Recombinant Aeromonas hydrophila subsp. hydrophila Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is mtgA and what is its role in bacterial cell wall synthesis?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) is a critical enzyme in Aeromonas hydrophila that catalyzes the polymerization of lipid II precursors to form peptidoglycan glycan strands, an essential component of bacterial cell walls. Unlike bifunctional penicillin-binding proteins (PBPs) that possess both transglycosylase and transpeptidase activities, mtgA specifically performs the transglycosylation reaction without transpeptidation capabilities.

The enzyme functions by forming glycosidic bonds between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues of peptidoglycan precursors. This process is fundamental for maintaining cell wall integrity, bacterial shape, and protection against osmotic pressure .

How is mtgA characterized in laboratory settings?

Several methods have been developed to characterize mtgA activity, each with distinct advantages:

When characterizing mtgA, researchers should consider that in vitro conditions significantly affect enzyme activity. Factors including temperature, DMSO concentration, detergent types, and divalent cations can dramatically impact observed activity levels .

How does mtgA contribute to Aeromonas hydrophila virulence and pathogenicity?

While not directly mentioned in the literature for mtgA specifically, research on related virulence factors in A. hydrophila provides insight into how cell wall biosynthetic enzymes like mtgA may contribute to pathogenicity:

• Structural integrity maintenance: By ensuring proper cell wall formation, mtgA likely contributes to bacterial survival within hosts, particularly under stress conditions encountered during infection.

• Immune evasion: Proper peptidoglycan structure can help bacteria evade host immune recognition or resist antimicrobial peptides.

• Growth and persistence: Efficient cell wall synthesis enables A. hydrophila to multiply rapidly in host environments, particularly in aquatic settings .

Research on A. hydrophila virulence demonstrates that this pathogen causes significant disease in multiple species, including fish, amphibians, reptiles, and humans. It's particularly problematic in aquaculture settings, where it causes motile Aeromonas septicemia (MAS) with symptoms including reddened fins, hemorrhages, and abdominal swelling .

What structural and functional differences exist between mtgA and other peptidoglycan transglycosylases?

Unlike bifunctional PBPs that contain both transglycosylase and transpeptidase domains, mtgA functions solely as a monofunctional transglycosylase. This specialization may allow for more sophisticated regulation of cell wall synthesis.

The structural architecture of mtgA likely includes:

• A transmembrane anchor domain

• A catalytic domain containing conserved motifs characteristic of glycosyltransferases

• Substrate-binding regions specific for lipid II interaction

Transmembrane regions play crucial roles in substrate binding and enzymatic activity. Studies have shown that full-length enzymes typically demonstrate higher activity levels compared to truncated forms lacking transmembrane portions, suggesting these regions are not merely anchors but actively participate in substrate recognition and catalysis .

What are the most effective assays for measuring mtgA activity?

Several assay systems have been developed for measuring transglycosylase activity, each with distinct applications:

• Fluorometric continuous assays: This approach utilizes lipid II substrates labeled with dansyl fluorophores, which exhibit increased quantum yield in hydrophobic environments. As transglycosylation proceeds, the environment of the fluorophore changes, resulting in measurable fluorescence changes. This method has been successfully adapted to multi-well formats for high-throughput screening .

• Gel-based assays: Using Tricine-SDS-PAGE, researchers can visualize the polymeric products of transglycosylase activity. This technique allows for the separation of glycan chains of different lengths, providing unique insights into enzyme processivity .

• HPLC-based methods: These provide quantitative analysis of transglycosylation products with medium sensitivity and are particularly useful for detailed product characterization .

For optimal results when working with recombinant mtgA:

• Maintain enzyme in appropriate detergent micelles

• Include necessary cofactors (particularly divalent cations)

• Control temperature and DMSO concentrations carefully

• Consider using full-length constructs rather than truncated versions

How can researchers effectively produce and purify recombinant mtgA?

While specific protocols for mtgA are not detailed in the provided literature, general approaches for membrane proteins like transglycosylases typically include:

• Expression system selection: E. coli-based systems are commonly used, but careful consideration of expression conditions is required for membrane proteins. Factors to optimize include:

  • Induction temperature (often lowered to 16-25°C)

  • Inducer concentration

  • Expression duration

• Solubilization strategies: Since mtgA is a membrane-associated protein, effective solubilization using detergents is critical. Common detergents include:

  • DDM (n-Dodecyl β-D-maltoside)

  • CHAPS

  • Triton X-100

• Purification approach:

  • Initial capture using affinity chromatography (His-tag purification)

  • Further purification via ion exchange or size exclusion chromatography

  • Maintaining detergent throughout the purification process

• Activity preservation: Including stabilizing agents such as glycerol and maintaining cold temperatures throughout purification helps preserve enzyme activity .

What techniques are available for detecting and identifying A. hydrophila expressing mtgA?

Recent advances in pathogen detection have led to the development of sophisticated methods for identifying A. hydrophila:

• CRISPR-Cas12a based detection: A rapid, sensitive visual detection method (dRAA-CRISPR/Cas12a) has been developed that integrates dualplex recombinase-assisted amplification with CRISPR/Cas12a systems. This method can detect as few as 2 copies of genomic DNA per reaction in approximately 45 minutes .

• Biochemical characterization: Traditional methods involve testing for characteristic enzymatic activities including:

  • DNase activity: A. hydrophila typically forms clear zones on DNase agar after 24 hours of incubation at 37°C

  • Gelatinase activity: A. hydrophila exhibits positive gelatinase activity, causing gelatin liquefaction in nutrient gelatin media after 72 hours incubation

• Genetic identification: PCR-based methods targeting specific genes, including virulence factors and metabolic enzymes, are widely used for definitive identification.

How can researchers differentiate between pathogenic and non-pathogenic strains expressing mtgA?

Multilocus sequence typing (MLST) and whole-genome sequencing (WGS) provide valuable tools for differentiating between pathogenic and non-pathogenic A. hydrophila strains:

• MLST analysis: Sequence types (STs) can be determined through analysis of housekeeping genes. For example, ST251 is considered a virulent strain clonal group responsible for recent MAS outbreaks .

• Whole-genome sequencing: This approach offers much higher resolution than MLST and has become more accessible for routine monitoring. WGS allows accurate diagnoses and facilitates disease outbreak investigations .

• Virulence gene profiling: Detection of specific virulence genes, such as those encoding hemolysins (hlyA) and aerolysin (aerA), can help identify pathogenic strains. Researchers have developed methods that can specifically detect A. hydrophila expressing these virulence genes .

How is mtgA being studied as a potential target for novel antibiotics?

Peptidoglycan transglycosylases represent attractive targets for antibiotic development for several reasons:

• Essential function: Transglycosylases are critical for bacterial cell wall synthesis, making them excellent targets for antibacterial agents.

• Surface accessibility: These enzymes function at the cell surface, potentially allowing easier access for inhibitors.

• Conservation: The enzymatic mechanism is conserved across bacterial species, potentially allowing for broad-spectrum activity.

Research approaches for mtgA inhibitor discovery include:

• High-throughput screening: The development of fluorescence-based assays has enabled screening of compound libraries against transglycosylase activity.

• Structure-based design: Understanding the structural features of the enzyme's active site can inform rational design of inhibitors.

• Natural product investigation: Compounds like moenomycin, a natural product that inhibits transglycosylases, provide valuable starting points for inhibitor development .

What is the relationship between mtgA and other bacterial virulence mechanisms?

In A. hydrophila, several virulence mechanisms have been identified that may work in concert with cell wall biosynthetic enzymes like mtgA:

• Type VI Secretion System (T6SS): This system delivers effector proteins to both eukaryotic and prokaryotic cells. Research has shown that VasH, a σ54-transcriptional activator, is required for T6SS functionality in A. hydrophila. This system contributes to bacterial cytotoxicity, resistance against host immune cleaning, and systemic dissemination during infection .

• Cell wall integrity: Properly functioning mtgA ensures cell wall integrity, which is essential for bacterial survival during infection and may indirectly support other virulence mechanisms by maintaining cellular viability under stress conditions.

Understanding the interplay between these systems can provide a more comprehensive picture of A. hydrophila pathogenesis and potentially identify multiple targets for therapeutic intervention .