Recombinant Bartonella henselae UDP-N-acetylglucosamine--N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (murG)

What is the function of MurG in Bartonella henselae?

MurG is an essential glycosyltransferase that catalyzes a critical step in peptidoglycan biosynthesis, forming the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the bacterial cell wall . In B. henselae, this enzyme plays a crucial role in maintaining cell wall integrity, which is essential for bacterial survival during infection processes. B. henselae, the causative agent of cat scratch disease and more serious conditions in immunocompromised individuals, relies on proper cell wall synthesis for both structural integrity and host interaction . The enzyme belongs to the GT-B superfamily of glycosyltransferases and represents an attractive antimicrobial target due to its essential nature and absence in mammalian cells.

How does B. henselae MurG relate to cell wall biosynthesis pathways?

B. henselae MurG functions as part of the complex peptidoglycan biosynthesis pathway. The enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to lipid-linked intermediates, specifically to the C4 hydroxyl of GlcNAc . This creates the glycosidic linkage that is fundamental to peptidoglycan structure. The process occurs at the cytoplasmic face of the bacterial membrane, where MurG interacts with its membrane-bound substrate. Based on structural studies of E. coli MurG, the enzyme contains two domains with a cleft between them where catalysis occurs . The C-terminal domain contains a conserved sequence motif that is found in most members of the GT-B superfamily and is involved in binding the UDP-GlcNAc donor substrate.

What expression systems are suitable for recombinant B. henselae MurG production?

The optimal expression system depends on research objectives:

• E. coli systems: BL21(DE3) strains are commonly used due to their high yield. Based on techniques used for other Bartonella proteins, in-frame fusion with glutathione S-transferase (GST) in vectors like pMX (a modified pGEX-2T vector) has proven successful .

• Purification approaches: Typically involve affinity chromatography (His-tag or GST-tag), followed by ion exchange and size exclusion chromatography.

• Expression conditions: Optimization of induction parameters (temperature, IPTG concentration, duration) is essential for maximizing soluble protein yield.

• Protein solubility: Addition of fusion partners like MBP (maltose-binding protein) or SUMO can improve solubility if aggregation issues are encountered.

What are the key considerations for designing primers for cloning B. henselae murG?

When designing primers for cloning B. henselae murG:

• Include appropriate restriction enzyme sites that are absent within the gene sequence

• Consider codon optimization if expressing in heterologous systems like E. coli

• Design primers with appropriate melting temperatures (55-65°C)

• Add extra bases beyond restriction sites to ensure efficient enzyme digestion

• Verify primer specificity using BLAST to avoid non-specific amplification

Based on techniques used for other Bartonella genes, PCR conditions typically include initial denaturation at 95°C for 15 min when using HotStarTaq, followed by 30 cycles of: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min .

How does substrate specificity of B. henselae MurG differ from other bacterial species?

Understanding the substrate specificity of B. henselae MurG requires comparative analysis with other bacterial MurG enzymes. Based on studies of E. coli MurG, key differences may exist in the binding pockets for both donor and acceptor substrates .

Key binding site interactions in E. coli MurG include:

• Hydrogen bonds between the invariant residue E269 and the ribose 2′ and 3′ hydroxyls

• Interactions between the backbone amides of L265 and T266 with alpha phosphate oxygens

• Contact from the backbone amide of A264 to the C4 hydroxyl of GlcNAc

• Interactions from the side chain amide of Q288 to both C3 and C4 hydroxyls of GlcNAc

To characterize B. henselae MurG specificity:

• Perform sequence alignments to identify potentially variant residues in binding sites

• Use homology modeling based on the E. coli MurG crystal structure

• Conduct site-directed mutagenesis of predicted key residues

• Measure kinetic parameters (Km, kcat) with various substrate analogs

What structural features of B. henselae MurG determine its catalytic mechanism?

While the specific crystal structure of B. henselae MurG remains uncharacterized, insights from E. coli MurG suggest:

• The enzyme likely adopts the characteristic two-domain structure of GT-B glycosyltransferases

• The catalytic site is located in the cleft between domains

• Both domains contribute residues to substrate binding and catalysis

• The C-terminal domain primarily interacts with the nucleotide portion of UDP-GlcNAc

• The N-terminal domain likely interacts with the lipid-linked acceptor substrate

Mutation studies of E. coli MurG demonstrate that altering E269 to either alanine or aspartate increases the Km of UDP-GlcNAc by more than an order of magnitude, with minimal change in kcat . This suggests that this conserved glutamate plays a key role in donor substrate binding but not in catalysis. Similar structure-function relationships likely exist in B. henselae MurG.

How does membrane association affect B. henselae MurG function?

MurG's membrane association is critical for accessing its lipid-linked substrate. Investigating this relationship requires:

• Liposome reconstitution experiments with defined lipid compositions

• Surface plasmon resonance (SPR) with immobilized lipid bilayers

• Fluorescence-based assays to monitor protein-membrane interactions

• Mutagenesis of putative membrane-interaction domains

• Comparative activity studies between soluble and membrane-associated forms

When designing experiments, consider:

• The lipid composition of B. henselae membranes may differ from model organisms

• Different detergents may have varying effects on enzyme stability and activity

• Specific lipids might enhance enzymatic activity through allosteric effects

• Membrane association could affect substrate specificity or catalytic efficiency

What role does MurG play in B. henselae pathogenesis?

As an essential enzyme in peptidoglycan synthesis, MurG likely contributes significantly to B. henselae pathogenesis:

• MurG inhibition would affect cell wall integrity, potentially reducing bacterial viability during infection

• Changes in peptidoglycan structure may affect host immune recognition

• Cell wall integrity is crucial for resistance to host defense mechanisms

• The enzyme may be differentially regulated during different infection stages

To investigate this relationship:

• Develop conditional mutants to modulate MurG levels in vivo

• Study the effects of subinhibitory concentrations of MurG inhibitors on virulence

• Analyze changes in peptidoglycan composition during different growth conditions

• Assess whether MurG expression changes during intracellular growth in host cells

• Examine how MurG activity differs between actively growing and dormant bacteria

What are the optimal conditions for culturing B. henselae?

Based on published research, optimal conditions for B. henselae culture include:

• Use of Brucella broth histidine-hematin (BBH-H) medium

• pH control within 6.8 to 7.2 using 100 mM HEPES buffer

• Incubation at 37°C with shaking (180 rpm)

• Growth under aerobic conditions (21% O2)

• Addition of hematin conjugated to histidine for improved solubility

• Monitoring to prevent overgrowth, as stationary phase cultures can undergo phage induction

Under these optimized conditions, B. henselae exhibits a doubling time of approximately 3 hours and can reach cell densities of 5 × 10^8 to 1 × 10^9 CFU/ml . This represents a significant improvement over previously reported growth rates.

How can I develop an effective enzymatic assay for B. henselae MurG?

A comprehensive enzymatic assay should include:

Assay optimization should include:

• Determination of Km values for both substrates (typically 0.05-0.1 mM for UDP-GlcNAc based on E. coli MurG )

• Evaluation of pH and temperature optima

• Assessment of metal ion requirements

• Identification of potential inhibitors or activators

What approaches are useful for structural studies of B. henselae MurG?

Structural studies require:

• Protein preparation: High-purity (>95%) protein samples in stable buffer conditions

• Crystallization: Screening various conditions (pH, precipitants, additives)

• Co-crystallization: Including substrates or substrate analogs to stabilize active conformation

• Data analysis: Molecular replacement using E. coli MurG as a search model

• Alternative approaches: Cryo-electron microscopy for membrane-associated forms

Critical factors for successful crystallization include:

• Protein homogeneity and stability

• Removal of flexible regions that might impede crystal formation

• Testing both vapor diffusion and batch crystallization methods

• Surface entropy reduction mutations to promote crystal contacts

• Seeding techniques to improve crystal quality

How can site-directed mutagenesis illuminate B. henselae MurG function?

Site-directed mutagenesis provides powerful insights:

• Target conserved residues identified through sequence alignment with other MurG proteins

• Focus on residues predicted to be involved in substrate binding (e.g., equivalents to E. coli MurG E269, L265, T266, Q288)

• Create conservative mutations to subtly alter function

• Perform alanine-scanning mutagenesis to identify essential residues

The technical approach should include:

• PCR-based mutagenesis protocols

• Verification of mutations by DNA sequencing

• Expression and purification under identical conditions to wild-type enzyme

• Comprehensive kinetic analysis comparing mutant and wild-type enzymes

• Thermal stability testing to ensure mutations don't simply destabilize the protein

How should kinetic data for B. henselae MurG be analyzed?

Proper analysis of kinetic data requires:

• Fitting initial velocity data to appropriate enzyme kinetic models

• Determining key parameters (Km, Vmax, kcat, kcat/Km) for both substrates

• Using appropriate software like GraphPad Prism or DynaFit

• Creating double-reciprocal plots to identify potential inhibition mechanisms

• Comparing parameters with those of MurG from other species

Example data from E. coli MurG shows substrate kinetics:

These values demonstrate the importance of the 2′-hydroxyl for substrate recognition and the inability of the enzyme to use TDP-GlcNAc .

How can contradictory experimental results be reconciled when studying B. henselae MurG?

When facing contradictory data:

• Systematically review experimental conditions for variables that might explain differences

• Consider the impact of different expression systems or purification methods

• Evaluate reagent quality and purity, particularly substrates

• Assess enzyme stability under different assay conditions

• Implement orthogonal assay methods to validate findings

Based on contradiction detection frameworks:

• Identify the type of contradiction (self-contradictory data, contradicting data pairs, or conditional contradictions)

• Assess the importance of the conflicting statements to determine priority

• Consider the relative positions of conflicting information within datasets

• Apply chain-of-thought reasoning to resolve apparent contradictions

What approaches help determine whether an observed effect is MurG-specific?

To confirm MurG-specific effects:

• Use multiple, structurally diverse inhibitors targeting MurG

• Include appropriate control experiments with related enzymes

• Perform rescue experiments with overexpression of MurG

• Employ genetic approaches (if available) to modulate MurG levels

• Use complementary biochemical and cellular assays

• Examine effects on multiple MurG-dependent processes

• Compare results with known MurG inhibitors

How might comparative studies between B. henselae MurG and other bacterial MurGs inform antimicrobial development?

Comparative studies can reveal:

• Unique structural features of B. henselae MurG that could be selectively targeted

• Conserved regions that might serve as broad-spectrum targets

• Differences in substrate binding that could be exploited for selectivity

• Potential resistance mechanisms based on natural variations

• Synergistic targets in the peptidoglycan synthesis pathway

• Species-specific regulatory mechanisms

These insights could inform the development of:

• Selective inhibitors targeting unique features of B. henselae MurG

• Broad-spectrum agents targeting highly conserved regions

• Combination therapies targeting multiple steps in peptidoglycan synthesis

• Novel screening approaches focused on B. henselae-specific features

What potential exists for developing B. henselae MurG inhibitors as therapeutic agents?

Development strategies include:

• Structure-based design utilizing homology models and crystal structures

• Fragment-based screening approaches

• High-throughput screening of diverse chemical libraries

• Natural product screening, particularly from sources with known antimicrobial properties

• Peptidomimetic approaches targeting the active site or substrate binding regions

Challenges to address:

• Achieving selectivity over human glycosyltransferases

• Developing compounds that effectively penetrate the bacterial envelope

• Balancing broad-spectrum activity with B. henselae specificity

• Translating enzymatic inhibition to whole-cell antimicrobial activity

How could improved animal models advance B. henselae MurG research?

Based on current murine models, advancements could include:

• Development of models that better mimic human infection patterns

• Transgenic mouse models expressing human receptors relevant to B. henselae infection

• Models to study the effect of MurG inhibition in vivo

• Imaging techniques to monitor B. henselae infection in real-time

• Reporter systems to monitor bacterial cell wall synthesis in living animals

In existing murine models, B. henselae infection causes granulomatous inflammation in liver tissue, with the highest intensity during the fourth week of infection and resolving within 12 weeks post-infection . While cultivatable bacteria are cleared within 6 days, B. henselae DNA can be detected in liver tissue for at least 3 months, suggesting potential persistence mechanisms .

What emerging technologies could transform B. henselae MurG research?

Several cutting-edge approaches show promise:

• Cryo-electron microscopy for structural studies of membrane-associated forms

• Native mass spectrometry for studying protein complexes and substrate interactions

• Single-molecule enzymology to reveal mechanistic details

• Advanced computational methods for drug design

• CRISPR-Cas9 systems adapted for precise genetic manipulation of B. henselae

• Quantitative proteomics to study MurG expression under different conditions

• Time-resolved crystallography to capture catalytic intermediates