Recombinant Pseudomonas putida Acyl-homoserine lactone acylase pvdQ (pvdQ), partial

What is PvdQ and what are its primary functions in bacterial systems?

PvdQ is an acylase enzyme originally identified in Pseudomonas aeruginosa PAO1 that exhibits a dual functional role in bacterial physiology. Primarily, it functions as a quorum-quenching enzyme that hydrolyzes the amide bond in long-chain N-acylhomoserine lactones (AHLs), particularly 3-oxo-C12-HSL, effectively degrading these quorum sensing signal molecules . This degradation leads to decreased virulence factor expression in Pseudomonas species. Additionally, PvdQ is involved in iron homeostasis pathways, with its expression being upregulated under iron starvation conditions . The enzyme plays a critical role in the biosynthesis pathways related to iron acquisition, though the exact mechanisms remain under investigation. Through these dual roles, PvdQ functions as a key regulatory enzyme influencing both bacterial communication and metal homeostasis.

What is the structural characterization of PvdQ and how does it compare to other acylases?

PvdQ exhibits a typical α/β heterodimeric N-terminal nucleophile (Ntn) hydrolase fold, sharing structural similarities with other clinically relevant acylases such as penicillin G acylase and cephalosporin acylase . The enzyme contains a distinctive, unusually large hydrophobic binding pocket that is ideally structured to accommodate and recognize C12 fatty acid-like chains of AHLs .

The active site architecture includes:

• Serβ1 functioning as the nucleophile in the catalytic mechanism

• Asnβ269 and Valβ70 forming the oxyanion hole for stabilizing reaction intermediates

• A substrate-binding pocket that undergoes conformational changes upon ligand binding, with volume reduction from approximately 1000 ų to 260 ų

These structural characteristics explain PvdQ's preference for long-chain AHLs, particularly those with 10-14 carbon acyl chains. Binding of substrate analogues such as dodecanoic acid or 3-oxo-C12 fatty acid induces subtle but significant conformational changes to the binding pocket, demonstrating the enzyme's structural flexibility to accommodate aliphatic chains of varying lengths .

What methodologies are recommended for detecting PvdQ expression and activity in bacterial cultures?

For comprehensive analysis of PvdQ expression and activity in bacterial cultures, researchers should implement a multi-technique approach:

Expression Detection:

• Western blot analysis using polyclonal PvdQ antibodies provides specific detection of protein expression patterns. Cultures should be sampled at various time points (0-24h) to track expression dynamics, with proteins separated on 4-12% polyacrylamide gels before transfer to nitrocellulose membranes for immunodetection .

• qRT-PCR for pvdQ gene expression quantification, particularly when comparing expression levels under varying iron concentrations or between wild-type and mutant strains.

Activity Assays:

• AHL degradation assay: Incubate purified PvdQ with synthetic AHLs (particularly 3-oxo-C12-HSL) followed by HPLC or LC-MS analysis to quantify remaining substrate and released products (homoserine lactone and fatty acid).

• Reporter strain assay: Use bacterial biosensor strains (e.g., Agrobacterium tumefaciens NTL4) containing a reporter gene fused to an AHL-responsive promoter to measure quorum-quenching activity in biological samples.

• Enzymatic kinetic analysis: Determine key parameters (Kcat, Km) using spectrophotometric detection of p-nitroaniline release from synthetic chromogenic substrates.

When analyzing PvdQ activity under different iron concentrations, CAA medium (5 g/L low-iron Bacto Casamino Acids, 1.54 g/L K₂HPO₄·3H₂O, 0.25 g/L MgSO₄·7H₂O) is recommended for controlled iron-limited conditions .

How can PvdQ be engineered to modify its substrate specificity?

Engineering PvdQ for altered substrate specificity requires strategic modifications to the enzyme's binding pocket. Based on successful variant generation, the following methodological approach is recommended:

1. Structure-guided rational design:

• Target residues lining the hydrophobic binding pocket that interact with the acyl chain of AHLs

• Key positions for modification include Lα146 and Fβ24, which have been successfully altered to create variants with shifted specificity

2. Recommended mutation strategy:

• For shifting specificity from 3-oxo-C12-HSL toward C8-HSL (Burkholderia-specific signal), introduce the double mutation Lα146W,Fβ24Y

• For other chain-length preferences, consider combinatorial mutagenesis of residues in the hydrophobic pocket

3. Screening methodology:

• Employ a two-tier screening approach:
  a) Initial high-throughput colorimetric assay with synthetic p-nitrophenyl esters of target chain lengths
  b) Secondary validation using native AHL substrates analyzed by HPLC or LC-MS

4. Kinetic characterization:

• Compare substrate specificity constants (kcat/Km) across multiple AHLs of varying chain lengths

• Analyze changes in binding pocket conformation using X-ray crystallography of enzyme-substrate complexes

The successful engineering of PvdQ variant Lα146W,Fβ24Y demonstrates that strategic mutations can effectively switch substrate preference from long-chain AHLs to medium-chain AHLs, creating enzymes with targeted activity against specific bacterial pathogens .

What experimental approaches can be used to evaluate PvdQ efficacy in infection models?

Evaluating PvdQ efficacy in infection models requires a systematic approach using both in vitro and in vivo systems:

In vitro models:

• Biofilm formation assays: Quantify biofilm biomass using crystal violet staining in the presence/absence of PvdQ or its variants, using static or flow cell systems.

• Virulence factor production: Measure pyocyanin, elastase, and rhamnolipid production in P. aeruginosa cultures treated with PvdQ.

• Swarming motility assays: Plate-based assays to assess bacterial swarming behavior, which is typically inhibited by PvdQ treatment under low iron conditions .

In vivo models:

• Galleria mellonella (wax moth) larval infection model:

  • Inject larvae with bacterial suspension (10³-10⁴ CFU)

  • Administer purified PvdQ enzyme (5-20 μg) via separate injection

  • Monitor survival rates over 72 hours

  • This model has successfully demonstrated PvdQ variant efficacy against Burkholderia infections

• Mouse pulmonary infection model:

  • Instill bacteria into mouse lungs via intranasal route

  • Administer nebulized PvdQ enzyme (0.5-2 mg/ml)

  • Evaluate bacterial load, inflammatory markers, and tissue damage

Analytical parameters:

• Bacterial load (CFU counting from tissue homogenates)

• Inflammatory cytokine profiling

• Histopathological analysis

• Survival rates and clinical scoring

When designing in vivo experiments, researchers should carefully consider dosing strategies, enzyme stability in physiological conditions, potential immunogenicity of the recombinant enzyme, and compatibility with existing antimicrobial treatments for combination therapy approaches.

How does PvdQ activity under iron-limiting conditions influence bacterial virulence mechanisms?

PvdQ demonstrates a complex regulatory relationship between iron availability, quorum sensing, and virulence in Pseudomonas aeruginosa. Under iron-limiting conditions, the following interconnected mechanisms have been observed:

Direct effects on quorum sensing:

• PvdQ expression is upregulated during iron starvation, as demonstrated by Western blot analysis of cultures grown in CAA medium compared to iron-replete LB medium

• Enhanced PvdQ activity leads to decreased 3-oxo-C12-HSL levels, disrupting the hierarchical quorum sensing network in P. aeruginosa

• This reduction in quorum sensing signal molecules subsequently affects downstream virulence factors controlled by the las system

Impacts on motility and biofilm formation:

• PvdQ deletion strains exhibit significantly different swarming motility patterns under iron-limiting conditions

• Wild-type P. aeruginosa PA14 showed normal swarming behavior, while ΔpvdQ mutants displayed complete absence of swarming motility on low-iron media

• This phenotype was independently confirmed in both deletion mutants and transposon mutants, suggesting a critical role for PvdQ in iron-limited mobility

Pyoverdine production regulation:

• PvdQ controls pyoverdine production, a major siderophore involved in iron acquisition

• The regulatory mechanism appears to function via the pyoverdine/iron pathway, ultimately influencing virulence expression

• This represents a critical adaptation mechanism allowing bacteria to coordinate virulence factor production with environmental iron availability

The complex interplay between PvdQ, iron availability, and virulence suggests that targeting this enzyme could provide a multi-faceted approach to virulence attenuation, particularly in iron-limited infection environments such as the cystic fibrosis lung.

What are the key structural determinants of PvdQ substrate specificity and how can this inform inhibitor design?

PvdQ exhibits distinct structural features that determine its substrate specificity, providing critical insights for rational inhibitor design:

Binding pocket architecture:

• The enzyme contains an unusually large hydrophobic binding pocket (initial volume ~1000 ų) that accommodates long acyl chains of AHLs

• Upon substrate binding, the pocket undergoes significant conformational changes, reducing to approximately 260 ų

• This induced-fit mechanism suggests that transition-state analogs could be particularly effective as inhibitors

Key substrate interaction residues:

• Nucleophilic residue: Serβ1 directly participates in catalysis by attacking the amide bond

• Oxyanion hole: Asnβ269 and Valβ70 stabilize the tetrahedral intermediate during catalysis

• Acyl chain accommodation: The hydrophobic pocket lined with aliphatic and aromatic residues determines chain length preference

Structural determinants for engineering:

• Mutations at positions Lα146 and Fβ24 have been demonstrated to alter substrate specificity, as exemplified by the successful development of variant PvdQ Lα146W,Fβ24Y with enhanced activity toward C8-HSL

• These positions represent critical "hotspots" for rational inhibitor design

Inhibitor design strategies:

• Transition-state mimetics incorporating features of the tetrahedral intermediate

• Non-hydrolyzable substrate analogs with modifications at the amide bond

• Compounds exploiting the induced-fit conformational changes

• Peptidomimetic inhibitors targeting the active site with specificity-determining residues

Understanding these structural determinants can guide the development of selective PvdQ inhibitors that could serve as therapeutic agents to restore quorum sensing in certain contexts or as research tools to probe PvdQ function in complex biological systems.

What is the comparative efficacy of PvdQ variants against different bacterial pathogens?

PvdQ variants demonstrate pathogen-specific efficacy profiles based on their engineered substrate preferences. The comparative performance of native PvdQ and its engineered variants reveals important considerations for targeted antimicrobial applications:

Key research findings:

• Wild-type PvdQ shows highest specificity toward long-chain AHLs (C10-C14), with particular preference for 3-oxo-C12-HSL produced by P. aeruginosa

• The engineered variant PvdQ Lα146W,Fβ24Y demonstrates a dramatic substrate switch, with enhanced hydrolytic activity toward C8-HSL, the primary signaling molecule in Burkholderia species

• In vivo protection studies in the G. mellonella infection model showed that while wild-type PvdQ offered minimal protection against B. cenocepacia infection, the engineered PvdQ Lα146W,Fβ24Y variant significantly increased survival rates

• The substrate switch achieved through strategic mutations demonstrates that PvdQ's scaffold is amenable to engineering approaches targeting diverse bacterial communication systems

These findings highlight the potential for developing pathogen-specific quorum-quenching enzymes based on the PvdQ scaffold, allowing for targeted intervention in polymicrobial infections where selective modulation of quorum sensing is desired.

What methodological challenges exist in the production and purification of recombinant PvdQ for research applications?

Producing recombinant PvdQ presents several methodological challenges that researchers must address to obtain functional enzyme for experimental applications:

Expression system optimization:

• Post-translational processing requirements: As an Ntn-hydrolase, PvdQ requires precise autoproteolytic processing to generate the active α/β heterodimeric form from a single precursor protein

• Expression host selection:

  • E. coli systems may yield inclusion bodies requiring refolding protocols

  • Pseudomonas-based expression systems may provide better processing but lower yields

  • Eukaryotic systems might be necessary for complex glycosylation if required for stability

Purification challenges:

• Heterodimeric nature: The α/β heterodimeric structure of processed PvdQ complicates purification strategies

• Recommended purification protocol:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Intermediate purification: Ion exchange chromatography to separate properly processed enzyme

  • Polishing: Size exclusion chromatography to ensure homogeneity and remove aggregates

• Quality control metrics:

  • SDS-PAGE analysis should confirm the presence of both α (~18-24 kDa) and β (~60 kDa) subunits

  • Western blot verification using polyclonal PvdQ antibodies

  • Activity assays with synthetic substrates to confirm functionality

Stability considerations:

• Buffer optimization: PvdQ activity and stability are affected by pH, ionic strength, and metal ion presence

• Storage conditions:

  • Short-term: 4°C in buffered solutions containing glycerol (10-20%)

  • Long-term: Flash-frozen aliquots at -80°C with cryoprotectants

Analytical validation:

• Kinetic characterization with model substrates

• Mass spectrometry to confirm proper processing

• Thermal shift assays to evaluate stability under different conditions

Addressing these methodological challenges is essential for producing high-quality recombinant PvdQ suitable for both basic research and potential therapeutic applications.

What are the potential applications of PvdQ in polymicrobial infection contexts?

Polymicrobial infections present unique therapeutic challenges that PvdQ-based approaches could potentially address through selective modulation of bacterial communication:

Targeted quorum sensing disruption:

• PvdQ variants with engineered substrate specificity could selectively target specific pathogens within polymicrobial communities

• Wild-type PvdQ preferentially degrades long-chain AHLs (C10-C14) common in P. aeruginosa, while engineered variants like PvdQ Lα146W,Fβ24Y target medium-chain AHLs (C8) in Burkholderia species

• This selective targeting allows for precision modulation of microbial communication networks

Potential clinical applications:

• Cystic fibrosis lung infections: Where P. aeruginosa and Burkholderia species often co-exist

• Chronic wound infections: Characterized by complex polymicrobial biofilms

• Urinary tract infections: Where quorum sensing plays a role in pathogenesis

Delivery strategies for polymicrobial contexts:

• Combination enzyme therapy using multiple PvdQ variants with different specificities

• Immobilized enzyme approaches on wound dressings or implant surfaces

• Inhalation delivery for respiratory infections via nebulization or dry powder formulations

Research priorities:

• Characterization of enzyme stability and activity in complex biological fluids

• Development of multi-specific PvdQ variants capable of degrading multiple AHL types

• Assessment of PvdQ impact on beneficial members of polymicrobial communities

• Investigation of potential synergistic effects when combined with conventional antimicrobials

The ability to engineer PvdQ specificity creates opportunities for precise manipulation of bacterial communication in complex microbial communities, potentially allowing suppression of virulence without disrupting beneficial microbiota or driving resistance development.

How might PvdQ be integrated into antimicrobial resistance mitigation strategies?

PvdQ represents a promising non-traditional antimicrobial approach that could contribute to combating antimicrobial resistance through several mechanisms:

Anti-virulence strategy advantages:

• Unlike conventional antibiotics, quorum-quenching enzymes like PvdQ target virulence rather than growth, potentially reducing selection pressure for resistance development

• By degrading AHL signal molecules, PvdQ interferes with pathogenicity without directly killing bacteria, allowing host defenses to clear infection

• This approach could preserve beneficial microbiota while selectively targeting pathogen virulence factors

Potential integration strategies:

• Adjuvant therapy with conventional antibiotics:

  • PvdQ could enhance antibiotic efficacy by disrupting biofilm formation

  • Lower effective doses of antibiotics might be achievable through combinatorial approaches

  • Reduced biofilm formation could improve antibiotic penetration

• Biotherapeutic applications:

  • Direct administration of purified enzyme to infection sites

  • Probiotic delivery systems expressing recombinant PvdQ

  • Immobilized enzyme approaches for medical devices or implant surfaces

Research priorities for resistance mitigation:

• Long-term evolution studies to assess potential resistance development to quorum-quenching approaches

• Investigation of PvdQ variant combinations to target multiple quorum sensing systems simultaneously

• Exploration of synergistic effects between PvdQ and immune system components

• Development of formulations that protect enzyme activity in diverse clinical environments

Anticipated challenges:

• Potential emergence of alternative communication pathways in bacteria

• Host immunogenic responses to repeated enzyme administration

• Delivery challenges in certain infection contexts

• Regulatory pathway development for novel antimicrobial approaches

The integration of PvdQ into antimicrobial strategies represents an innovative approach to address the growing challenge of antimicrobial resistance, particularly for chronic infections where conventional antibiotics have limited efficacy due to biofilm formation and resistance development.

What are the most significant recent advances in PvdQ research and applications?

Recent advances in PvdQ research have expanded our understanding of this enzyme's potential as both a research tool and therapeutic agent. Key developments include:

• Structural elucidation of PvdQ's mechanism of action, identifying Serβ1 as the nucleophile and Asnβ269 and Valβ70 as the oxyanion hole residues in AHL degradation

• Successful engineering of PvdQ variants with altered substrate specificity, as demonstrated by the PvdQ Lα146W,Fβ24Y variant capable of effectively hydrolyzing C8-HSL produced by Burkholderia species

• Demonstration of in vivo efficacy in infection models, particularly the ability of engineered PvdQ variants to rescue Galleria mellonella larvae upon infection with Burkholderia cenocepacia

• Elucidation of PvdQ's dual role in quorum sensing and iron homeostasis, revealing complex regulatory interactions in Pseudomonas aeruginosa virulence mechanisms

• Development of improved expression and purification protocols facilitating the production of functional recombinant enzyme for research applications