Recombinant Enterococcus faecalis D-alanyl-D-alanine carboxypeptidase (vanYB)

What is the primary function of VanYB in Enterococcus faecalis?

VanYB functions as a D,D-carboxypeptidase that hydrolyzes the terminal D-alanine from peptidoglycan precursors ending in D-alanyl-D-alanine. This enzymatic activity contributes to glycopeptide resistance by reducing the availability of the natural target for vancomycin in the bacterial cell wall. When introduced along with other vancomycin resistance genes, VanY carboxypeptidase can lead to a significant increase in vancomycin MIC (minimum inhibitory concentration), as demonstrated in complementation studies where its addition resulted in a fourfold increase from 16 to 64 μg/ml . This enzymatic action works synergistically with other resistance proteins in the VanB-type resistance pathway.

How does VanYB differ structurally and functionally from other Van-type proteins?

VanYB belongs to the VanB-type resistance system and differs from other Van-type carboxypeptidases in several key aspects:

• Unlike VanY in VanA-type resistance, VanYB is specifically associated with the VanB operon and contributes to vancomycin resistance without affecting teicoplanin susceptibility

• VanYB works in concert with VanB ligase, which produces D-Ala-D-Lac-ending precursors, whereas VanN, VanC, VanE, VanG, and VanL proteins produce D-Ala-D-Ser-ending precursors

• The VanYB carboxypeptidase is part of a resistance mechanism that can confer various levels of resistance specifically to vancomycin while maintaining susceptibility to teicoplanin, unlike VanA and VanD systems that confer resistance to both glycopeptides

What is the genetic context of vanYB within the vanB operon?

The vanYB gene resides within the vanB gene cluster, which is organized differently from other vancomycin resistance operons. Based on the available data:

• The vanB operon typically contains vanR, vanS, vanY, vanW, vanH, vanB, and vanX genes

• VanYB functions within this gene cluster to eliminate terminal D-Ala residues from cell wall precursors

• In the first reported VanB-type VRE outbreak in Japan, Southern blot analysis revealed that VanB-type determinants (including vanYB) resided on a 110-kbp plasmid in 19 out of 20 isolated strains

• Unlike some other resistance determinants, vanB gene clusters (including vanYB) can be plasmid-borne and transmissible, allowing for horizontal transfer of resistance

What are the optimal conditions for recombinant expression of VanYB?

While the search results don't provide specific optimized conditions for VanYB expression, researchers should consider these general approaches based on related Van-type protein research:

• Expression system selection: E. coli expression systems using vectors with inducible promoters (such as T7) are commonly employed for Van proteins

• Membrane protein considerations: As VanYB interacts with cell membrane components, expression systems maintaining a native-like membrane environment may improve functionality

• Nanodiscs approach: Similar to the study of VanSB, reconstitution of VanYB into nanodiscs might provide a native-like membrane environment while allowing purification and functional studies

• Detergent sensitivity: Van proteins can be sensitive to detergents; VanSB showed almost no activity in the presence of dodecyl maltoside (DDM) or lauryl dimethyl amine oxide (LDAO), suggesting careful selection of membrane mimetics is critical

• Activity assessment: D,D-carboxypeptidase activity can be monitored by measuring the release of terminal D-alanine or by analyzing changes in peptidoglycan precursor composition

How can researchers quantitatively assess VanYB enzymatic activity?

Several methodological approaches can be employed to measure VanYB D,D-carboxypeptidase activity:

• HPLC analysis of peptidoglycan precursors: Quantify the ratio of pentapeptide (UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala) to tetrapeptide (UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala) precursors, as performed in studies of VanY's contribution to resistance

• D-alanine release assay: Measure free D-alanine released by the carboxypeptidase activity using coupled enzymatic reactions

• Mass spectrometry: Analyze the structures of peptidoglycan precursors to detect modifications resulting from VanYB activity

• Antibiotic susceptibility testing: Assess the functional impact of VanYB expression by determining changes in vancomycin MIC, as demonstrated in complementation studies where VanY D,D-carboxypeptidase increased resistance fourfold (from 16 to 64 μg/ml)

• Fluorescent substrate analogs: Design fluorogenic substrates based on D-Ala-D-Ala termini that change properties upon hydrolysis

What strategies can be used to study VanYB contribution to vancomycin resistance in isolation?

To study VanYB's specific contribution to vancomycin resistance, researchers can employ these methodological approaches:

• Genetic complementation: Introduce vanYB alone or in combination with other van genes into a vancomycin-susceptible strain, similar to studies where VanY complementation of vanR, vanS, vanH, vanA, and vanX genes led to increased vancomycin resistance

• Gene knockout studies: Create isogenic mutants with vanYB deletion to assess the specific contribution to resistance levels

• Controlled expression systems: Use inducible promoters to modulate VanYB expression levels and correlate with resistance phenotypes

• Peptidoglycan analysis: Compare the composition of peptidoglycan precursors in strains with and without VanYB expression to quantify the specific modifications

• Competitive growth assays: Assess fitness costs or benefits of VanYB expression under varying vancomycin concentrations

How does VanYB interact with other components of the VanB resistance system?

VanYB functions within the complex VanB-type resistance system, interacting with multiple components:

• Functional relationship with VanSB sensor kinase: VanSB detects vancomycin through direct binding and activates the two-component regulatory system . VanYB expression is likely regulated downstream of this signaling cascade.

• Complementary activity with VanXB: While VanXB functions as a D,D-dipeptidase that hydrolyzes D-Ala-D-Ala dipeptides, VanYB acts as a D,D-carboxypeptidase removing terminal D-Ala from peptidoglycan precursors. These activities work together to eliminate susceptible precursors.

• Coordination with VanB ligase: VanB ligase produces modified D-Ala-D-Lac precursors, while VanYB removes natural D-Ala-D-Ala precursors, collectively leading to cell wall composition with reduced vancomycin binding affinity.

• Regulation within the VanB operon: Expression studies indicate that VanB-type resistance genes can be constitutively expressed, unlike some other resistance types that require induction .

What mechanisms underlie differential expression of VanYB in clinical isolates?

The expression of VanYB can vary among clinical isolates, potentially contributing to different resistance levels:

• Plasmid copy number effects: When carried on plasmids, differences in copy number can affect VanYB expression levels, as suggested by studies showing plasmid-borne resistance determinants in clinical isolates

• Promoter variations: Different clinical isolates may contain variations in the VanB operon promoter region, affecting transcription efficiency

• Regulatory system sensitivity: Mutations in the VanSB sensor kinase can alter its sensitivity to vancomycin, affecting downstream regulation of resistance genes including VanYB

• Integration site effects: When integrated into the chromosome, the genomic context can influence expression levels through local DNA topology or regulatory elements

• Post-transcriptional factors: Differences in mRNA stability, translation efficiency, or protein turnover could contribute to variable VanYB levels among isolates

How can structural modifications of VanYB alter its substrate specificity and catalytic efficiency?

Research on VanYB structural modifications could explore:

• Active site mutations: Targeted modifications of catalytic residues could alter substrate recognition or catalytic rate

• Domain swapping experiments: Exchanging domains between VanYB and other D,D-carboxypeptidases may create hybrid enzymes with novel properties

• Substrate binding pocket modifications: Alterations that affect the binding of D-Ala-D-Ala termini could modify specificity for different cell wall precursors

• Allosteric site engineering: Creating modified allosteric sites could enable regulation of VanYB activity by non-native molecules

• Membrane interaction domains: Modifications that alter membrane association could affect access to substrate pools within the cell

What are the optimal techniques for detecting and quantifying vanYB genes in clinical samples?

Based on methodologies developed for detecting vancomycin resistance genes, researchers can employ these approaches for vanYB detection:

• Real-time PCR assays: Quantitative PCR using vanYB-specific primers can detect and quantify gene presence in clinical samples. This approach has been validated for vanA and vanB genes, achieving detection limits of 46.9 dcp/ml for vanA and 60.8 dcp/ml for vanB

• Multiplex PCR: Detection of multiple van genes simultaneously, including vanYB, can improve efficiency in screening clinical isolates

• Next-generation sequencing: Targeted or whole-genome sequencing can identify and characterize vanYB genes and their genetic context

• Digital PCR: This technique can provide absolute quantification of vanYB gene copies without requiring standard curves

The performance characteristics of a PCR-based approach for vanB detection (which would include vanYB as part of the operon) include:

dcp/ml = DNA copies per milliliter

How can researchers distinguish between active and inactive forms of VanYB in experimental systems?

To differentiate between active and inactive VanYB:

• Activity-based protein profiling: Use chemical probes that selectively bind to active enzyme forms

• Enzymatic assays: Measure D,D-carboxypeptidase activity directly using synthetic substrates or natural peptidoglycan precursors

• Conformational antibodies: Develop antibodies that specifically recognize active or inactive protein conformations

• Mass spectrometry-based approaches: Monitor post-translational modifications that might regulate enzyme activity

• In situ peptidoglycan analysis: Analyze the actual modifications to cell wall components as direct evidence of VanYB activity

What are the challenges in detecting VanYB-mediated resistance in polymicrobial samples?

Detection of VanYB-mediated resistance in polymicrobial samples presents several challenges:

• Cross-reactivity with non-target organisms: Related D,D-carboxypeptidases in other species might cross-react in molecular assays

• False positives with vanB detection: Studies have shown that vanB detection can have lower specificity (92.2%) compared to vanA detection (100%), with false positives potentially due to the presence of vanB in non-enterococcal species

• Variable gene expression: Detection of the gene does not necessarily correlate with expression levels or functional resistance

• Sample preparation considerations: Inhibitory substances in clinical samples can affect assay performance, requiring optimal sample dilution (1:6 dilution reduced invalid results compared to 1:2 dilution)

• Distinguishing colonization from infection: Detection of vanYB-containing strains doesn't necessarily indicate clinical significance

How does VanYB compare functionally with other Van-type carboxypeptidases from different resistance phenotypes?

VanYB can be compared with other Van-type carboxypeptidases across several parameters:

VanYB works in concert with the VanB ligase system to produce resistance to vancomycin only, unlike the VanA and VanD D-Ala-D-Lac operons that confer resistance to both vancomycin and teicoplanin . The VanYB system provides various levels of resistance specifically to vancomycin, working through a different mechanism than D-Ala-D-Ser systems (VanC, VanE, VanG, and VanL) which typically confer lower resistance levels .

What is the evolutionary relationship between VanYB and other carboxypeptidases?

While the search results don't provide direct phylogenetic information about VanYB, its evolutionary relationships can be inferred:

• VanYB likely evolved within the context of the VanB resistance cluster, which has a distinct organization from other van operons

• The differential specificity of VanB-type resistance (vancomycin only) versus VanA-type (vancomycin and teicoplanin) suggests evolutionary divergence in response to different selective pressures

• The transferability of VanB-type resistance elements on plasmids suggests a history of horizontal gene transfer that has shaped the evolution of these resistance determinants

• Comparison with other D,D-carboxypeptidases, such as those in the d-Ala-d-Ser resistance types (VanC, VanE, VanG, and VanL) , could provide insights into the evolutionary diversification of these enzymes

How do experimental conditions affect VanYB activity in reconstituted systems?

Based on studies of related Van proteins, several experimental conditions likely influence VanYB activity:

• Membrane environment: Like VanSB, VanYB may be sensitive to the specific membrane mimetic used. VanSB showed essentially no activity in the presence of detergents like DDM or LDAO but maintained functionality in nanodiscs

• pH and ion requirements: D,D-carboxypeptidases typically have optimal pH ranges and may require specific metal ions for catalysis

• Substrate concentration: The kinetics of VanYB activity would be affected by the availability of peptidoglycan precursors

• Presence of vancomycin: The substrate-modifying activity of VanYB might be influenced by the presence of vancomycin, particularly if there are allosteric effects

• Synergy with other Van proteins: Reconstituted systems containing multiple Van proteins may show altered activities compared to isolated VanYB

What are the potential applications of VanYB in developing new strategies to combat vancomycin resistance?

Understanding VanYB could lead to several therapeutic strategies:

• Inhibitor development: Creating specific inhibitors of VanYB could potentially restore vancomycin sensitivity in VanB-type resistant strains

• Combination therapies: Drugs targeting VanYB in combination with vancomycin might overcome resistance mechanisms

• Diagnostic applications: VanYB-specific detection methods could improve identification of resistant strains and guide treatment decisions

• Resistance evolution modeling: Studying VanYB could help predict how resistance might evolve in response to new glycopeptide antibiotics

• Structure-based drug design: Detailed structural information about VanYB could inform the development of new antibiotics that remain effective against resistant strains

How might studying VanYB inform our understanding of bacterial cell wall remodeling during antibiotic stress?

Research on VanYB provides insights into cell wall adaptations:

• Stress response mechanisms: VanYB activity represents a specific bacterial adaptation to vancomycin pressure, illuminating how bacteria modify essential cellular structures in response to antibiotics

• Cell wall plasticity: The ability to substitute modified precursors while removing susceptible ones demonstrates the remarkable adaptability of bacterial cell walls

• Metabolic costs: Quantifying the energy requirements and growth penalties associated with VanYB-mediated resistance could reveal the biological trade-offs in resistance acquisition

• Coordination of cell wall enzymes: VanYB functions within a complex network of enzymes involved in peptidoglycan synthesis and modification, providing a model for studying enzyme coordination

• Transition dynamics: Studying how quickly VanYB can remodel existing peptidoglycan provides insights into the kinetics of resistance development

What role might VanYB play in the horizontal transfer and spread of vancomycin resistance?

VanYB contributes to the transferability and spread of resistance:

• Mobile genetic elements: VanB-type resistance determinants (including vanYB) have been found on plasmids , facilitating horizontal transfer between bacteria

• Transmission dynamics: Understanding VanYB's contribution to resistance levels could help model the selective advantages driving the spread of resistant strains

• Host range factors: Studying how VanYB functions in different bacterial backgrounds could reveal factors limiting or enabling resistance transfer between species

• Co-selection pressures: Investigating genetic linkages between vanYB and other resistance determinants could explain patterns of co-selection in clinical settings

• Fitness costs: Quantifying the metabolic burden of VanYB expression could help explain persistence or loss of resistance in the absence of selection pressure