Recombinant Neisseria meningitidis serogroup C Peptide deformylase (def)

What is peptide deformylase (def) and why is it a promising target for antimicrobial development?

Peptide deformylase (EC 3.5.1.31) is an essential bacterial enzyme that catalyzes the removal of the N-formyl group from N-terminal methionine following protein translation. This enzyme is encoded by the def gene, which is present in all pathogenic bacteria, including Mycoplasma and Chlamydia species . The critical feature making peptide deformylase an attractive antimicrobial target is that it does not share a functionally equivalent gene in mammalian cells, providing a unique opportunity for selective targeting of bacterial systems .

Peptide deformylase is essential for bacterial growth and survival, as validated in organisms like Streptococcus pneumoniae and Escherichia coli . The enzyme contains three highly conserved catalytic domains and belongs to the matrix metallo-protease (MMP) family of enzymes . Inhibition of this enzyme produces effects similar to other protein synthesis inhibitors, including tetracyclines, macrolides, streptogramins, lincosamides, and chloramphenicol.

What are the structural and functional characteristics of N. meningitidis serogroup C?

Neisseria meningitidis serogroup C, particularly the representative strain FAM18 from the ST-11/ET-37 complex, has been a major cause of meningococcal disease worldwide throughout the last century . The genome of strain FAM18 contains 1,976 predicted genes, of which 60 do not have orthologues in previously sequenced serogroup A or B strains .

This strain belongs to the ST-11/ET-37 complex, which despite having low carriage rates, continues to be associated with sporadic disease outbreaks worldwide . Serogroup C meningococci present distinctive surface structures that contribute to their virulence, including specific capsular polysaccharides and outer membrane proteins .

The genetic makeup of N. meningitidis is characterized by high plasticity due to natural transformability, which allows for extensive recombination and horizontal gene transfer . This recombination contributes significantly to the diversification of surface structures and may affect the expression and structure of proteins including peptide deformylase .

How does peptide deformylase inhibition affect bacterial protein synthesis?

Inhibition of peptide deformylase results in the accumulation of N-terminal formylated peptides and proteins within the bacterial cell . This accumulation occurs in a time-dependent manner, with different peptides/proteins showing varying degrees of formylation .

The blockade of bacterial peptide deformylase ultimately produces inhibition of protein synthesis through a mechanism conceptually similar to other protein synthesis inhibitors, though it acts at a different stage of the process .

What are effective approaches for recombinant expression of N. meningitidis peptide deformylase?

Based on research with other bacterial peptide deformylases, effective recombinant expression typically requires careful consideration of several factors:

• Expression system selection: For meningococcal proteins, E. coli expression systems are commonly used, though protein folding and solubility challenges may arise .

• Gene optimization: The gene sequence should be analyzed for rare codons and optimized if necessary, particularly given the different GC content between N. meningitidis and common expression hosts .

• Fusion tags: Expression with N- or C-terminal His-tags facilitates purification while maintaining enzymatic activity. For peptide deformylase specifically, N-terminal tags may interfere with activity assessment and may require removal .

• Metal ion incorporation: Since peptide deformylase is a metalloenzyme that coordinates with active-site metal atoms (typically nickel), expression conditions should include appropriate metal supplementation .

• Induction conditions: Temperature, inducer concentration, and duration significantly impact the yield of soluble and active recombinant peptide deformylase. Lower temperatures (16-25°C) often favor proper folding .

Studies on related proteins from B. pseudomallei indicate that preliminary small-scale expression trials are crucial for determining optimal conditions before scaling up production .

What purification strategies maintain the enzymatic activity of recombinant peptide deformylase?

Effective purification of active recombinant peptide deformylase requires:

• Buffer optimization: Buffers containing stabilizing agents such as glycerol (10-20%) and reducing agents like DTT or β-mercaptoethanol help maintain enzyme stability .

• Metal coordination: Since peptide deformylase coordinates with metal ions at its active site, purification buffers may need to be supplemented with appropriate metal ions (Ni2+ or Fe2+) to maintain activity .

• Chromatography approach: A multi-step purification typically involves:

  • Initial capture using immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography to ensure monodispersity and remove aggregates

  • Ion exchange chromatography for final polishing if needed

• Stability assessment: Differential scanning fluorimetry can be used to assess thermal stability of the purified enzyme and optimize buffer conditions .

• Activity preservation: Storage in small aliquots with cryoprotectants at -80°C helps preserve enzymatic activity for extended periods.

Size exclusion chromatography coupled to laser light scattering detection provides valuable information about the monodispersity of the purified enzyme, which is critical for structural studies and activity assays .

How can researchers assess peptide deformylase inhibition kinetics?

Inhibition kinetics of peptide deformylase can be assessed through several approaches:

• Time-dependent inhibition analysis: Some peptide deformylase inhibitors like actinonin and BB-3497 show time-dependent inhibition, where binding occurs in two steps - an initial encounter complex followed by a tightening into a final encounter complex with slow dissociation rates .

• Dissociation rate determination: The half-life for dissociation can be measured (≥0.77 days for actinonin and ≥1.9 days for BB-3497) .

• IC50 determination: Inhibitor concentration causing 50% enzyme inhibition provides a standard measure of potency. For example, PDF-611 and BB-3497 showed IC50 values of 69.5 nM and 24.9 nM, respectively, against M. bovis peptide deformylase .

• Enzyme-inhibitor complex analysis: Crystallography studies of enzyme-substrate complexes at resolutions of 2Å or less provide detailed insights into inhibitor binding modes. Many peptide deformylase inhibitors are hydroxamic acid derivatives that coordinate with the active-site metal atom .

• Proteome analysis: Two-dimensional electrophoresis can track the accumulation of N-terminal formylated peptides/proteins during inhibitor exposure, providing an indirect measure of inhibition efficiency in vivo .

How can recombinant peptide deformylase be used to screen potential inhibitors against N. meningitidis?

Recombinant peptide deformylase provides a valuable tool for screening potential inhibitors through:

• High-throughput enzymatic assays: Spectrophotometric or fluorescence-based assays using synthetic peptide substrates can rapidly screen compound libraries for inhibitory activity.

• Structure-guided screening: Crystal structures of peptide deformylase in complex with known inhibitors like actinonin or BB-3497 can guide structure-based drug design efforts .

• Selectivity profiling: Comparing inhibition of N. meningitidis peptide deformylase with mammalian enzymes ensures target selectivity. PDIs are selective for bacterial enzymes and exhibit activity against similar mammalian enzymes only at extremely high concentrations .

• Hydroxamic acid derivatives testing: Given that many effective PDIs are hydroxamic acid derivatives that coordinate with the active-site metal atom, this chemical class should be prioritized in screening efforts .

• Time-dependent inhibition assessment: Since some PDIs show time-dependent inhibition with extremely slow dissociation rates, kinetic studies should include appropriate time-course analyses .

• Whole-cell validation: Promising inhibitors identified in enzymatic screens should be tested against whole N. meningitidis cells to confirm antimicrobial activity and cell penetration.

What is the relationship between genetic variation in N. meningitidis and potential impact on peptide deformylase as a drug target?

The relationship between genetic variation and peptide deformylase targeting involves several critical considerations:

How does the genomic context of the def gene in N. meningitidis compare to other bacterial pathogens?

Understanding the genomic context of the def gene provides insights into potential regulatory mechanisms and evolutionary relationships:

• Comparative genomics: While specific information about the def gene's location in N. meningitidis is not provided in the search results, in most bacteria, the def gene is part of essential gene clusters involved in protein synthesis and processing.

• Core genome classification: Computational screening for signs of recombination has revealed that about 40% of meningococcal core genes are affected by recombination, primarily within metabolic genes and genes involved in DNA replication and repair . Understanding whether the def gene falls within frequently recombining regions would inform its potential variability.

• Conservation across species: The peptide deformylase enzyme contains three highly conserved catalytic domains, suggesting strong functional constraints despite the high recombination rates observed in the N. meningitidis genome .

• Regulatory elements: Analysis of upstream and downstream regions of the def gene may reveal regulatory mechanisms that could differ between N. meningitidis and other pathogens, potentially affecting expression levels and response to stress conditions.

• Association with mobile genetic elements: The N. meningitidis genome contains numerous mobile genetic elements and prophages that shape its population structure . Determining whether the def gene is associated with such elements would provide insights into its potential for horizontal transfer.

What are the major challenges in using recombinant N. meningitidis peptide deformylase for structural studies?

Structural studies of recombinant N. meningitidis peptide deformylase face several challenges:

• Protein stability: As a metalloenzyme, peptide deformylase requires appropriate metal ion incorporation for stability and activity. Metal loss during purification or crystallization can lead to structural changes or loss of function .

• Expression and purification optimization: Like other meningococcal proteins, recombinant expression may require extensive optimization of conditions to obtain sufficient quantities of properly folded protein .

• Crystallization challenges: Obtaining high-quality crystals suitable for X-ray diffraction studies requires screening numerous conditions, often complicated by the need to maintain metal coordination and prevent oxidation.

• Conformational heterogeneity: Peptide deformylase may exist in multiple conformational states, particularly in the presence of different inhibitors or substrates, requiring specific approaches to capture relevant structural states .

• Complex formation with inhibitors: For co-crystallization with inhibitors, the time-dependent nature of some inhibitor interactions necessitates careful timing of crystallization trials .

These challenges are reflected in studies of related proteins, such as those from B. pseudomallei, where significant efforts were required to produce protein suitable for biophysical analyses .

How might peptide deformylase inhibitors be integrated into combination therapies against drug-resistant N. meningitidis?

Peptide deformylase inhibitors offer promising opportunities for combination therapy approaches:

• Novel mechanism of action: PDIs inhibit protein synthesis through a mechanism distinct from other antibiotics, making them potentially effective against strains resistant to conventional antibiotics .

• Synergistic potential: Combining PDIs with antibiotics that target other aspects of bacterial metabolism or cell integrity could enhance efficacy and reduce the emergence of resistance.

• Resistance management: The essential nature of peptide deformylase and its absence in mammalian cells suggests a potentially high barrier to resistance development. Combination therapy could further elevate this barrier.

• Outer membrane considerations: Studies of N. meningitidis outer membrane composition reveal a complex structure with numerous proteins . PDIs would need to penetrate this barrier, potentially requiring combination with agents that enhance permeability.

• Post-antibiotic effect: PDIs like NVP LBM 415 demonstrate prolonged post-antibiotic effects in vitro when maintained at sub-MIC concentrations . This characteristic could be advantageous in designing dosing regimens for combination therapy.

• Activity spectrum considerations: Some PDIs have shown activity against pathogenic Neisseria species. For example, LBM415 (NVP PDF-713) demonstrated activity against both N. gonorrhoeae and N. meningitidis with MIC90 values of 8 μg/mL and 2 μg/mL, respectively .

What emerging technologies could enhance research on N. meningitidis peptide deformylase?

Several emerging technologies could significantly advance research in this area:

• Cryo-electron microscopy: This rapidly advancing technique could enable structural determination of peptide deformylase without the need for crystallization, potentially capturing multiple conformational states relevant to function.

• Nanobody-based approaches: Developing nanobodies against specific conformational states of peptide deformylase could stabilize these states for structural studies and provide insights into the dynamics of enzyme-substrate interactions.

• Single-molecule enzymology: Techniques to study individual enzyme molecules could reveal heterogeneity in catalytic behavior and provide insights into the mechanism of peptide deformylase that are obscured in bulk measurements.

• Genome editing in N. meningitidis: Advanced genome editing tools like CRISPR-Cas systems adapted for N. meningitidis could enable precise modification of the def gene to study structure-function relationships in vivo.

• High-throughput screening platforms: Microfluidic systems and droplet-based assays could dramatically increase the throughput of inhibitor screening against recombinant peptide deformylase.

• Proteomics approaches: Advanced proteomics techniques could provide comprehensive analysis of the effects of peptide deformylase inhibition on the N. meningitidis proteome, building upon the two-dimensional electrophoresis approaches previously used in S. aureus and S. pneumoniae .

• In silico modeling: Computational approaches incorporating molecular dynamics simulations could predict inhibitor binding and resistance-conferring mutations, guiding rational design of improved inhibitors.

How does peptide deformylase activity in N. meningitidis compare with other bacterial pathogens?

Comparative analysis of peptide deformylase across bacterial species reveals important insights:

• Inhibitor sensitivity: While specific data for N. meningitidis is limited in the search results, studies with LBM415 (NVP PDF-713) showed different MIC values against N. gonorrhoeae (MIC90 of 8 μg/mL) and N. meningitidis (MIC90 of 2 μg/mL), indicating species-specific differences in susceptibility .

• Structural conservation: Peptide deformylase enzymes across bacterial species contain three highly conserved catalytic domains, suggesting functional similarity despite species differences .

• Metal coordination: Peptide deformylases typically coordinate with metal ions (Ni or Fe) at their active sites, though the preferred metal may vary between species, affecting inhibitor binding and catalytic efficiency .

• Inhibition kinetics: Two peptide deformylase inhibitors, actinonin and BB-3497, demonstrate time-dependent inhibition with extremely slow rates of dissociation (half-lives for dissociation ≥0.77 days and ≥1.9 days, respectively) . Similar kinetic parameters would be expected for N. meningitidis peptide deformylase, though species-specific variations may exist.

• Activity spectrum: The peptide deformylase inhibitor LBM415 was tested against multiple pathogenic Neisseria species, demonstrating activity against both N. gonorrhoeae and N. meningitidis, though with different potency profiles .

This comparative data highlights the importance of species-specific testing when developing peptide deformylase inhibitors as potential antimicrobial agents.

What insights can be gained from studying peptide deformylase across different N. meningitidis serogroups?

Analysis across different N. meningitidis serogroups provides valuable comparative insights:

• Genomic context: The genome of N. meningitidis serogroup C strain FAM18 contains 60 genes without orthologues in serogroup A or B strains . While the def gene is likely present in all serogroups due to its essential function, regulatory elements or associated genes may differ.

• Evolutionary pressure: Different serogroups have evolved under various selective pressures, potentially leading to subtle variations in housekeeping genes like def. Comparative analysis could reveal serogroup-specific adaptations.

• Expression levels: Transcriptomic studies comparing def gene expression across serogroups under various conditions could identify differential regulation patterns that might influence susceptibility to inhibitors.

• Association with virulence: Serogroup C strains, particularly the ST-11/ET-37 complex represented by FAM18, have been major causes of meningococcal disease worldwide . Studying whether peptide deformylase characteristics correlate with virulence potential could provide insights into pathogenesis mechanisms.

• Recombination patterns: About 40% of the meningococcal core genes are affected by recombination . Determining whether the def gene shows evidence of recombination across serogroups could inform its evolutionary history and potential for developing resistance.