Recombinant Burkholderia cenocepacia Acyl carrier protein (acpP)

What is the role of Acyl Carrier Protein (acpP) in B. cenocepacia quorum sensing systems?

Acyl Carrier Protein (acpP) in B. cenocepacia serves as an essential component in quorum sensing (QS) pathways by providing acylated acyl-carrier protein (acyl-ACP) substrates. These acyl-ACP molecules interact with acyl homoserine lactone (AHL) synthases such as CepI, which uses them alongside S-adenosyl methionine (SAM) to synthesize N-octanoyl-homoserine lactone (C8-HSL) and smaller amounts of N-hexanoyl-homoserine lactone (C6-HSL) . These signaling molecules are fundamental to the CepIR QS system that regulates virulence factors, biofilm formation, and pathogenicity in B. cenocepacia.

How does acpP structurally interact with AHL synthases?

Molecular docking studies suggest that acpP forms specific interactions with AHL synthases via arginine residues in the synthase protein. Research on CepI has identified "putative binding sites with predicted strong stabilization energies... near the Arg residues involved in interaction with ACP" . These interactions occur outside the SAM and acyl substrate binding sites, which explains the non-competitive inhibition patterns observed with certain inhibitors. The specific three-dimensional arrangement facilitates the transfer of the acyl group from acpP to the AHL synthase during signal molecule biosynthesis.

What is the relationship between acpP and virulence in B. cenocepacia infections?

The acpP protein indirectly contributes to B. cenocepacia virulence by enabling QS signal production, which regulates multiple virulence factors. When QS systems are impaired, either through mutation or inhibition, B. cenocepacia shows significantly reduced production of proteases, siderophores, and impaired biofilm formation . In vivo studies using C. elegans infection models have demonstrated that disruption of the QS system reduces pathogenicity, with survival rates increasing from 65% in untreated infections to 91% when QS inhibitors are administered .

What are effective strategies for recombinant expression of B. cenocepacia acpP?

Successful heterologous expression of B. cenocepacia proteins has been achieved using E. coli systems. For optimal expression of GC-rich Burkholderia genes, codon optimization is recommended, as demonstrated with the expression of dCas9 in Burkholderia species . The following expression protocol is suggested:

• Clone the codon-optimized acpP gene into an expression vector with an N-terminal affinity tag

• Transform into E. coli BL21(DE3) or similar expression strain

• Culture at 30°C to mid-log phase before induction with 0.5-1.0 mM IPTG

• Continue expression at lower temperature (16-25°C) to improve protein solubility

• Purify using affinity chromatography followed by size exclusion chromatography

To ensure proper post-translational modification, co-expression with a phosphopantetheinyl transferase may be necessary to produce the holo-form of acpP.

How can CRISPRi technology be applied to study acpP function in B. cenocepacia?

CRISPRi provides a powerful tool for investigating acpP function through controlled gene silencing. Based on recent developments, the following methodological approach is recommended:

• Integration of codon-optimized dCas9 into the B. cenocepacia chromosome using the mini-CTX system with φCTX integrase

• Place dCas9 under control of a rhamnose-inducible promoter for tunable expression

• Design multiple gRNAs targeting different regions:

  • Non-template strand near the promoter

  • Non-template strand near the start codon

  • Template strand near coding regions

• Express gRNAs from plasmid-borne constructs

• Induce with variable rhamnose concentrations (0.005% to 0.2%) to achieve tunable repression

• Confirm knockdown efficiency using RT-qPCR

What assays can quantify the functional implications of acpP manipulation?

Following manipulation of acpP expression, several phenotypic assays can quantify the functional outcomes:

• Protease production: Use casein or gelatin degradation assays to measure protease activity in culture supernatants

• Siderophore production: The Chrome Azurol S (CAS) assay can detect and quantify siderophore production

• Biofilm formation: Crystal violet staining can quantify biofilm biomass; confocal microscopy can visualize biofilm architecture and thickness

• Virulence assessment: C. elegans infection models provide a reliable system to measure pathogenicity, with survival rates serving as a quantitative measure of virulence

• Growth kinetics: Monitor growth on different carbon sources to assess metabolic capabilities

How can protein-protein interactions between acpP and partner enzymes be characterized?

Characterizing the interactions between acpP and its partner enzymes requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP): Using tagged versions of acpP to pull down interaction partners

• Bacterial two-hybrid assays: To screen for potential interaction partners in vivo

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics

• Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of binding

• Crosslinking coupled with mass spectrometry: To identify interaction interfaces

• Fluorescence resonance energy transfer (FRET): For studying interactions in living cells

Importantly, when analyzing these interactions, consider that acpP may form different complexes depending on its acylation state and the presence of regulatory molecules.

What approaches are most effective for developing inhibitors targeting acpP-dependent pathways?

Developing inhibitors that target acpP-dependent pathways represents a promising anti-virulence strategy. Based on successful approaches with related targets, consider:

• Structure-based design: Using computational modeling to design molecules that interfere with acpP-enzyme interactions

• Scaffold-hopping from known inhibitors: Diketopiperazines have proven effective against CepI with IC50 values in the micromolar range

• High-throughput screening: Adaptation of enzymatic assays for screening compound libraries

• Fragment-based drug discovery: Building inhibitors from small molecular fragments with weak binding affinity

Validation should include in vitro enzymatic assays, virulence factor production assays, biofilm formation assays, and in vivo infection models such as C. elegans .

How does the role of acpP differ across Burkholderia species and in different environmental conditions?

Understanding species-specific and condition-dependent variations in acpP function requires comparative analysis:

• Cross-species comparison: While core functions are conserved, differences exist in regulation and interaction partners. For example, B. cenocepacia J2315 possesses "two complete AHL QS systems (CepIR and CciIR) and one orphan (CepR2) plus the Burkholderia Diffusible Signal Factor (BDSF)-based system" , whereas other species may have different configurations.

• Environmental regulation:

  • Iron availability significantly affects QS regulation: "iron increases cepR expression"

  • Carbon source impacts QS-dependent phenotypes, as demonstrated by differential growth on phenylacetic acid upon QS disruption

  • Oxygen limitation may alter acpP expression and function

• Host environment adaptation: During infection, especially in cystic fibrosis patients, B. cenocepacia adapts to host conditions, potentially altering acpP expression and function.

• Methodological approach for comparative studies:

  • RT-qPCR to quantify expression across conditions

  • Transcriptomics to identify condition-specific regulatory networks

  • Cross-complementation studies to test functional conservation

What are common pitfalls in recombinant acpP production and how can they be resolved?

Several challenges frequently arise when working with recombinant acpP:

• Post-translational modification issues:

  • Problem: Insufficient conversion to holo-acpP

  • Solution: Co-express with phosphopantetheinyl transferase or perform in vitro modification

• Protein solubility problems:

  • Problem: Aggregation during expression

  • Solution: Lower induction temperature (16°C), use solubility-enhancing tags (MBP, SUMO), optimize buffer conditions

• Activity verification challenges:

  • Problem: Difficult to confirm functional activity

  • Solution: Mass spectrometry to confirm modification status, functional assays with partner enzymes

• Stability concerns:

  • Problem: Protein degradation during purification

  • Solution: Include protease inhibitors, minimize freeze-thaw cycles, optimize storage conditions

How can contradictory results in acpP-related experiments be interpreted?

When faced with conflicting data, consider these methodological approaches:

• Strain variation effects: Different B. cenocepacia strains can yield different results. For example, "B. cenocepacia H111 (which lacks the CciIR system)" behaves differently from the J2315 strain regarding QS regulation .

• Experimental condition differences:

  • Media composition affects QS activity, particularly iron concentration

  • Growth phase significantly impacts QS-dependent phenotypes

  • Temperature and oxygen availability alter gene expression patterns

• Overlapping regulatory networks: B. cenocepacia possesses multiple QS systems that may compensate for each other, as "the BDSF system controls the AHL-based QS system" .

• Resolution approach:

  • Standardize experimental conditions

  • Include appropriate strain-specific controls

  • Perform comprehensive genetic complementation

  • Use multiple phenotypic readouts to confirm findings

What strategies can overcome challenges in studying acpP in biofilm contexts?

Investigating acpP function within biofilms presents unique challenges:

• Heterogeneity issues:

  • Problem: Variable gene expression within biofilm structures

  • Solution: Single-cell approaches, spatial transcriptomics, reporter constructs

• Extraction difficulties:

  • Problem: Challenging to extract proteins or RNA from biofilms

  • Solution: Optimize extraction protocols with appropriate matrix-degrading enzymes

• Visualization challenges:

  • Problem: Distinguishing acpP activity within biofilm architecture

  • Solution: Fluorescent protein fusions, activity-based probes, correlative microscopy

• Quantification inconsistencies:

  • Problem: Variable results in biofilm quantification

  • Solution: Multiple complementary methods (crystal violet, confocal microscopy, biomass measurement)

How might systems biology approaches enhance our understanding of acpP function?

Integrative systems biology approaches offer powerful frameworks for understanding acpP's role:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to map the effect of acpP perturbation across cellular systems

• Network analysis: Constructing interaction networks to identify:

  • Hub proteins connected to acpP

  • Regulatory motifs controlling acpP expression

  • Feedback mechanisms in QS networks

• Predictive modeling: Developing mathematical models of QS systems incorporating acpP to predict:

  • Effects of environmental perturbations

  • Outcomes of genetic manipulations

  • Potential therapeutic targets

• Emerging technologies to apply:

  • Single-cell RNA-seq to capture heterogeneity

  • CRISPRi screens to systematically identify genetic interactions

  • Machine learning to predict phenotypic outcomes of acpP manipulation

What unexplored roles might acpP play beyond quorum sensing in B. cenocepacia?

While acpP's role in QS is well-established, several potential functions remain under-explored:

• Stress response: acpP may contribute to adaptation to host-imposed stresses during infection

• Metabolic flexibility: Beyond fatty acid biosynthesis, acpP might participate in alternative metabolic pathways under nutrient limitation

• Interspecies communication: acpP-dependent products might mediate interactions within polymicrobial communities, particularly relevant in cystic fibrosis lung microbiomes

• Host-pathogen interactions: acpP-derived molecules might directly modulate host immune responses

• Experimental approaches to explore these roles:

  • Conditional knockdown using CRISPRi in diverse environments

  • Metabolomics to identify novel acpP-dependent products

  • Co-culture experiments to examine interspecies effects

  • Host cell infection models with acpP-manipulated strains

How can acpP research contribute to novel therapeutic strategies for B. cenocepacia infections?

Research on acpP offers several promising avenues for therapeutic development:

• Anti-virulence approaches: Targeting acpP function to attenuate virulence without imposing selection pressure for resistance. This aligns with the finding that "therapies directed at inhibiting QS (as well as other anti-virulence treatments) do not directly kill the bacteria, making the development of drug resistance less likely" .

• Combination therapies: QS inhibitors targeting acpP pathways could enhance antibiotic efficacy, although initial studies with compounds 6a and 8b did not show synergy with conventional antibiotics .

• Immunomodulatory strategies: Understanding how acpP-dependent processes interact with host immunity could lead to host-directed therapies

• Diagnostic applications: acpP expression patterns might serve as biomarkers for B. cenocepacia adaptation during infection

• Translational considerations:

  • Delivery strategies for acpP inhibitors to infection sites

  • Patient-specific approaches based on B. cenocepacia strain characteristics

  • Biofilm penetration requirements for effective therapy

  • Potential for narrow-spectrum therapeutics with reduced impact on beneficial microbiota