Recombinant Enterococcus faecalis UDP-N-acetylenolpyruvoylglucosamine reductase (murB)

What is the function of MurB in E. faecalis cell wall biosynthesis and why is it significant for antimicrobial research?

MurB (UDP-N-acetylenolpyruvoylglucosamine reductase) catalyzes the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramate, the second step in peptidoglycan biosynthesis. This reaction is essential for bacterial cell wall formation as it produces a key precursor for subsequent steps in the pathway. In E. faecalis, MurB is part of a protein interaction network with other Mur enzymes (MurC, MurAA, MurAB, MurF, MurE, etc.) that collectively synthesize peptidoglycan .

The significance of MurB in antimicrobial research stems from several factors. First, E. faecalis is an opportunistic pathogen capable of causing serious infections, including endocarditis, urinary tract infections, wound infections, and bacteremia . Second, this organism exhibits intrinsic resistance to multiple antibiotics, including most β-lactams such as cephalosporins . Third, peptidoglycan biosynthesis enzymes like MurB are absent in mammalian cells, making them selective targets for antimicrobial development with potentially minimal side effects on human cells.

MurB inhibitors have shown antibacterial activity against vancomycin-resistant E. faecalis and other gram-positive bacteria, demonstrating the therapeutic potential of targeting this enzyme . Understanding MurB's structure and function could lead to the development of novel antibiotics against multidrug-resistant E. faecalis strains.

What methods are most effective for recombinant expression and purification of E. faecalis MurB?

Based on established protocols for recombinant expression of bacterial MurB enzymes, the following optimized methodology is recommended for E. faecalis MurB:

Expression System Design:

• Amplify the E. faecalis murB gene using PCR with primers containing appropriate restriction sites (e.g., NdeI/XhoI or NcoI/BamHI)

• Clone the PCR product into an expression vector such as pET-28b for C-terminal His-tagged fusion or pET-29A as used for S. aureus MurB

• Transform the recombinant plasmid into E. coli BL21(λDE3) for protein expression

Protein Expression Protocol:

• Grow transformed E. coli in LB medium with appropriate antibiotic at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with IPTG (typically 0.5-1 mM)

• Continue cultivation at 16-25°C for 16-20 hours to enhance soluble protein production

Purification Strategy:

• Harvest cells by centrifugation and resuspend in buffer containing protease inhibitors

• Lyse cells by sonication or French press

• Clarify lysate by centrifugation at 20,000×g for 30 minutes

• Purify His-tagged MurB using Ni-NTA affinity chromatography

• Further purify by ion exchange and size exclusion chromatography

For site-directed mutagenesis studies, researchers can use a BsaI-based cloning strategy to introduce specific mutations, as demonstrated for other E. faecalis proteins . Protein quality can be assessed using SDS-PAGE, Western blotting, and enzymatic activity assays measuring NADPH oxidation spectrophotometrically.

This expression and purification protocol has been successful for other MurB enzymes and can be adapted for E. faecalis MurB with minor modifications based on protein-specific characteristics.

How can the enzymatic activity of recombinant E. faecalis MurB be measured in vitro?

The enzymatic activity of recombinant E. faecalis MurB can be measured using several complementary approaches:

Spectrophotometric NADPH Oxidation Assay:

This is the most common method for measuring MurB activity, tracking the decrease in NADPH absorbance at 340 nm. The reaction mixture typically contains:

• 50 mM HEPES buffer (pH 7.5)

• 10 mM MgCl2

• 100-200 μM NADPH

• 50-100 μM UDP-N-acetylenolpyruvylglucosamine (substrate)

• Purified recombinant MurB enzyme (0.1-1 μg)

The reaction is monitored continuously at 25°C, and the initial velocity is calculated from the slope of the absorbance decrease. Enzyme kinetic parameters (Km, kcat, and kcat/Km) can be determined by varying substrate concentrations.

Fluorescence-Based Binding Assay:

For inhibitor studies, a fluorescence-binding assay can be used to determine binding affinities. When inhibitors bind to MurB, they can cause changes in intrinsic protein fluorescence or displacement of fluorescent probe molecules. This approach has been used successfully to demonstrate tight binding of 3,5-dioxopyrazolidine compounds to E. coli MurB with a dissociation constant of 260 nM .

HPLC Analysis of Reaction Products:

To directly quantify the formation of UDP-N-acetylmuramate, HPLC analysis can be employed. After stopping the reaction with acid or heat, the products can be separated on a C18 reverse-phase column and detected by UV absorbance at 260 nm.

Coupled Enzyme Assay:

MurB activity can also be measured in a coupled enzyme system where the product of MurB serves as a substrate for the next enzyme in the peptidoglycan biosynthesis pathway (MurC). This approach can be particularly useful for screening inhibitors that might affect multiple steps in the pathway.

When performing these assays, it's important to include appropriate controls, such as reaction mixtures without enzyme or substrate, to account for background rates and non-enzymatic reactions.

How does E. faecalis MurB contribute to antibiotic resistance mechanisms?

While direct evidence specific to MurB's role in E. faecalis antibiotic resistance is limited, several mechanisms can be inferred from related research:

Coordination with Resistance Regulatory Systems:

The CroRS two-component system in E. faecalis upregulates cell envelope biosynthesis genes in response to antimicrobials like teixobactin . This coordinated response likely includes MurB regulation as part of the cell wall biosynthesis pathway, contributing to antimicrobial tolerance.

Interaction with Vancomycin Resistance Mechanisms:

The vanB gene, which encodes a D-alanine--D-lactate ligase involved in vancomycin resistance, appears in the same protein interaction network as MurB in E. faecalis . This suggests potential coordination between MurB function and the vancomycin resistance mechanism, which involves modification of peptidoglycan precursors.

Biofilm Formation Support:

E. faecalis biofilm formation plays a key role in its virulence and drug resistance . As a cell wall biosynthesis enzyme, MurB likely contributes to the cell wall properties that facilitate biofilm formation. Proteins associated with metabolic processes and stress responses are differentially expressed in biofilms , which may include changes in MurB expression or activity.

Understanding MurB's specific contributions to antibiotic resistance mechanisms could provide insights for developing combination therapies that target both the resistance mechanisms and the essential peptidoglycan synthesis pathway.

What structural characteristics make MurB a potential target for novel antimicrobials against E. faecalis?

Several structural features make MurB an attractive target for antimicrobial development:

Essential Catalytic Domain:

MurB contains a critical FAD cofactor binding domain that is essential for its reductase activity. Crystal structures of E. coli MurB complexed with inhibitors show that compounds can interact with both active site residues and the FAD cofactor . These interactions provide multiple opportunities for inhibitor design targeting the conserved catalytic mechanism.

Substrate Binding Interactions:

Key substrate interactions in MurB involve specific residues that coordinate the substrate carboxylate (e.g., Arg159 and Glu325 in E. coli MurB) and diphosphate moiety (e.g., Tyr190, Lys217, Asn233, and Glu288) . These interaction sites offer potential for designing mimetics that competitively inhibit substrate binding.

Species-Specific Binding Pockets:

While the active site of MurB is largely conserved across bacterial species, there are likely species-specific differences in the binding pocket that could be exploited for selective inhibition of E. faecalis MurB over human enzymes or MurB from beneficial bacteria.

Absence in Human Cells:

The peptidoglycan synthesis pathway, including MurB, is absent in mammalian cells, making it an ideal selective target with potentially minimal off-target effects in humans.

Validated Target Class:

The success of 3,5-dioxopyrazolidines as MurB inhibitors with activity against vancomycin-resistant E. faecalis (MICs of 0.25 to 16 μg/ml) validates MurB as a druggable target.

For researchers developing novel antimicrobials against E. faecalis, comparative modeling of E. faecalis MurB based on known structures from other bacteria (such as the 2.4 Å resolution structure of E. coli MurB complexed with inhibitors ) could guide structure-based drug design efforts.

How does the CroRS two-component system regulate cell wall biosynthesis genes, including murB, in E. faecalis?

The CroRS two-component system has been identified as a master regulator of cell envelope homeostasis in E. faecalis . While specific regulation of murB is not directly mentioned in the available data, the following regulatory mechanisms have been established:

Global Regulation of Cell Envelope Biosynthesis:

RNA-seq analysis of wild-type and croRS mutant strains revealed a 132-gene CroRS regulon that is activated in response to antimicrobials like teixobactin. CroRS "upregulates biosynthesis of all major components of the enterococcal cell envelope in response to teixobactin" , likely including peptidoglycan synthesis enzymes such as MurB.

Mevalonate Pathway Coordination:

CroRS strongly upregulates the mevalonate (MVA) pathway genes (2.9–4.6 fold-log2) in response to antimicrobial challenge . This pathway produces isoprenoid precursors essential for cell wall lipid carrier undecaprenyl pyrophosphate (UPP), which transports peptidoglycan precursors (including MurB products) across the membrane.

Penicillin-Binding Protein Regulation:

CroRS upregulates expression of six penicillin-binding proteins in E. faecalis , which are involved in the final stages of peptidoglycan assembly following the reactions catalyzed by MurB and other Mur enzymes. This suggests coordinated regulation of the entire peptidoglycan synthesis pathway.

Sensor Kinase Activation:

The CroS sensor kinase likely detects cell envelope stress caused by antimicrobials, leading to phosphorylation of the CroR response regulator, which then activates expression of cell wall biosynthesis genes.

Based on these observations, researchers studying E. faecalis MurB should examine its expression patterns in wild-type versus croRS mutant strains under antimicrobial stress conditions to establish direct evidence of CroRS regulation. Promoter analysis of the murB gene could also reveal potential CroR binding sites.

What methods can be used to investigate MurB inhibitors as potential antibiotics against E. faecalis?

A comprehensive approach to investigating MurB inhibitors includes:

In Vitro Enzyme Inhibition Assays:

• Primary screening using spectrophotometric NADPH oxidation assays with purified recombinant E. faecalis MurB

• Determination of IC50 values for promising compounds

• Kinetic analysis to identify inhibition mechanisms (competitive, non-competitive, uncompetitive)

• Fluorescence-binding assays to measure binding affinities (Kd), as demonstrated for 3,5-dioxopyrazolidines with E. coli MurB (Kd of 260 nM)

Structural Studies:

• Co-crystallization of E. faecalis MurB with inhibitors for X-ray diffraction analysis

• Structure determination at high resolution (≤2.5 Å) to identify specific interactions, as achieved for E. coli MurB-inhibitor complexes at 2.4 Å resolution

• In silico docking and molecular dynamics simulations to screen virtual compound libraries

Antimicrobial Activity Assessment:

• Determination of Minimum Inhibitory Concentration (MIC) against E. faecalis clinical isolates, including vancomycin-resistant strains

• Time-kill kinetics to assess bactericidal versus bacteriostatic effects

• Combination studies with existing antibiotics to identify synergistic effects, similar to the synergy observed between mitoxantrone and vancomycin against vancomycin-resistant strains

Mode of Action Validation:

• Peptidoglycan biosynthesis studies using radioactive precursors to confirm on-target effects

• Analysis of peptidoglycan composition changes using HPLC or mass spectrometry

• Resistant mutant generation and whole-genome sequencing to identify resistance mechanisms

Advanced Preclinical Evaluation:

• Cytotoxicity assessment against mammalian cell lines

• Pharmacokinetic and pharmacodynamic studies in animal models

• Efficacy in E. faecalis infection models

Table 1: Example MIC Data for MurB Inhibitors Against E. faecalis

VREF: Vancomycin-resistant E. faecalis; VSE: Vancomycin-sensitive E. faecalis

How do mutations in cell wall biosynthesis genes affect E. faecalis virulence and biofilm formation?

Mutations in cell wall biosynthesis genes have significant effects on E. faecalis virulence and biofilm formation:

Impact on Antibiotic Resistance:

The deletion of murAA in E. faecalis leads to increased susceptibility to cephalosporins, while deletion of its homolog murAB does not affect resistance . This demonstrates that specific peptidoglycan synthesis enzymes have distinct roles in antibiotic resistance, which is a key virulence factor for hospital survival.

Biofilm Formation Effects:

Biofilm formation is a critical virulence attribute of E. faecalis that contributes to both pathogenicity and drug resistance . Proteomics analysis of strong versus weak biofilm formers reveals that "the major differences in biofilm formation arise from differences in metabolic activity levels" . Cell wall biosynthesis enzymes likely affect biofilm structure and stability through their impact on cell surface properties.

Stress Response Activation:

Proteins related to stress responsiveness and gluconeogenesis are upregulated in E. faecalis biofilms exposed to antimicrobial compounds . This suggests that cell wall stress caused by mutations or inhibition of biosynthesis enzymes may trigger compensatory mechanisms affecting virulence.

Cell Wall Composition Changes:

Mutations in cell wall biosynthesis genes can alter peptidoglycan structure, potentially affecting the presentation of surface virulence factors. In vancomycin-resistant strains, cell wall remodeling leads to altered permeability that affects susceptibility to other antimicrobials .

Immune Response Evasion:

E. faecalis can subvert immune responses through various mechanisms, including the secretion of gelatinase that cleaves complement components . Changes in cell wall composition due to mutations in biosynthesis genes may affect recognition by host immune receptors and subsequent immune responses.

These findings suggest that targeting MurB and other cell wall biosynthesis enzymes could not only directly inhibit bacterial growth but also reduce virulence and biofilm formation, providing multiple beneficial effects in treating E. faecalis infections.

What genetic manipulation techniques can be applied to study murB function in E. faecalis?

Several genetic manipulation techniques have been developed for E. faecalis that can be applied to study murB function:

Gene Deletion and Replacement Methods:

• Markerless Allelic Exchange: This approach uses plasmids like pCJK47 or its derivative pJRG32 for in-frame deletion of target genes without leaving antibiotic markers . For murB studies, this would allow clean deletion to assess essentiality and phenotypic effects.

• Cre-lox System: This two-step process involves:

  • Initial positive selection for double crossover with an antibiotic cassette

  • Subsequent removal of the marker using a temperature-sensitive cre-expressing plasmid, leaving only frt site scars

• Positive Selection Systems:

  • upp System: Utilizes the toxicity of 5-FU mediated by Upp (uracil phosphoribosyl transferase) to select for second crossover events

  • PheS System*: Makes cells sensitive to p-chloro-phenylalanine in the presence of a mutated phenylalanine tRNA synthetase allele

Expression Systems:

• Constitutive Expression: Plasmids like pDL278p23 can be used for constitutive expression of wild-type or mutant murB alleles

• Inducible Expression: For tight control of murB expression, especially if the gene is essential

• Protein Tagging: Addition of affinity or fluorescent tags to MurB for localization or interaction studies

Site-Directed Mutagenesis:

BsaI-based cloning strategy can be used to introduce specific mutations in murB to study structure-function relationships . This approach allows seamless fusion of PCR amplicons carrying desired mutations.

Overcoming DNA Transfer Barriers:

To efficiently transform E. faecalis, researchers must address barriers such as restriction-modification systems:

• Use of DC10B E. coli strain (lacking dcm gene) to prevent cytosine methylation

• Application of improved allelic exchange vectors like pIMAY-Z with blue/white screening

• Conjugation from permissive donor strains, such as E. faecalis CK111 containing pCF10-101

These techniques allow comprehensive functional characterization of murB, from essentiality testing to structure-function analysis and regulatory studies.

How does isoprenoid biosynthesis interact with peptidoglycan synthesis in E. faecalis antimicrobial tolerance?

The relationship between isoprenoid biosynthesis and peptidoglycan synthesis represents a key intersection in E. faecalis antimicrobial tolerance:

Shared Precursor Pathways:

In E. faecalis, the mevalonate (MVA) pathway synthesizes isoprenoid precursors farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP), which are essential for two critical components :

• Undecaprenyl pyrophosphate (UPP) - the lipid carrier that transports peptidoglycan precursors (including MurB products) across the membrane

• Demethylmenaquinone (DMK) - a quinone electron carrier used in respiratory electron transport

Coordinated Regulation:

The CroRS two-component system upregulates both pathways in response to antimicrobial stress:

• Four of the five MVA pathway genes are strongly upregulated (2.9–4.6 fold-log2) by CroRS in response to teixobactin

• Cell wall biosynthesis genes are similarly upregulated, suggesting synchronized production of both peptidoglycan precursors and their transport machinery

Key Role in Antimicrobial Tolerance:

Experimental evolution studies revealed that "truncation of HppS, a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone, was sufficient to rescue tolerance in the croRS deletion strain" . This surprising finding highlights how redirecting isoprenoid flux away from respiration and toward cell wall biosynthesis can enhance antimicrobial tolerance.

Proposed Model:

The CroRS system appears to function as "a master regulator of cell envelope biogenesis and a gate-keeper between isoprenoid biosynthesis and respiration to ensure tolerance against antimicrobial challenge" . This suggests that under antimicrobial stress, E. faecalis prioritizes cell wall integrity over respiratory function by directing isoprenoid precursors toward UPP synthesis rather than quinone production.

This intricate relationship suggests that targeting both pathways simultaneously could be an effective strategy against E. faecalis. Inhibitors of both MurB and MVA pathway enzymes might show synergistic effects by disrupting both peptidoglycan synthesis and its essential lipid carrier system.

What omics approaches can be used to understand the role of MurB in E. faecalis physiology?

Integrated omics approaches offer powerful tools to elucidate MurB's role in E. faecalis physiology:

Transcriptomics:

• RNA-seq Analysis: Compare gene expression profiles of wild-type and murB mutant strains under various conditions. This approach successfully identified the 132-gene CroRS regulon in response to teixobactin .

• Quantitative RT-PCR: Validate expression changes of murB and functionally related genes under different antimicrobial stresses.

• Transcription Start Site Mapping: Identify murB promoters and potential regulatory elements to understand its expression control.

Proteomics:

• Quantitative Proteomics: Use iTRAQ-based or label-free quantitative proteomics to identify changes in protein abundance in response to murB mutation or inhibition, similar to the approach used for biofilm studies .

• Protein-Protein Interaction Studies: Employ pull-down assays or cross-linking mass spectrometry to identify MurB interaction partners, validating and expanding the predicted interactions shown in STRING database .

• Phosphoproteomics: Determine if MurB undergoes post-translational modifications under different conditions.

Metabolomics:

• Targeted Metabolite Analysis: Measure levels of peptidoglycan precursors and intermediates in murB mutants or after MurB inhibition.

• Untargeted Metabolomics: Identify broader metabolic changes associated with altered MurB function.

Genomics:

• Whole-Genome Sequencing: Identify compensatory mutations that arise in response to murB mutations or MurB inhibition, similar to the approach used for evolved antibiotic-resistant strains .

• Comparative Genomics: Analyze murB sequence and genomic context across different E. faecalis strains and related species.

Systems Biology Integration:

These approaches would provide complementary insights into MurB's functional role, regulation, and importance in E. faecalis physiology, particularly under antimicrobial stress conditions.

How can crystallographic data of MurB be used to design potential inhibitors targeting E. faecalis?

Crystallographic data provides crucial structural insights for rational inhibitor design targeting E. faecalis MurB:

Structure-Based Drug Design Process:

• Template Selection: While no E. faecalis MurB crystal structure is directly mentioned in the search results, structures of E. coli MurB (including a 2.4 Å resolution structure complexed with an inhibitor ) can serve as templates for homology modeling.

• Homology Modeling: Generate a three-dimensional model of E. faecalis MurB based on sequence alignment with crystallized MurB proteins from other bacteria.

• Active Site Analysis: Identify the NADPH binding site, substrate binding site, and FAD cofactor binding region. Key interactions observed in E. coli MurB include :

  • Carboxylate interactions with residues equivalent to Arg159 and Glu325

  • Diphosphate moiety interactions with residues equivalent to Tyr190, Lys217, Asn233, and Glu288

  • FAD cofactor interactions with inhibitor compounds

• Virtual Screening: Use the modeled structure for in silico screening of compound libraries to identify potential binders.

Design Strategies Based on Known Inhibitors:

• Diphosphate Mimetic Approach: Design compounds that mimic the diphosphate moiety of the natural substrate, as this approach led to the identification of trisubstituted thiazolidinones as MurB inhibitors .

• 3,5-Dioxopyrazolidine Scaffold: Build upon the 3,5-dioxopyrazolidine scaffold that has shown activity against vancomycin-resistant E. faecalis (MICs of 0.25-16 μg/ml) .

• Fragment-Based Design: Identify small molecular fragments that bind to different sub-pockets of the active site and link them to create high-affinity inhibitors.

Optimization Considerations:

• Species Selectivity: Identify unique features of the E. faecalis MurB binding site to design inhibitors with enhanced selectivity.

• Pharmacokinetic Properties: Optimize inhibitors for bacterial penetration, particularly considering the cell wall structure of E. faecalis.

• Resistance Barrier: Design inhibitors that interact with highly conserved residues to minimize the development of resistance.

Table 2: Key Features for Structure-Based Design of E. faecalis MurB Inhibitors

This structure-based approach has been successful for developing inhibitors of other bacterial enzymes and could lead to novel antimicrobials specifically targeting E. faecalis MurB.