Recombinant Salmonella paratyphi C Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is Salmonella paratyphi C Monofunctional Biosynthetic Peptidoglycan Transglycosylase (mtgA)?

Salmonella paratyphi C Monofunctional Biosynthetic Peptidoglycan Transglycosylase (mtgA) is a 242-amino acid protein involved in bacterial cell wall biosynthesis. This enzyme catalyzes the polymerization of glycan strands during peptidoglycan assembly, a critical process for maintaining bacterial cell wall integrity. The recombinant version is typically expressed with an N-terminal His-tag in E. coli expression systems, allowing for purification via affinity chromatography .

What are the optimal storage and handling conditions for recombinant mtgA?

For optimal stability, recombinant Salmonella paratyphi C mtgA should be stored at -20°C to -80°C upon receipt. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided. The protein is typically supplied as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0. For reconstitution, it is recommended to briefly centrifuge the vial before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% as default) is advised for long-term storage .

What expression systems are most effective for producing recombinant Salmonella paratyphi C mtgA?

E. coli expression systems are the standard choice for recombinant production of Salmonella paratyphi C mtgA, offering high yields and simplified purification when combined with affinity tags. The protein is typically expressed with an N-terminal His-tag to facilitate purification using metal affinity chromatography . This approach parallels recombinant protein expression methods used for other bacterial proteins, where the coding regions are cloned into expression plasmids that allow for unlimited production under controlled conditions .

How can researchers verify the activity of purified recombinant mtgA?

Researchers can verify mtgA activity through multiple complementary approaches:

• Enzymatic assays: Measuring transglycosylase activity using synthetic peptidoglycan precursors labeled with fluorescent or radioactive markers.

• Complementation studies: Testing the ability of recombinant mtgA to restore peptidoglycan synthesis in mtgA-deficient bacterial strains.

• Structural integrity assessment: Using circular dichroism or thermal shift assays to confirm proper protein folding.

• Binding studies: Evaluating interaction with known substrates or inhibitors using techniques such as surface plasmon resonance.

These verification methods are consistent with approaches used for other recombinant enzymes in bacterial systems .

What purification strategies maximize yield and purity of recombinant mtgA?

This multi-step purification approach is designed to achieve protein purity greater than 90% as required for structural and functional studies .

How does mtgA contribute to Salmonella paratyphi C pathogenicity?

Salmonella paratyphi C mtgA plays a crucial role in bacterial pathogenicity through its function in peptidoglycan synthesis. As a monofunctional transglycosylase, mtgA contributes to cell wall integrity, which is essential for bacterial survival during infection processes. The cell wall provides protection against host immune defenses and environmental stresses encountered during pathogenesis. Disruption of mtgA function could potentially attenuate virulence by compromising cell wall integrity, making it an important target for understanding Salmonella pathogenicity mechanisms .

What experimental approaches are most effective for studying mtgA's role in cell wall synthesis?

To investigate mtgA's role in cell wall synthesis, researchers can employ these approaches:

• Gene knockout/complementation studies: Creating mtgA deletion mutants followed by phenotypic characterization and complementation with recombinant mtgA.

• Fluorescent D-amino acid labeling: Visualizing peptidoglycan synthesis in real-time using fluorescent D-amino acids to track incorporation patterns in wild-type versus mtgA-mutant strains.

• Electron microscopy: Examining ultrastructural changes in the cell wall architecture resulting from mtgA modification.

• Muropeptide analysis: Using HPLC and mass spectrometry to characterize peptidoglycan composition differences attributable to mtgA activity.

• Inhibitor studies: Employing specific transglycosylase inhibitors to examine the resulting phenotypic effects on bacteria.

These approaches provide complementary insights into mtgA function within the complex process of bacterial cell wall assembly.

How can recombinant mtgA be utilized in vaccine development research?

Recombinant Salmonella paratyphi C mtgA holds potential for vaccine development through several research applications:

• Antigen discovery: As a surface-exposed enzyme involved in cell wall synthesis, mtgA could represent a target for protective antibodies.

• Attenuated live vaccine strains: Engineering Salmonella strains with modified mtgA expression could create attenuated strains for live vaccine candidates.

• Adjuvant development: Recombinant mtgA could be investigated as a potential molecular adjuvant to enhance immune responses to other antigens.

• Challenge models: Similar to approaches used with Salmonella Paratyphi A, controlled human infection models could be developed to evaluate vaccine candidates targeting mtgA or related pathways .

The implementation of these approaches would require extensive preclinical validation before advancing to clinical studies.

What structural features of mtgA might be exploited for antimicrobial development?

Key structural features of mtgA that could be exploited for antimicrobial development include:

• Catalytic domain: The active site responsible for transglycosylase activity presents a potential target for small molecule inhibitors.

• Substrate binding pocket: The region that binds peptidoglycan precursors could be targeted by competitive inhibitors.

• Protein-protein interaction surfaces: Interfaces where mtgA interacts with other cell wall synthesis machinery components could be disrupted by peptide inhibitors.

• Allosteric sites: Regions distant from the active site that influence enzyme activity could be targeted for allosteric modulation.

Targeting these features could lead to novel antimicrobials against multidrug-resistant Salmonella paratyphi C infections, which is particularly relevant given the rising concern of antimicrobial resistance in enteric pathogens .

How does mtgA from Salmonella paratyphi C compare with orthologs from other enteric pathogens?

Comparative analysis of mtgA orthologs across enteric pathogens reveals both conserved and variable regions that may influence species-specific functions:

• Catalytic domain conservation: The core transglycosylase domain typically shows high sequence conservation across species, reflecting the essential nature of the enzymatic function.

• N-terminal region variation: The signal peptide and membrane association domains often show greater sequence divergence, potentially influencing localization patterns.

• Species-specific insertions/deletions: These structural differences might affect substrate specificity or interactions with other cell wall synthesis components.

• Post-translational modification sites: Variation in phosphorylation or glycosylation sites could influence enzyme regulation in different bacterial species.

Understanding these evolutionary relationships provides context for interpreting experimental results with recombinant mtgA and may guide the development of species-specific inhibitors.

What are the common challenges in expressing and purifying active recombinant mtgA?

Researchers frequently encounter several challenges when working with recombinant Salmonella paratyphi C mtgA:

• Membrane association: As mtgA contains hydrophobic regions, it may form inclusion bodies or aggregate during expression. Solution: Optimizing expression temperature (typically lowering to 16-20°C) and including detergents or solubilizing agents in purification buffers.

• Proteolytic degradation: The protein may be susceptible to proteolysis. Solution: Including protease inhibitors throughout purification and minimizing handling time.

• Activity loss during purification: The enzyme may lose activity during purification steps. Solution: Incorporating stabilizing agents such as glycerol or specific substrate analogs in storage buffers .

• Proper folding: Ensuring correct disulfide bond formation if present. Solution: Expression in specialized E. coli strains that facilitate disulfide bond formation in the cytoplasm .

These challenges parallel those encountered with other recombinant proteins requiring specific optimization strategies.

How can researchers effectively utilize recombinant antibodies against mtgA for localization studies?

Recombinant antibodies offer significant advantages for mtgA localization studies compared to conventional antibodies:

• Defined specificity: Recombinant antibodies with sequence-verified binding domains ensure consistent epitope recognition across experiments.

• Engineered formats: Researchers can generate full-length IgG (R-mAbs) or smaller fragments like ScFVs depending on the experimental needs.

• Multiplex compatibility: Recombinant antibodies can be engineered with different constant regions allowing simultaneous detection with subclass-specific secondary antibodies.

• Direct labeling options: Addition of epitope tags or sites for direct conjugation enables flexible detection strategies .

When developing immunofluorescence protocols, researchers should validate antibody specificity using appropriate controls, including mtgA knockout strains, and optimize fixation conditions to preserve epitope accessibility while maintaining cellular ultrastructure.

How might genomic surveillance approaches be applied to tracking mtgA evolution in Salmonella paratyphi C?

Genomic surveillance of mtgA in Salmonella paratyphi C could adapt approaches similar to those developed for Salmonella Paratyphi A:

These surveillance approaches could be implemented using open-source bioinformatic tools similar to the Paratype framework developed for Salmonella Paratyphi A, allowing standardized analysis across research groups .

What role might mtgA play in bacterial interactions with bacteriophages?

Emerging research suggests potential roles for mtgA in bacteriophage-bacteria interactions:

• Phage receptor modification: Changes in peptidoglycan structure mediated by mtgA could alter phage binding sites on the bacterial surface.

• Phage-encoded inhibitors: Some bacteriophages encode proteins that specifically target transglycosylases like mtgA to disrupt bacterial cell wall synthesis during infection.

• Prophage integration effects: As suggested by search result , pathogenicity-associated prophages in Salmonella paratyphi C might influence mtgA expression or function.

• Cell wall stress responses: Phage infection could trigger stress responses that modify mtgA activity as part of bacterial defense mechanisms.

These interactions represent an emerging area of research that connects bacterial cell wall synthesis machinery with phage biology and bacterial pathogenicity .

What are the most promising future research directions for Salmonella paratyphi C mtgA?

Future research on Salmonella paratyphi C mtgA should prioritize:

• Structure-function relationships: Determining high-resolution structures of mtgA in complex with substrates or inhibitors to guide rational drug design.

• In vivo dynamics: Developing tools to monitor mtgA activity during different stages of infection using biosensors or activity-based probes.

• Regulatory networks: Elucidating how mtgA expression and activity are regulated in response to environmental stresses encountered during infection.

• Host-pathogen interactions: Investigating potential interactions between mtgA or its products and host immune receptors.

• Combination therapy approaches: Exploring synergistic effects between mtgA inhibitors and existing antibiotics.

These research directions build upon current knowledge of mtgA while addressing critical gaps that may lead to novel therapeutic interventions for Salmonella paratyphi C infections.

How might emerging technologies advance our understanding of mtgA function?

Several emerging technologies hold promise for advancing mtgA research:

• Cryo-electron microscopy: Enabling visualization of mtgA in complex with other cell wall synthesis machinery components at near-atomic resolution.

• CRISPR-Cas9 genome editing: Facilitating precise genetic manipulation to create reporter fusions and conditional expression systems in Salmonella.

• Single-molecule enzymology: Allowing real-time observation of individual mtgA molecules during catalysis to resolve mechanistic details.

• Artificial intelligence for protein design: Enabling the rational design of mtgA variants with altered specificity or activity for biotechnological applications.

• Microfluidic cell wall biosensors: Developing high-throughput systems to screen potential mtgA inhibitors against living bacteria.