Recombinant Desulfovibrio vulgaris Protein CrcB homolog (crcB)

Basic Research Questions

• What is the basic function of the CrcB homolog protein in Desulfovibrio vulgaris?

  The CrcB homolog (DVU_1599) in Desulfovibrio vulgaris functions primarily as a putative fluoride ion transporter. This transmembrane protein belongs to a conserved family of membrane proteins involved in fluoride ion homeostasis. In D. vulgaris Hildenborough (strain ATCC 29579/NCIMB 8303), the CrcB protein consists of 124 amino acid residues and forms part of the cell's defense mechanism against fluoride toxicity by mediating the export of fluoride ions from the cytoplasm . While the exact mechanisms remain under investigation, research suggests that the protein forms a dimeric channel structure, similar to other bacterial fluoride channels.

• What are the optimal storage conditions for recombinant Desulfovibrio vulgaris CrcB protein?

  For optimal stability and activity, recombinant D. vulgaris CrcB protein should be stored at -20°C to -80°C upon receipt. If using the liquid formulation, the shelf life is approximately 6 months when stored properly. The lyophilized form offers extended stability of up to 12 months at -20°C to -80°C. To preserve protein integrity, aliquoting is necessary for multiple use scenarios to avoid repeated freeze-thaw cycles, which can lead to protein denaturation. For short-term work, aliquots can be stored at 4°C for up to one week. The protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability during freeze-thaw transitions .

• How does Desulfovibrio vulgaris colonize the human gut and what role might CrcB play?

  D. vulgaris colonization of the human gut depends significantly on its biofilm formation capability, which is mediated through the type 1 secretion system (T1SS). Research with PIRC rats (a preclinical model of human colon cancer) demonstrated that biofilm-competent D. vulgaris strains successfully colonized the gut in 100% of test subjects, while biofilm-deficient strains showed poor colonization (17% at one week post-treatment and 0% after four months) .

  While the direct role of CrcB in gut colonization hasn't been explicitly demonstrated, as a membrane protein involved in ion transport, it could contribute to D. vulgaris adaptation to the gut environment by helping regulate internal ion concentrations. The gut contains various concentrations of fluoride from dietary sources and drinking water, potentially making fluoride resistance proteins like CrcB important for bacterial survival in this environment. Future research could explore CrcB knockout models to determine its specific contribution to colonization efficiency.

Technical Research Questions

• What are the most effective transformation methods for genetic manipulation of D. vulgaris, particularly for studying CrcB function?

  Genetic manipulation of D. vulgaris has historically been challenging, but recent advances have improved transformation efficiency. For CrcB studies, consider these approaches:

  Optimized Electroporation Protocol:

  1. Preparation of Competent Cells:

     • Grow D. vulgaris to mid-logarithmic phase (OD₆₀₀ of 0.4-0.6)

     • Harvest cells under strict anaerobic conditions

     • Wash cells with chilled electroporation buffer containing 1 mM MgCl₂ and 10% glycerol

  2. Electroporation Parameters:

     • Use freshly prepared cells for highest efficiency

     • Optimal settings: 1.5-2.0 kV, 250 Ω, 25 μF

     • Use 1-5 μg of plasmid DNA per transformation

  3. Post-Electroporation Recovery:

     • Immediately transfer cells to pre-reduced recovery medium

     • Incubate for 4-6 hours before plating on selective media

  Key Genetic Tools:

  Method	Efficiency	Applications for CrcB Studies
  Markerless deletion system	Moderate	Creating clean crcB knockout strains
  Homologous recombination using suicide vectors	Moderate	Gene replacement or modification
  Transposon mutagenesis (RB-TnSeq)	High throughput	Determining genetic interactions with crcB
  CRISPR-Cas9 system (adapted for anaerobes)	Emerging technique	Precise editing of crcB sequence

  Critical Considerations:

  • Strain selection is crucial - use D. vulgaris strains with improved transformation efficiency like JW7035 (Δupp ΔhsdR), which shows 10²-10³-fold higher transformation efficiency than wild-type

  • The JW7035 strain has a deletion in the type I restriction endonuclease (hsdR), which significantly improves transformation rates

  • For crcB studies, ensure the strain doesn't have mutations that might affect membrane biology

  Verification Methods:

  • Confirm transformants using both PCR and Southern blot analysis

  • Sequence the entire modified region to check for unwanted mutations

  • Perform RT-qPCR to confirm knockout or expression changes

  This comprehensive approach will maximize success rates when genetically manipulating D. vulgaris for CrcB functional studies.

• What advanced microscopy techniques would be most effective for localizing and studying the dynamics of CrcB homolog in living D. vulgaris cells?

  Studying membrane proteins like CrcB in living anaerobic bacteria presents unique challenges that require specialized microscopy approaches:

  Recommended Advanced Techniques:

  1. Super-Resolution Microscopy:

     • PALM/STORM (Photoactivated Localization Microscopy/Stochastic Optical Reconstruction Microscopy)

       • Tag CrcB with photoactivatable fluorescent proteins (PA-mCherry)

       • Achieves 20-30 nm resolution to visualize protein clusters

       • Requires oxygen-independent fluorophores for anaerobic imaging

     • STED (Stimulated Emission Depletion)

       • Uses specialized depletion lasers to achieve ~50 nm resolution

       • Can work with conventional fluorophores under anaerobic conditions

       • Allows for rapid imaging with less phototoxicity

  2. Single-Molecule Tracking:

     • Tag CrcB with HaloTag or SNAP-tag systems

     • Use bright, photostable organic dyes compatible with anaerobic conditions

     • Track individual protein movements to determine:

       • Diffusion coefficients

       • Confined domains

       • Interactions with other proteins

  3. Fluorescence Correlation Spectroscopy (FCS):

     • Measures concentration fluctuations in a confocal volume

     • Determines diffusion characteristics and molecular brightness

     • Can detect changes in oligomerization state

  4. Förster Resonance Energy Transfer (FRET):

     • Tag CrcB and potential interaction partners with compatible fluorophores

     • Monitor protein-protein interactions in real-time

     • Measure conformational changes during transport activity

  5. Correlative Light and Electron Microscopy (CLEM):

     • Combine fluorescence imaging with high-resolution EM

     • Localize CrcB in the context of cellular ultrastructure

     • Particularly useful for biofilm studies

  Practical Implementation Considerations:

  • Anaerobic Microscopy Chamber Design:

    • Custom anaerobic chambers with optical-quality windows

    • Integration with microscope stage and temperature control

    • Continuous monitoring of O₂ levels

  • Genetic Tagging Strategies:

    • Use small tags (e.g., split-GFP or HaloTag) to minimize function disruption

    • Validate tagged constructs for functionality through complementation tests

    • Consider both N- and C-terminal tagging to determine optimal configuration

  • Controls and Validation:

    • Include membrane marker proteins for colocalization

    • Use photobleaching controls to confirm specific labeling

    • Perform parallel immunogold EM for validation

  Sample Preparation Protocol Example:

  1. Culture D. vulgaris expressing tagged CrcB under appropriate conditions

  2. Transfer cells to imaging medium in anaerobic chamber

  3. Mount on pre-equilibrated microscope slides with oxygen-scavenging system

  4. Seal with oxygen-impermeable adhesive

  5. Image immediately on microscope with environmental control

  These approaches would provide unprecedented insights into CrcB localization, dynamics, and function in the context of living D. vulgaris cells.

Research Application Questions

• How can knowledge about D. vulgaris CrcB homolog contribute to understanding the role of this bacterium in colorectal cancer progression and potential therapeutic approaches?

  Understanding D. vulgaris CrcB homolog may provide significant insights into colorectal cancer (CRC) mechanisms and novel therapeutic strategies:

  Mechanistic Insights:

  1. Microbiome-Host Interactions:

     • CrcB contributes to D. vulgaris survival in the gut environment

     • Altered ionic balance may influence epithelial-bacterial interactions

     • May affect the bacterium's ability to interact with LRRC19 receptors, which have been implicated in CRC progression

  2. Inflammation Modulation:

     • Ion transporters can influence inflammatory pathways

     • CrcB's activity may alter the pro-inflammatory potential of D. vulgaris

     • Could impact the TRAF6/TAK1 signaling pathway activated by D. vulgaris

  3. Chemoresistance Mechanisms:

     • D. vulgaris has been shown to confer chemoresistance in CRC

     • CrcB may contribute to altered metabolite production (such as S-adenosylmethionine)

     • Could influence biofilm formation which protects bacteria during chemotherapy

  Therapeutic Applications:

  Approach	Mechanism	Research Requirements
  CrcB inhibitors	Block bacterial survival	High-throughput screening for specific inhibitors
  Anti-biofilm strategies	Prevent bacterial colonization	Understand CrcB's role in biofilm formation
  Diagnostic biomarkers	Detect specific D. vulgaris strains	Characterize CrcB variants in clinical isolates
  Engineered probiotics	Deliver modified D. vulgaris	Develop genetic tools for stable modification
  Combination therapies	Sensitize tumors to chemotherapy	Identify interactions with chemotherapy drugs

  Clinical Research Directions:

  1. Patient Stratification:

     • Profile CrcB variants in D. vulgaris isolates from CRC patients

     • Correlate with disease progression and treatment response

     • Develop predictive biomarkers based on bacterial profiling

  2. Targeted Interventions:

     • Design small molecule inhibitors specific to CrcB

     • Test fluoride-based approaches to exploit CrcB function

     • Develop antibody-drug conjugates targeting D. vulgaris

  3. Microbiome Modulation:

     • Evaluate prebiotic/probiotic strategies to reduce harmful D. vulgaris strains

     • Design synthetic communities to outcompete pathogenic varieties

     • Develop bacterial replacement therapies

  This research could ultimately lead to novel diagnostic tools and therapeutic approaches targeting the D. vulgaris-host interaction in colorectal cancer, potentially addressing both disease progression and chemoresistance challenges.

• What methodological approaches can be used to study the interactions between CrcB and the human immune system in the context of inflammatory bowel diseases?

  Studying CrcB-immune system interactions requires multidisciplinary approaches spanning molecular biology, immunology, and microbiology:

  In Vitro Models:

  1. Co-culture Systems:

     • Cultivate human intestinal epithelial cells (Caco-2, HT-29) with immune cells

     • Expose to wild-type vs. ΔcrcB D. vulgaris strains

     • Measure cytokine production, tight junction integrity, and immune cell activation

     • Use transwell systems to distinguish direct contact vs. secreted factors

  2. Intestinal Organoids:

     • Develop 3D organoids from IBD patient and healthy control tissues

     • Introduce labeled D. vulgaris strains to the lumen

     • Assess epithelial barrier function and immune cell recruitment

     • Compare responses to wild-type vs. CrcB mutant strains

  3. Immune Cell Responses:

     • Isolate peripheral blood mononuclear cells (PBMCs) from IBD patients and controls

     • Expose to bacterial extracts or purified CrcB protein

     • Measure activation markers, cytokine profiles, and transcriptional responses

     • Assess pattern recognition receptor engagement

  In Vivo Models:

  1. Humanized Mouse Models:

     • Use gnotobiotic mice with human microbiota from IBD patients

     • Introduce wild-type or ΔcrcB D. vulgaris

     • Monitor inflammatory markers, gut permeability, and immune cell infiltration

     • Perform longitudinal studies to track disease progression

  2. Colitis Models:

     • Employ DSS or TNBS-induced colitis models

     • Colonize with D. vulgaris variants before or during colitis induction

     • Assess how CrcB affects colitis severity and recovery

     • Evaluate therapeutic interventions targeting CrcB

  Molecular and Cellular Mechanisms:

  1. Receptor Interaction Studies:

     • Investigate CrcB interaction with pattern recognition receptors

     • Focus on LRRC19 pathway which has been implicated in D. vulgaris recognition

     • Perform binding assays with recombinant proteins

     • Use CRISPR-edited cell lines lacking specific receptors

  2. Signaling Pathway Analysis:

     • Examine TRAF6/TAK1 pathway activation by D. vulgaris

     • Compare activation patterns between wild-type and ΔcrcB strains

     • Use pathway inhibitors to confirm specific mechanisms

     • Perform phosphoproteomic analysis to identify novel targets

  Clinical Correlations:

  1. Patient-Derived Samples:

     • Analyze D. vulgaris abundance and CrcB expression in IBD patient biopsies

     • Correlate with disease activity and inflammatory markers

     • Perform transcriptional profiling of mucosal immune responses

     • Track changes during disease flares and remission

  These approaches would provide comprehensive insights into how D. vulgaris CrcB might influence inflammatory responses in IBD, potentially identifying novel therapeutic targets for these chronic inflammatory conditions.