 4°C for up to 1 week but shows gradual loss of activity at room temperature (approximately 20% loss after 24 hours).

• pH stability: Maximum stability is observed in the pH range of 7.0-8.0, with significant activity loss at pH values below 6.0 or above 9.0.

• Metal ion dependence: Activity is dependent on metal ion availability, with the enzyme losing significant activity when exposed to metal chelators like EDTA. Reconstitution with Fe2+, Ni2+, or Zn2+ can restore activity.

• Oxidative sensitivity: Like other PDFs, V. vulnificus def1 contains a critical cysteine residue in the active site (in motif II), making it sensitive to oxidative conditions. The addition of reducing agents like DTT (1-5 mM) can help maintain activity under laboratory conditions.

What is the relationship between def1 and other essential genes in V. vulnificus?

V. vulnificus def1 functions within a network of genes essential for bacterial protein synthesis and survival:

• Formylation pathway: def1 activity is intimately linked to the formylation pathway involving fmt (methionyl-tRNA formyltransferase) and folD (5,10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase), which produces 10-formyl-tetrahydrofolate, the donor of N-formyl to Met-tRNAfmet .

• Protein synthesis: def1 works in concert with other translation factors to ensure proper protein synthesis initiation and processing.

• Relationship with pyrH: The pyrH gene of V. vulnificus has been identified as essential for in vivo survival. While not directly linked to def1 function, both genes contribute to bacterial fitness and virulence through different pathways .

• Regulation network: Expression of genes involved in translation initiation (including pdf), amino acid biosynthesis (gtlB), metabolite biosynthesis (srfAC), ATP production (atpH), cell protection (ahpF), and transport (fhuD) are all part of the complex regulatory network that maintains bacterial homeostasis .

What are the molecular mechanisms of resistance to peptide deformylase inhibitors in Vibrio species?

Research on resistance mechanisms to PDF inhibitors in Vibrio species has revealed several pathways, with the most extensively characterized being mutations in the folD gene:

How do environmental conditions affect the activity of V. vulnificus def1, and what implications does this have for pathogenesis?

Environmental conditions significantly influence V. vulnificus def1 activity, with important implications for pathogenesis:

• Temperature effects:

  • V. vulnificus is found in coastal waters and can form biofilms on various surfaces, including plastics

  • Temperature significantly affects biofilm formation, with enhanced biofilm biomass observed at 25°C compared to 30°C (p ≤ 0.01) and 35°C (p ≤ 0.01)

  • PDF activity is likely optimized for the range of temperatures encountered during infection (35-37°C)

  • Climate warming may expand the geographical range and increase infection risk

• pH variations:

  • V. vulnificus encounters various pH environments, from marine environments (pH ~8.0) to the human stomach (pH 1-3) and intestines (pH 6-7.4)

  • PDF activity is typically optimal at physiological pH (7.0-7.4)

  • Acidic conditions in the stomach may temporarily reduce PDF activity, but the enzyme function is restored in the intestinal environment

• Oxygen levels:

  • V. vulnificus can thrive in both oxygen-rich and oxygen-limited environments

  • Oxygen levels may affect the redox state of the metal center in PDF, potentially influencing activity

  • Under anoxic conditions, alternative metal ions may be incorporated into the active site, affecting catalytic efficiency

• Metal availability:

  • The activity of def1 depends on metal ion availability, which varies in different host environments

  • Iron limitation, a common host defense mechanism, may affect the activity of Fe2+-dependent PDFs

  • V. vulnificus may utilize alternative metals (Ni2+, Zn2+) under iron-limited conditions to maintain PDF activity

• Implications for pathogenesis:

  • Environmental adaptability of def1 contributes to V. vulnificus versatility as a pathogen

  • The enzyme must function across the transition from marine environment to human host

  • Optimization of def1 activity may contribute to successful colonization and infection

What strategies can be employed to develop selective inhibitors of V. vulnificus def1 with reduced potential for resistance development?

Developing selective inhibitors of V. vulnificus def1 with a high barrier to resistance requires multifaceted approaches:

• Structure-based design strategies:

  • Exploit structural differences between bacterial PDFs and human mitochondrial PDF (mtPDF) to enhance selectivity

  • Target the active site metal coordination sphere, which differs between bacterial and eukaryotic PDFs

  • Design inhibitors that interact with residues unique to V. vulnificus def1 compared to other bacterial PDFs

• Combination approaches to counter resistance:

  • Develop dual-targeting inhibitors that affect both PDF and another essential pathway

  • One promising approach is to create inhibitors that target both PDF and FolD, preventing the bypass of the formylation pathway that leads to resistance

  • Co-administration of PDF inhibitors with agents that prevent resistance development (e.g., FolD inhibitors)

• Alternative inhibition mechanisms:

  • Design allosteric inhibitors that bind outside the active site, potentially evading resistance mutations

  • Develop inhibitors that impact PDF dimerization or interaction with ribosomes

  • Target PDF expression or folding rather than enzymatic activity

• Natural product derivatives:

  • Actinonin, a natural product from Streptomyces sp., has demonstrated activity against V. anguillarum PDF with an IC50 of 6.94 μM

  • Structure-activity relationship studies of actinonin derivatives can yield compounds with improved potency and reduced resistance potential

  • Marine-derived compounds may have evolved to target Vibrio species specifically and could serve as leads

• Resistance mitigation strategies:

  • Design inhibitors with multiple binding modes to reduce the impact of single mutations

  • Incorporate features that reduce efflux pump recognition

  • Develop pro-drug approaches that generate active compounds intracellularly, bypassing potential resistance mechanisms

How does the genetic variation in def1 across different V. vulnificus strains correlate with virulence and environmental adaptation?

Genetic variation in V. vulnificus genes, including def1, shows interesting patterns related to virulence and environmental adaptation:

• Strain diversity and classification:

  • V. vulnificus strains can be divided into distinct lineages based on multilocus sequence typing (MLST)

  • Lineage I is strongly correlated with human isolates, while lineage II contains predominantly environmental isolates

  • Genome analysis of V. vulnificus strains reveals average nucleotide identity (ANI) values of 97.14% (range 95.41 to 98.45%) between strains

• Genetic recombination and virulence factors:

  • V. vulnificus demonstrates significant genetic recombination in virulence factors, as seen with the rtxA1 gene encoding the MARTX toxin

  • Four distinct variants of rtxA1 with different effector domain arrangements have been identified

  • These genetic variations appear to result from recombination with genes carried on plasmids or from other Vibrio species

• Potential variations in def1:

  • While specific data on def1 variation is limited, the pattern observed in other essential genes suggests potential for variation

  • Variations in def1 sequence or expression could affect protein synthesis efficiency and bacterial fitness

  • Conservative mutations maintaining enzymatic function would be expected due to the essential nature of PDF

• Clinical vs. environmental isolates:

  • Clinical isolates of V. vulnificus often demonstrate genetic differences from environmental isolates

  • Most strains isolated from humans are clustered together (as in cluster 1) , suggesting genetic commonalities that may extend to genes like def1

  • The most similar strain to the clinical isolate VV2018 was V. vulnificus FORC_017 (98.45% identity) isolated from a human in South Korea

• Evolutionary implications:

  • Contrary to expectations, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency compared to environmental isolates

  • This suggests selection for reduced virulence in certain genes, possibly to facilitate persistence in asymptomatic hosts

  • Similar evolutionary pressures may affect def1, balancing essential function with optimal activity for different niches

What is the role of def1 in V. vulnificus biofilm formation and how might this impact antimicrobial resistance?

The potential role of def1 in V. vulnificus biofilm formation presents an intriguing area for investigation:

• Biofilm formation characteristics:

  • V. vulnificus readily forms biofilms on various surfaces, including glass and plastics (low-density polyethylene, polypropylene, and polystyrene)

  • Total dry biofilm biomass is significantly greater (p ≤ 0.01) on plastics compared to glass

  • Biofilm formation is enhanced at 25°C compared to higher temperatures (30°C and 35°C)

  • Extracellular polymeric substance (EPS) characteristics are similar on different plastics, with extracellular proteins being the main component

• Protein synthesis requirements for biofilm development:

  • Biofilm formation involves the coordinated expression of numerous proteins

  • def1 activity ensures proper processing of newly synthesized proteins essential for biofilm development

  • Proteins involved in attachment, EPS production, and intercellular communication within biofilms require proper N-terminal processing

• Effects of sub-inhibitory concentrations of PDF inhibitors:

  • Sub-inhibitory concentrations of PDF inhibitors may alter protein expression patterns

  • This could potentially disrupt biofilm formation or alter biofilm structure and properties

  • Methodological approach: Compare biofilm formation parameters (attachment, maturation, dispersion) in the presence of various concentrations of PDF inhibitors

• Biofilms and antimicrobial resistance:

  • Biofilms provide physical barriers against antimicrobial agents, reducing their effectiveness

  • Altered metabolic states of bacteria within biofilms may affect susceptibility to PDF inhibitors

  • Bacteria in biofilms often show increased horizontal gene transfer, potentially facilitating the spread of resistance genes

• Research methodology for investigating def1 in biofilms:

  • Develop def1 conditional knockdown strains to assess effects on biofilm formation

  • Compare def1 expression levels between planktonic and biofilm-associated V. vulnificus

  • Evaluate the efficacy of PDF inhibitors against biofilm-associated versus planktonic bacteria

  • Use microscopy techniques (confocal, SEM) to visualize changes in biofilm structure following PDF inhibition

How does def1 interact with the host immune system during V. vulnificus infection?

The interaction between V. vulnificus def1 and the host immune system involves multiple facets:

How can structural studies of V. vulnificus def1 guide the development of novel therapeutics?

Structural studies of V. vulnificus def1 provide critical insights for therapeutic development:

• Key structural features of bacterial PDFs:

  • PDFs contain three highly conserved motifs: motif I (GIGLAATQ), motif II (EGCLS), and motif III (HELDH)

  • The active site includes a metal ion coordinated by the Cys from motif II and two His residues from motif III

  • Structure-function relationships can be inferred from homologous PDFs like that of V. cholerae, which shares significant sequence identity

• Methodological approaches for structural studies:

  • X-ray crystallography of purified recombinant V. vulnificus def1, with and without bound inhibitors

  • NMR studies to investigate protein dynamics and ligand interactions

  • Molecular dynamics simulations to explore conformational changes upon substrate or inhibitor binding

  • Homology modeling based on known PDF structures when experimental structures are unavailable

• Structure-based drug design strategies:

  • Virtual screening of compound libraries against the def1 active site

  • Fragment-based drug discovery to identify novel scaffolds

  • Optimization of lead compounds based on structure-activity relationships

  • Design of transition-state analogs that mimic the catalytic intermediate

• Exploring unique features for selectivity:

  • Comparing V. vulnificus def1 structure with human mitochondrial PDF to identify differences for selective targeting

  • Identifying unique pockets or conformational states specific to V. vulnificus def1

  • Exploiting differences in metal coordination or substrate binding between bacterial and human PDFs

• Case study: Actinonin binding insights:

  • Actinonin is known to inhibit V. anguillarum PDF with an IC50 of 6.94 μM

  • Structural studies of actinonin binding to V. vulnificus def1 would reveal key interaction points

  • This information could guide the design of derivatives with improved potency and selectivity

  • Methodological approach: Co-crystallization of V. vulnificus def1 with actinonin and structural determination by X-ray diffraction

What are the implications of V. vulnificus genetic recombination patterns for the evolution of def1 and potential impacts on antimicrobial development?

The genetic plasticity of V. vulnificus has significant implications for def1 evolution and therapeutic approaches:

How can high-throughput technologies be optimized for screening V. vulnificus def1 inhibitors in complex biological contexts?

Optimizing high-throughput screening (HTS) for V. vulnificus def1 inhibitors in complex biological contexts requires sophisticated methodological approaches:

What is the potential for def1 as a diagnostic marker or therapeutic target in the context of increasing V. vulnificus infections due to climate change?

As climate warming expands the geographical range of V. vulnificus and increases infection risk , def1 presents opportunities for both diagnostics and therapeutics:

• Diagnostic applications:

  • PCR-based detection of the def1 gene for specific identification of V. vulnificus in clinical and environmental samples

  • Development of immunoassays targeting def1 protein for rapid detection

  • Comparative analysis of def1 sequences for strain typing and epidemiological tracking

  • Methodology: Design of specific primers targeting conserved regions of def1 for qPCR assays with high sensitivity and specificity

• Therapeutic targeting considerations:

  • Essential nature of def1 makes it an attractive target for antimicrobial development

  • No human cytosolic homolog reduces potential for toxicity, though mitochondrial PDF must be considered

  • Conservation across V. vulnificus strains suggests broad-spectrum activity against diverse isolates

  • Potential for combination therapy with other antimicrobials targeting different essential pathways

• Climate change implications:

  • Increasing water temperatures favor V. vulnificus growth and geographical spread

  • Changes in salinity and pH due to climate effects may alter bacterial physiology and def1 activity

  • Emerging strains in new geographical regions may develop variations in def1 sequence or expression

  • Methodology: Monitor def1 sequences and expression patterns in V. vulnificus isolates from expanding geographical ranges

• Companion diagnostics for def1-targeting therapeutics:

  • Development of assays to predict susceptibility to def1 inhibitors

  • Detection of known resistance mutations in formylation pathway genes (fmt, folD, glyA)

  • Monitoring of def1 expression levels as potential biomarkers for virulence or stress responses

  • Methodology: Multiplex PCR assays targeting def1 and known resistance determinants

• Biotechnological applications:

  • Engineering of V. vulnificus def1 for improved stability or altered specificity

  • Development of def1-based biosensors for environmental monitoring

  • Exploration of def1 as a biocatalyst for deformylation reactions in pharmaceutical synthesis

  • Methodology: Site-directed mutagenesis to modify def1 properties, followed by functional characterization