Recombinant Haemophilus ducreyi Protein CrcB homolog (crcB)

What are the optimal conditions for expressing recombinant H. ducreyi CrcB homolog protein in E. coli?

Expressing membrane proteins like CrcB homolog requires careful optimization. Based on successful expression approaches with other H. ducreyi proteins, the following methodology is recommended:

Expression system selection should prioritize vectors with tight regulation. The pET expression system with T7 promoter often yields good results for membrane proteins like CrcB. Consider using pET28a with an N-terminal His-tag for purification, similar to approaches used for expressing other H. ducreyi proteins . For host strains, E. coli C41(DE3) or C43(DE3) are engineered specifically for membrane protein expression and typically give better yields than standard BL21(DE3).

Growth conditions should be carefully controlled with initial cultures in LB medium at 37°C until OD600 reaches 0.6-0.8, followed by induction with 0.1-0.5 mM IPTG (lower concentrations often yield better results for membrane proteins). Post-induction temperature should be reduced to 16-20°C for 16-20 hours, as slower expression reduces inclusion body formation.

Several culture additives can improve yield and quality:

• 1% glucose to reduce basal expression

• 10 mM betaine and 1 M sorbitol as osmolytes to improve folding

For extraction optimization, use mild detergents like DDM (n-Dodecyl β-D-maltoside) or LMNG (Lauryl Maltose Neopentyl Glycol) for membrane extraction, and include 20% glycerol in extraction buffers to stabilize the protein.

How can I verify the identity and purity of recombinant H. ducreyi CrcB homolog after purification?

Comprehensive verification requires multiple complementary approaches:

SDS-PAGE analysis should be performed to visualize protein bands using Coomassie blue staining. The expected molecular weight of CrcB homolog is approximately 12-15 kDa, though it may run aberrantly due to hydrophobicity. Assess purity by the absence of contaminating bands.

Western blotting provides specific detection via anti-His antibody if using His-tagged constructs. Consider developing specific antibodies against CrcB for improved specificity, similar to the approach used for detecting CDT proteins in H. ducreyi .

Mass spectrometry verification through tryptic digest followed by LC-MS/MS analysis provides peptide mass fingerprinting for sequence coverage analysis. Intact protein mass determination by ESI-MS can confirm the complete protein sequence.

Functional assays are crucial and should include fluoride ion transport assays using reconstituted proteoliposomes, fluoride-selective electrode measurements, and fluorescence-based ion flux assays to confirm activity.

Structural integrity assessment using circular dichroism spectroscopy confirms secondary structure elements, while thermal stability assays using differential scanning fluorimetry assess protein stability.

This multi-faceted approach ensures both the identity and functional integrity of the purified protein, similar to verification methods employed for other H. ducreyi recombinant proteins .

What methods are available for studying membrane protein topology of H. ducreyi CrcB homolog?

Several complementary methods can elucidate the membrane topology of CrcB homolog:

Computational prediction provides the foundation using TMHMM, MEMSAT, and TOPCONS algorithms to predict transmembrane domains, SignalP to identify potential signal peptides, and consensus modeling from multiple prediction algorithms.

Cysteine scanning mutagenesis offers experimental verification by systematically replacing residues with cysteine throughout the protein, treating with membrane-permeable and impermeable sulfhydryl reagents, and using differential labeling to identify residues accessible from each side of the membrane.

Reporter fusion analysis involves creating fusion constructs with reporter proteins (GFP, PhoA, LacZ) as C-terminal fusions at various truncation points. Reporter activity indicates cytoplasmic (GFP) or periplasmic (PhoA) localization of the fusion point.

Protease protection assays using inside-out and right-side-out membrane vesicles treated with proteases can identify protected fragments by mass spectrometry or Western blotting. Protected regions indicate membrane-embedded or inaccessible domains.

For high-resolution structural determination, cryo-EM or X-ray crystallography may be employed, though these techniques may require fusion with stabilizing proteins (e.g., T4 lysozyme) or antibody fragments, with detergent screening critical for crystallization success.

How is crcB gene expression regulated in H. ducreyi under different growth conditions?

Understanding crcB regulation requires systematic investigation of expression patterns:

Transcriptional analysis should employ quantitative RT-PCR to measure crcB transcript levels under various conditions and RNA-seq to place crcB regulation within the context of global gene expression patterns. Based on the search results, H. ducreyi shows distinct transcriptional profiles under anaerobic vs. aerobic conditions , suggesting crcB may be similarly regulated.

Promoter characterization using 5' RACE to identify transcription start sites, reporter fusions (luciferase, GFP) to the promoter region for activity monitoring, and DNA footprinting to identify protein binding sites is essential. This approach is similar to the putative promoter analysis performed for the cdtABC genes .

Transcription factor identification through electrophoretic mobility shift assays (EMSA) can identify proteins binding to the crcB promoter, while ChIP-seq identifies transcription factors binding in vivo. Bacterial one-hybrid or yeast one-hybrid screens can also reveal potential regulators.

Environmental condition testing should examine expression under varying oxygen levels (aerobic vs. anaerobic growth, as studied for other H. ducreyi genes ), pH values, nutrient limitations, fluoride concentrations, and host cell contact conditions.

Post-transcriptional regulation investigation should include mRNA stability under different conditions, identification of potential small RNAs, and ribosome profiling to assess translation efficiency.

What vector systems are most effective for cloning and expressing the H. ducreyi crcB gene?

Selecting the appropriate vector system depends on specific experimental goals:

For basic expression and purification, pET series vectors (pET28a, pET24d) with T7 promoter provide high-level expression. Include N-terminal or C-terminal His-tag for purification. The T7 expression system with IPTG induction provides controlled expression. From the search results, similar approaches were used successfully for expressing H. ducreyi cdtABC genes .

For structural studies, vectors with fusion partners that aid crystallization (e.g., SUMO, MBP, or T4 lysozyme) are valuable. Consider pMal-c2X for MBP fusions or pET-SUMO for SUMO fusions, with cleavable tags using precision protease sites.

For functional studies in E. coli, the pBAD series with arabinose-inducible promoter provides tight regulation. pACYC or pSC101 derivatives offer moderate copy number and are compatible with fluoride sensitivity assays in E. coli.

For complementation studies in H. ducreyi, shuttle vectors like pLS88 that replicate in both E. coli and H. ducreyi allow expression in the native organism. Include H. ducreyi native promoter region for physiological expression levels.

For protein-protein interaction studies, bacterial two-hybrid vectors (pKT25, pUT18C), split-GFP complementation vectors, or tandem affinity purification tag vectors are appropriate.

The search results demonstrate successful cloning of H. ducreyi genes using PCR amplification and subsequent expression in recombinant systems , suggesting similar approaches would work for crcB.

How does the expression of crcB in H. ducreyi change under anaerobic versus aerobic conditions?

Investigating crcB expression across oxygen conditions requires a systematic approach:

Transcriptomic analysis should include RNA-seq comparison between aerobic and anaerobic cultures, sampled at multiple time points (4h, 8h, 18h as in the referenced study ), with quantitative RT-PCR validation of crcB expression levels. The search results indicate that H. ducreyi shows distinct transcriptional profiles under anaerobic conditions , suggesting crcB expression may also be differentially regulated.

Protein-level verification through Western blot analysis of CrcB protein levels using specific antibodies and proteomic analysis using LC-MS/MS can quantify relative protein abundance. Pulse-chase labeling determines protein synthesis and turnover rates.

Promoter activity measurement using reporter gene constructs (luciferase, GFP) fused to the crcB promoter allows monitoring of activity in real-time during transition from aerobic to anaerobic conditions and identification of potential oxygen-responsive regulatory elements.

Regulatory network identification through ChIP-seq can identify transcription factors binding to the crcB promoter under different oxygen conditions and identify anaerobic response regulators (similar to FNR or ArcA) that might control crcB. The search results mention that H. ducreyi upregulates genes involved in anaerobic metabolism during infection .

Physiological relevance assessment should compare expression levels in vitro to those in human infection models and examine expression in tissue infection models with varying oxygen tensions. The search results indicate significant overlap between genes regulated by anaerobiosis in vitro and those differentially expressed during human infection .

What experimental approaches can be used to determine the ion selectivity of H. ducreyi CrcB homolog?

Determining ion selectivity of membrane transporters like CrcB requires multiple complementary approaches:

Reconstitution into proteoliposomes involves purifying CrcB homolog and reconstituting into liposomes, loading liposomes with fluorescent indicators sensitive to specific ions, monitoring fluorescence changes upon addition of different ions, and calculating transport rates for various ions to determine selectivity profile.

Electrophysiological measurements include planar lipid bilayer recordings with purified and reconstituted CrcB, patch-clamp analysis of CrcB expressed in giant liposomes or cell systems, and ion-selective electrodes to directly measure ion flux. These methods determine conductance, reversal potentials, and ion preference.

Fluoride-specific functional assays include bacterial growth assays with CrcB-expressing strains in the presence of fluoride, comparing growth inhibition by fluoride versus other halides, using fluoride-sensitive reporter systems (e.g., riboswitch-based reporters), and measuring intracellular ion concentrations using ion-specific fluorescent probes.

Binding studies using isothermal titration calorimetry (ITC) measure binding affinities for different ions, while microscale thermophoresis (MST) quantifies ion-protein interactions. Fluorescence-based binding assays with ion-sensitive fluorophores provide additional binding data.

Mutational analysis through generating point mutations in predicted ion coordination sites, measuring changes in transport specificity and efficiency, and performing evolutionary analysis identifies conserved residues likely involved in ion selectivity.

Computational modeling with molecular dynamics simulations of ion permeation, free energy calculations for different ions passing through the channel/transporter, and homology modeling based on related transporters with known structures provides theoretical support for experimental findings.

How can I establish the functional relationship between CrcB homolog and other H. ducreyi virulence factors?

Investigating functional relationships between CrcB and other virulence factors requires a multi-faceted approach:

Genetic interaction studies should generate single and double knockout mutants of crcB and other virulence genes, perform synthetic lethality/sickness screens, and evaluate changes in virulence phenotypes in combination mutants. Similar approaches were used to study H. ducreyi hemolysin and its relationship to other factors .

Transcriptomic analysis comparing gene expression profiles between wild-type and crcB mutants identifies co-regulated genes under different conditions. RNA-seq analysis reveals regulatory networks. The search results indicate H. ducreyi undergoes significant transcriptional changes during infection and anaerobiosis .

Protein-protein interaction studies through co-immunoprecipitation with CrcB-specific antibodies, bacterial two-hybrid screening, proximity-dependent biotin labeling (BioID), and cross-linking mass spectrometry identify interaction partners. Cross-referencing with known virulence factors like the CDT proteins described in the search results helps establish functional connections.

Phenotypic analysis in infection models using human infection models with wild-type and crcB mutant strains, microscopic examination of infected tissues, and cytotoxicity assays with host cells allows comparison with phenotypes of other virulence factor mutants. The search results mention human volunteer studies for identifying differentially expressed genes during infection .

Biochemical pathway analysis through metabolomic profiling of wild-type versus crcB mutants, examination of fluoride sensitivity in combination with other virulence factor mutations, and investigation of shared metabolic pathways affected by CrcB and other virulence factors provides mechanistic insights. The search results indicate H. ducreyi modifies its metabolism during infection .

What structural modeling approaches are most appropriate for predicting the tertiary structure of H. ducreyi CrcB homolog?

Predicting the structure of membrane proteins like CrcB requires specialized approaches:

Homology modeling identifies structural homologs using HHpred, Phyre2, or I-TASSER. CrcB homologs with solved structures (e.g., from E. coli or other bacteria) serve as templates. Building alignment-based models with specialized membrane protein modeling tools and refining models with membrane-specific force fields produces initial structural predictions.

Ab initio and deep learning approaches such as AlphaFold2 or RoseTTAFold provide template-free modeling, specifically effective for proteins like CrcB where homologs may have low sequence identity. Including biological constraints from evolutionary conservation data and membrane protein-specific modifications to these algorithms improves accuracy.

Hybrid experimental/computational approaches incorporate low-resolution experimental data (e.g., crosslinking constraints), distance constraints from NMR or mass spectrometry, and validate models using experimental approaches like disulfide crosslinking. These approaches are similar to those that could be used for structural determination of the CDT proteins described in the search results .

Molecular dynamics simulations embed predicted structures in explicit lipid bilayers, assess stability and conformational dynamics over 100+ ns simulations, identify potential ion binding sites and permeation pathways, and use membrane-specific force fields (CHARMM36, Martini) for accurate lipid interactions.

Model validation strategies include ProSA and QMEAN for general quality assessment, membrane protein-specific validation (e.g., hydrophobic residue positioning), functional predictions based on the model (ion coordination sites), and experimental validation through mutagenesis of predicted key residues.

What are the most effective strategies for generating crcB knockout mutants in H. ducreyi?

Creating gene knockouts in H. ducreyi requires careful consideration of its genetic manipulation challenges:

Allelic exchange methodology involves designing suicide vectors containing crcB flanking regions with antibiotic resistance cassette, implementing a two-step selection process (integration and resolution), and including counterselectable markers (sacB, rpsL) for efficient selection. Similar approaches would be used as for other H. ducreyi gene deletions.

PCR-based mutagenesis strategies use overlap extension PCR to create deletion constructs. Target regions for homologous recombination should be 500-1000 bp, with careful consideration of codon usage and regulatory elements when designing constructs. The search results mention PCR-based methods for amplifying H. ducreyi genes .

Electroporation optimization addresses H. ducreyi's specific requirements:

• Growth phase: mid-log phase cells

• Buffer: 10% glycerol with 1 mM HEPES

• Voltage: 1.8-2.5 kV

• Resistance: 200-400 Ω
  Pre-treatment with cell wall-weakening agents may improve efficiency.

Selection and verification include antibiotic selection based on resistance cassette (kanamycin, spectinomycin), PCR verification of deletion using primers outside the recombination region, Southern blot to confirm single integration at correct locus, and RT-PCR to confirm absence of crcB transcript. Similar verification approaches would be used as for other H. ducreyi gene deletions.

Complementation strategies involve shuttle vectors like pLS88 for H. ducreyi complementation, including native promoter region for physiological expression, and site-specific integration systems for single-copy complementation. The search results mention recombinant plasmids used for expressing H. ducreyi genes .

How can I elucidate the role of CrcB homolog in H. ducreyi pathogenesis during human infection?

Investigating CrcB's role in pathogenesis requires a comprehensive approach:

Human infection model studies should compare wild-type and crcB mutant strains in human volunteer studies, measure pustule formation rate, size, and bacterial recovery, and analyze host immune responses to infection. The search results mention human volunteer studies for H. ducreyi infection .

Transcriptomic and proteomic analysis comparing gene/protein expression between wild-type and crcB mutants, analyzing expression during infection versus in vitro growth, and identifying pathways affected by crcB deletion provides molecular insights. The search results describe RNA-seq approaches for H. ducreyi in different conditions .

Infection microenvironment analysis involves measuring ion concentrations (particularly fluoride) in infection sites, determining pH and oxygen levels in pustules, and comparing metabolite profiles between wild-type and mutant infections. The search results indicate H. ducreyi resides in anaerobic environments during infection .

Host-pathogen interaction studies examine adherence to and invasion of host cells, assess survival within human macrophages, measure resistance to antimicrobial peptides, and analyze effects on host cell viability and immune response.

Virulence factor expression analysis determines whether crcB mutation affects expression of known virulence factors, measures cytotoxin activity (such as CDT described in the search results ), and assesses hemolysin expression and activity.

What comparative genomic approaches can reveal the evolutionary conservation of CrcB across Pasteurellaceae?

Comprehensive comparative genomics of CrcB requires multiple analytical approaches:

Phylogenetic analysis collects CrcB homolog sequences from diverse Pasteurellaceae species, performs multiple sequence alignment using MUSCLE or MAFFT, constructs phylogenetic trees using maximum likelihood or Bayesian methods, and compares CrcB evolution to species phylogeny to identify horizontal gene transfer events.

Synteny analysis examines gene organization around crcB across Pasteurellaceae, identifies conserved gene neighborhoods and operon structures, and detects genomic rearrangements that may affect regulation. The search results mention potential transposon-associated elements near H. ducreyi genes , which may be relevant for crcB genomic context.

Selection pressure analysis calculates dN/dS ratios to identify positions under purifying or positive selection, performs branch-site tests to detect lineage-specific selection, and correlates selected sites with structural features and functional domains.

Structural conservation assessment maps sequence conservation onto predicted protein structures, identifies conserved residues in transmembrane domains and functional sites, and compares with other fluoride channels for functional conservation.

Regulatory element analysis examines promoter regions for conserved transcription factor binding sites, identifies potential regulatory RNAs associated with crcB, and compares expression patterns across different Pasteurellaceae species.

How can contradictory data regarding CrcB function in H. ducreyi be reconciled through experimental design?

Resolving contradictory data about CrcB function requires a systematic investigative approach:

Identification of experimental variables should catalog discrepant results and methodological differences, analyze growth conditions, strain variations, and measurement techniques, and consider oxygen availability (aerobic/anaerobic) as the search results indicate this significantly affects H. ducreyi gene expression . Standardizing experimental protocols eliminates technical variability.

Strain-specific functional analysis compares crcB sequences across H. ducreyi isolates used in different studies, creates isogenic mutants in multiple strain backgrounds, tests phenotypes under identical conditions across strains, and determines if strain-specific genetic context affects CrcB function.

Multi-condition phenotypic assessment tests CrcB function under varied conditions:

• Aerobic vs. anaerobic (particularly relevant based on search results )

• Different pH values

• Varying ion concentrations

• With/without host cell factors
  This approach determines condition-specific functionality that might explain discrepancies.

Biochemical characterization with purified protein involves expressing and purifying CrcB from different strains, directly measuring ion transport activity in reconstituted systems, comparing with activity of CrcB homologs from other species, and identifying post-translational modifications that might affect function.

Genetic interaction mapping screens for suppressor mutations that alleviate crcB mutant phenotypes, performs synthetic genetic array analysis to identify genetic interactions, and creates double mutants with genes in related pathways. This may reveal compensatory mechanisms active in some experimental systems.

What are the recommended protocols for studying protein-protein interactions involving CrcB homolog in H. ducreyi?

Investigating CrcB protein interactions requires multiple complementary approaches:

Co-immunoprecipitation (Co-IP) generates antibodies against CrcB or uses epitope-tagged versions, solubilizes membranes with mild detergents (DDM, LMNG), immunoprecipitates complexes and identifies partners by mass spectrometry, and includes appropriate controls (tag-only, irrelevant membrane protein). Similar approaches could be applied as those used for studying H. ducreyi CDT proteins .

Bacterial two-hybrid (B2H) analysis clones crcB into B2H vectors (e.g., pKT25, pUT18C), screens against H. ducreyi genomic library, validates positive interactions with direct B2H assays, and quantifies interaction strength using β-galactosidase assays.

Proximity-dependent labeling creates CrcB-BioID or CrcB-APEX2 fusion proteins, expresses in H. ducreyi or heterologous systems, activates labeling and identifies biotinylated proteins by mass spectrometry. This approach is particularly valuable for transient interactions.

Surface plasmon resonance (SPR) purifies CrcB with appropriate detergents or nanodiscs, immobilizes on SPR chip, tests binding with purified candidate interactors, and determines binding kinetics and affinities.

Crosslinking mass spectrometry treats intact cells or membrane preparations with crosslinking reagents, digests and analyzes by LC-MS/MS, identifies crosslinked peptides to map interaction interfaces, and provides spatial constraints for structural modeling.

For membrane proteins like CrcB, special consideration must be given to maintaining proper folding and membrane environment during sample preparation, similar to challenges that would be faced when studying membrane-associated virulence factors in H. ducreyi.

How can transcriptomic data be integrated with proteomic analysis to understand CrcB regulation networks?

Integrating multi-omics data for CrcB regulation analysis requires sophisticated analytical approaches:

Coordinated experimental design collects matched transcriptomic and proteomic samples under identical conditions, includes multiple time points to capture temporal regulation, compares wild-type and mutant strains, and tests various conditions (aerobic/anaerobic as in the search results , pH, fluoride levels).

RNA-seq analysis workflow requires high-depth sequencing (>20M reads) for comprehensive coverage, proper normalization and statistical analysis, identification of co-expressed gene clusters containing crcB, and inference of transcriptional regulators using motif analysis. The search results describe RNA-seq approaches for H. ducreyi under different conditions .

Proteomics approaches include both shotgun proteomics and targeted methods (PRM/SRM), membrane protein enrichment protocols to detect CrcB, post-translational modification analysis (phosphoproteomics), and protein turnover studies using pulse-chase SILAC.

Integrated data analysis calculates transcript-protein correlation coefficients, identifies discordant regulation suggesting post-transcriptional control, applies network inference algorithms (WGCNA, Bayesian networks), and constructs protein-protein interaction networks centered on CrcB.

Validation experiments include ChIP-seq for predicted transcriptional regulators, ribosome profiling to assess translation efficiency, targeted proteomics to verify key network components, and perturbation experiments (overexpression, CRISPR interference).

The search results indicate that H. ducreyi undergoes significant transcriptional changes during anaerobiosis and infection , suggesting that integrated -omics approaches would be valuable for understanding how CrcB regulation fits within these broader adaptive responses.