Recombinant Bordetella pertussis Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the amino acid sequence and structural characteristics of Bordetella pertussis MtgA?

Bordetella pertussis MtgA is a 242-amino acid protein with the sequence: MPKPTARRLNWFRVITAVIMAVLCIAILYQLWMFSLVVWYAYRDPGSSAIMRQELARLRERDPEAELKYQWVPYDRISNTLKQAVVASEDANFTEHDGVEWDAIRKAWEYNQRQAERGRTKMRGGSTITQQLAKNLFLSGSRSYLRKGQELVLAYMIEHVMPKERILELYLNVAEWGVGVFGAEAAARHYYNTSAARLGAGQAARLAAMLPNPRYYDRHRNTGYLNSRTATLTRRMRMVEIP . The protein contains hydrophobic regions characteristic of membrane association, consistent with its role in peptidoglycan synthesis at the cell membrane. When analyzing this sequence using hydropathy plots and secondary structure prediction tools, researchers should pay particular attention to the N-terminal region which likely contains the membrane anchor domain, while the C-terminal portion houses the catalytic domain responsible for transglycosylase activity.

What is the functional role of MtgA in Bordetella pertussis cell wall biosynthesis?

MtgA (Monofunctional biosynthetic peptidoglycan transglycosylase) in B. pertussis functions as a glycan polymerase, catalyzing the polymerization of lipid II precursors to form the glycan strands of peptidoglycan . In B. pertussis, peptidoglycan synthesis is particularly important for maintaining cell integrity despite the changing environmental conditions faced during infection. From a metabolic perspective, peptidoglycan synthesis represents a significant energy investment for the bacterium, requiring precise coordination with central carbon metabolism pathways . Researchers investigating MtgA function should consider its relationship with the tricarboxylic acid (TCA) cycle activity, as B. pertussis metabolic models have demonstrated functional connections between central metabolism and cell wall precursor synthesis.

What expression systems are most effective for producing recombinant B. pertussis MtgA?

For successful expression of recombinant B. pertussis MtgA, E. coli expression systems have proven effective as demonstrated in currently available recombinant protein products . When designing expression constructs, researchers should consider incorporating N-terminal His-tags for purification while preserving the native sequence (amino acids 1-242) . The optimal methodology involves using BL21(DE3) or similar E. coli strains with IPTG-inducible promoters for controlled expression.

For maximum protein yield, expression conditions should be optimized through factorial design experiments testing:

• Induction temperature (typically 18-30°C)

• IPTG concentration (0.1-1.0 mM)

• Expression duration (4-24 hours)

Membrane-associated proteins like MtgA often benefit from lower induction temperatures (18-20°C) to allow proper folding and prevent inclusion body formation.

What purification challenges are specific to MtgA and how can they be addressed?

Purifying MtgA presents several challenges due to its membrane-associated nature. A systematic purification approach should include:

• Cell lysis using either sonication or French press in buffer containing 20-50 mM Tris/PBS, pH 8.0

• Detergent selection trials (test CHAPS, DDM, Triton X-100) at concentrations above their critical micelle concentration

• IMAC purification using Ni-NTA resins with imidazole gradient elution (20-250 mM)

• Size exclusion chromatography for final polishing and detergent exchange

The purified protein should be stored in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 for optimal stability . Addition of 5-50% glycerol to the final formulation significantly improves long-term stability . Researchers should be cautious about repeated freeze-thaw cycles, which can substantially decrease enzyme activity; working aliquots should be stored at 4°C for up to one week, while long-term storage requires -20°C/-80°C conditions .

How does MtgA contribute to Bordetella pertussis virulence and pathogenesis?

MtgA's contribution to B. pertussis pathogenesis stems from its essential role in cell wall synthesis, which indirectly supports virulence factor production and bacterial persistence. While MtgA is not classified as a primary virulence factor like pertussis toxin, filamentous hemagglutinin (FHA), or pertactin , it plays a critical supporting role in maintaining bacterial fitness during infection.

Researchers investigating MtgA's role in pathogenesis should consider:

• Evaluating peptidoglycan structure in MtgA-deficient strains vs. wild-type

• Measuring stress resistance (osmotic, pH, temperature) in MtgA-attenuated strains

• Assessing adherence to respiratory epithelial cells when MtgA activity is altered

• Examining immune recognition of peptidoglycan fragments generated through MtgA activity

The metabolic interplay between MtgA activity and virulence factor production is particularly relevant, as peptidoglycan synthesis competes for carbon and energy resources that could otherwise support toxin production .

What is the potential of MtgA as a vaccine antigen or drug target against B. pertussis?

MtgA represents a potential novel target for B. pertussis vaccine development, distinctively different from current acellular vaccine components. Current acellular pertussis vaccines (aP) predominantly contain pertussis toxin, FHA, and pertactin , which have shown variable efficacy against different Bordetella species. Notably, these vaccines demonstrate limited cross-protection against B. parapertussis, with vaccine effectiveness calculations showing wide variability (from -15% to 66%) .

MtgA-based vaccine strategies could offer advantages through:

• Targeting of a highly conserved bacterial process essential for survival

• Potential cross-protection against multiple Bordetella species due to sequence conservation

• Combination with existing antigens to potentially enhance vaccine efficacy

As a drug target, MtgA is appealing because:

• Transglycosylase inhibitors would specifically target bacterial cell wall synthesis

• The enzyme has no human homolog, reducing potential toxicity

• Inhibition would likely synergize with β-lactam antibiotics that target transpeptidase activity

Researchers pursuing MtgA as a target should consider structure-based drug design approaches leveraging the crystal structures of homologous enzymes.

How can metabolic models be used to optimize experimental conditions for studying MtgA activity?

Genome-scale metabolic models of B. pertussis, such as the iBP1870 model, provide powerful frameworks for optimizing experimental conditions for MtgA studies . These models integrate 1,473 internal reactions and exchange reactions for 202 compounds, providing a comprehensive overview of B. pertussis metabolism .

To leverage these models for MtgA research, investigators should:

• Use flux balance analysis to predict metabolic states that maximize peptidoglycan precursor availability

• Identify key metabolites that may influence MtgA activity based on metabolic network connectivity

• Design minimal media compositions that support both growth and cell wall synthesis

The following experimental approach is recommended:

• Start with defined media (such as SS or CIT-NH3)

• Supplement with specific nutrients predicted by the model to enhance peptidoglycan synthesis

• Monitor growth parameters and correlate with MtgA activity measurements

A comparative analysis of growth conditions and their impact on MtgA activity can be structured as follows:

This approach allows researchers to systematically investigate how metabolic conditions influence MtgA function and peptidoglycan synthesis.

What advanced analytical techniques are most effective for characterizing MtgA enzymatic activity?

Characterizing MtgA enzymatic activity requires specialized techniques that can monitor the polymerization of peptidoglycan precursors. A comprehensive analytical approach should include:

• Radioactive assays: Using ¹⁴C-labeled lipid II to track incorporation into polymeric peptidoglycan

• HPLC-based methods: For analyzing reaction products and determining kinetic parameters

• Mass spectrometry: To characterize the structure of synthesized peptidoglycan fragments

• Fluorescence-based assays: Using dansylated or BODIPY-labeled lipid II analogues for continuous monitoring of enzyme activity

For kinetic characterization, researchers should determine:

• K_m for lipid II precursors

• V_max under varying ionic strength and pH conditions

• Inhibition profiles using known transglycosylase inhibitors (moenomycin derivatives)

When validating enzymatic activity, combinations of HPLC and mass spectrometry are particularly powerful for unambiguous product identification.

How does MtgA sequence and function vary across different B. pertussis strains?

MtgA sequence conservation across B. pertussis strains is generally high, but subtle variations may impact enzyme function or regulation. Genome-scale metabolic reconstructions of multiple B. pertussis strains have revealed that while the structural genes for key metabolic pathways (including those related to cell wall synthesis) are widely conserved, experimental validation shows strain-specific regulatory mechanisms shape actual metabolic capabilities .

When investigating strain variability in MtgA:

• Compare sequence alignments across clinical isolates, particularly those with ptxP1 vs. ptxP3 alleles

• Examine expression levels of MtgA in different strains under identical growth conditions

• Assess enzyme activity using standardized assays to identify functional differences

Researchers should note that the same genes may be differently regulated in various strains, as demonstrated by the observation that only some B. pertussis strains can utilize thiosulfate or exhibit fully functional TCA cycle activity despite containing the necessary genes .

How can contradicting experimental data regarding MtgA function be resolved through systems biology approaches?

Resolving contradictory data regarding MtgA function requires integrating systems biology approaches with targeted experimental validation. The genome-scale metabolic model iBP1870 provides a framework for this integration, helping to identify potential regulatory constraints beyond simple gene presence/absence .

A systematic approach to resolving contradictions includes:

• Multi-omics integration:

  • Correlate transcriptomics data with observed MtgA activity

  • Use proteomics to verify MtgA expression levels

  • Apply metabolomics to identify potential allosteric regulators

• Strain-specific regulatory analysis:

  • Compare growth of multiple strains in conditions requiring specific MtgA functionality

  • Identify regulatory elements affecting MtgA expression across strains

• Model refinement:

  • Update metabolic models to incorporate regulatory constraints

  • Test model predictions with targeted experiments

This approach recognizes that purely stoichiometric models have limitations in explaining strain-specific phenotypes, as regulatory mechanisms not considered in these models often explain experimental discrepancies . For example, while in silico analysis predicted growth on multiple amino acids requiring TCA cycle functionality, experimental validation showed strain-specific variations in this capability .

How might MtgA be explored in the context of improving pertussis vaccine production?

MtgA research has significant potential for improving pertussis vaccine production through metabolic engineering approaches. Current pertussis vaccine manufacturing faces challenges in optimizing antigen production, particularly pertussis toxin (PT) and filamentous hemagglutinin (FHA) .

A strategic approach for leveraging MtgA research in vaccine production includes:

• Metabolic balancing:

  • Modulate MtgA expression to optimize cell wall synthesis without diverting resources from antigen production

  • Adjust sulfur metabolism pathways (shown to impact PT production)

• Media optimization based on metabolic models:

  • Replace cysteine with thiosulfate as sulfur source (51% increase in PT yield)

  • Balance amino acid concentrations guided by metabolic flux analysis (29% increase in PT yield)

  • Optimize pH control strategies (48% increase with phosphoric acid vs. acetic acid)

The combination of these approaches has demonstrated significant improvements in vaccine antigen production:

These improvements underscore the value of metabolic engineering approaches informed by comprehensive understanding of B. pertussis physiology, including cell wall biosynthesis pathways involving MtgA .

What are the methodological challenges in studying MtgA interactions with other peptidoglycan synthesis enzymes?

Investigating MtgA interactions with other peptidoglycan synthesis enzymes presents several methodological challenges that require innovative experimental approaches. As a membrane-associated enzyme functioning in a complex multienzyme system, MtgA likely participates in protein-protein interactions that are technically difficult to characterize.

Key methodological challenges and potential solutions include:

• Membrane protein complexes:

  • Apply chemical crosslinking combined with mass spectrometry to capture transient interactions

  • Use bacterial two-hybrid systems modified for membrane proteins

  • Implement FRET-based approaches with fluorescently tagged enzyme pairs

• Reconstitution of multi-enzyme complexes:

  • Develop liposome-based reconstitution systems with purified components

  • Establish cell-free expression systems with defined membrane environments

  • Create chimeric constructs with solubilized domains for interaction studies

• In situ activity measurement:

  • Develop fluorescent D-amino acid derivatives to label newly synthesized peptidoglycan

  • Implement super-resolution microscopy to track enzyme localization during active synthesis

  • Utilize metabolic labeling combined with click chemistry for visualization

Researchers should note that these complex interactions are likely influenced by the metabolic state of the cell, as suggested by the finding that B. pertussis strains with the same gene content exhibit different metabolic capabilities due to regulatory differences .