Recombinant Flavobacterium psychrophilum Protein CrcB homolog (crcB)

What is Flavobacterium psychrophilum and why is it significant in research?

Flavobacterium psychrophilum is a significant fish pathogen affecting salmonid aquaculture worldwide, causing cold water disease (CWD) and rainbow trout fry syndrome (RTFS) . This gram-negative bacterium has substantial economic impacts on global aquaculture operations, particularly in freshwater environments. The bacterium belongs to the Flavobacteriaceae family and is characterized by its psychrophilic nature, exhibiting optimal growth at lower temperatures (15-20°C). Research into F. psychrophilum is critical due to its persistent nature and the challenges associated with controlling outbreaks in aquaculture settings. Genome analysis has revealed that F. psychrophilum possesses an open pan genome with at least 3373 genes, while its core genome contains approximately 1743 genes . This genomic plasticity likely contributes to the bacterium's adaptability and pathogenicity across different environmental conditions and host species.

How does F. psychrophilum genomic diversity influence protein expression studies?

The genomic diversity of F. psychrophilum significantly impacts protein expression studies. Comparative genomic analyses of 11 F. psychrophilum isolates revealed that while the core genome contains 1743 genes, each new genome added to the analysis contributes approximately 67 new genes, indicating an open pan genome . This genetic diversity presents several challenges for protein expression studies:

• Strain-specific variations: Researchers must carefully select representative strains when studying specific proteins.

• Genetic context effects: On average, 67 new genes were detected for every new genome added to analysis, indicating considerable strain variation .

• Regulatory differences: Genomic islands containing horizontally acquired DNA sequences may alter protein expression patterns.

A methodological approach to address these challenges includes:

When specifically targeting the CrcB homolog, researchers should examine conservation of this gene across the core genome and assess whether strain-specific variations exist that might affect protein function or expression.

What methods are recommended for identifying and analyzing the crcB gene in F. psychrophilum genomes?

To effectively identify and analyze the crcB gene in F. psychrophilum genomes, researchers should employ a systematic approach combining bioinformatic tools with experimental validation. The following methodological framework is recommended:

• Genome mining and homology searches: Utilize BLAST searches against the 11 fully sequenced F. psychrophilum genomes using known crcB sequences from related species as queries . The comparative genomic analysis approach that revealed an open pan genome with 3373 genes can be adapted for specific gene identification.

• Genomic context analysis: Examine genes flanking the putative crcB homolog, as genomic context is often conserved for functionally related genes. Similar to the analysis performed for serotype-specific genes in F. psychrophilum , researchers should look for conserved gene neighborhoods.

• Phylogenetic analysis: Construct phylogenetic trees to determine the evolutionary relationship of the crcB homolog among different F. psychrophilum strains and related species. This approach helped identify strain relationships and virulence factor distributions in previous studies .

• Primer design for amplification: Design PCR primers targeting conserved regions of the crcB gene for amplification from various strains. This methodology was successfully employed in developing a multiplex PCR-based serotyping scheme for F. psychrophilum .

• Sequence variation analysis: Analyze sequence polymorphisms among different strains to identify potential functional variations. The striking association between PCR-serotype and fish host species illustrates how genetic variations can correlate with biological characteristics .

A practical workflow would include initial in silico identification followed by PCR verification and sequencing of the gene from multiple isolates to confirm conservation and identify strain-specific variations.

How can researchers determine if crcB is part of the core or accessory genome of F. psychrophilum?

Determining whether crcB belongs to the core or accessory genome of F. psychrophilum requires a systematic pan-genome analysis approach. Based on methodologies employed in previous F. psychrophilum genomic studies, researchers should:

• Perform whole-genome sequence comparison: Analyze all available F. psychrophilum genomes (at least the 11 isolates from temporally and geographically distant populations previously studied) to identify the presence/absence of crcB across all strains .

• Calculate gene prevalence: A gene is typically considered part of the core genome if it is present in all or nearly all (>95%) of the strains analyzed. The core genome of F. psychrophilum was determined to contain 1743 genes through such analysis .

• Examine sequence conservation: For genes present across multiple strains, analyze sequence conservation levels. Core genes typically exhibit higher sequence conservation than accessory genes.

• Perform comparative synteny analysis: Examine the genomic context of crcB across strains to determine if its chromosomal location is conserved, similar to the analysis that identified Type-0, Type-1, Type-2, and Type-3 genomic organizations in the polysaccharide biosynthesis locus .

• Functional category assessment: Consider whether crcB belongs to functional categories typically associated with core genes (e.g., metabolism, replication) or accessory genes (e.g., adaptation, virulence).

This methodological framework will allow researchers to confidently classify crcB as either a core component essential to F. psychrophilum biology or an accessory gene potentially linked to niche adaptation or specific virulence mechanisms.

What is the relationship between CRISPRs and prophages in F. psychrophilum, and how might this affect crcB expression?

The relationship between CRISPR systems and prophages in F. psychrophilum represents a complex interaction that may influence gene expression, including potential effects on crcB. Based on the available research, the following insights and methodological approaches are relevant:

Two distinct CRISPR systems have been identified in F. psychrophilum strains: CRISPR1, which is widely distributed among isolates, and CRISPR2, which was found only in strain 4 . Interestingly, both CRISPR systems contain spacers that match sequences from the temperate bacteriophage 6H, but with variable numbers of 6H-specific spacers .

The prophage 6H appears in 5 out of 11 studied F. psychrophilum isolates, suggesting a widespread distribution of this temperate phage across F. psychrophilum populations . This phage-host relationship may affect gene expression through several mechanisms:

• Regulatory interactions: CRISPR systems may interact with prophage genes to regulate bacterial functions, as observed in P. aeruginosa where the CRISPR/Cas system interacts with prophage genes to inhibit biofilm formation .

• Genomic stability: The integration of prophages can influence genomic stability and potentially affect the expression of nearby genes, including potential effects on crcB if it is located in proximity to prophage integration sites.

• Horizontal gene transfer: Prophages may facilitate the transfer of genetic material between bacteria, potentially influencing the evolution and distribution of genes like crcB.

To investigate these relationships, researchers should:

The stability of CRISPR1 across temporally and geographically distant strains suggests it may serve alternative functions beyond phage defense , potentially including regulatory roles that could influence membrane protein expression.

What are the optimal methods for recombinant expression of F. psychrophilum CrcB homolog?

Successful recombinant expression of the F. psychrophilum CrcB homolog requires careful consideration of multiple factors, especially given that it is likely a membrane protein. Based on successful approaches with other F. psychrophilum proteins, the following methodological framework is recommended:

• Expression system selection: E. coli remains the most common expression system for initial attempts, but specialized strains designed for membrane protein expression (such as C41(DE3) or C43(DE3)) should be considered. For challenging membrane proteins, alternative systems such as Lactococcus lactis or cell-free expression systems may be more appropriate.

• Construct design considerations:

  • Include affinity tags (His6, GST, or MBP) for purification

  • Consider fusion partners that enhance solubility

  • Engineer constructs with and without predicted signal peptides

  • Optimize codon usage for the expression host

  • Include TEV or PreScission protease sites for tag removal

• Expression optimization:

  • Test multiple growth temperatures (15-30°C), with lower temperatures often favoring proper folding of psychrophilic proteins

  • Use reduced inducer concentrations to slow expression rate

  • Supplement with specific lipids if needed for membrane protein stability

  • Consider expression in the presence of specific chaperones

• Initial assessment:

  • Verify expression using Western blotting with either tag-specific or custom antibodies

  • Assess membrane localization through fractionation procedures

  • Evaluate protein solubility in various detergents

Previous immunoproteomic analysis of F. psychrophilum identified 15 immunogenic proteins using two-dimensional polyacrylamide gel electrophoresis and Western blotting , demonstrating the feasibility of expressing and detecting F. psychrophilum proteins. The immunogenic membrane protein OmpA (P60) was successfully expressed and characterized , providing a potential model for CrcB expression approaches.

What purification strategies are most effective for membrane proteins like CrcB from F. psychrophilum?

Purifying membrane proteins like the CrcB homolog from F. psychrophilum presents unique challenges that require specialized approaches. The following comprehensive purification strategy is recommended based on successful membrane protein purification methodologies:

• Membrane extraction and solubilization:

  • Extract membranes through differential centrifugation

  • Screen multiple detergents for optimal solubilization

  • Consider a systematic detergent screen including:

    • Mild detergents (DDM, LMNG)

    • Zwitterionic detergents (LDAO, FC-12)

    • Nonionic detergents (OG, DM)

  • Evaluate solubilization efficiency using Western blotting

• Affinity chromatography:

  • Utilize affinity tags engineered into the recombinant construct

  • Perform binding in batch mode to maximize recovery

  • Include detergent in all buffers at concentrations above CMC

  • Consider adding lipids or cholesteryl hemisuccinate for stability

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and assess oligomeric state

  • Ion exchange chromatography if additional purity is required

  • Validate protein homogeneity by SDS-PAGE and Western blotting

• Quality assessment:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal stability assays to optimize buffer conditions

  • Mass spectrometry for identity confirmation

  • Functional assays (e.g., fluoride transport for CrcB)

The immunoproteomic analysis of F. psychrophilum that successfully identified multiple proteins, including membrane proteins like OmpA (P60) , demonstrates that F. psychrophilum proteins can be isolated and characterized with appropriate methodologies. For CrcB specifically, researchers should prioritize maintaining the native conformation throughout the purification process to preserve functional activity.

How can researchers assess the proper folding and functionality of recombinant CrcB?

Assessing proper folding and functionality of recombinant CrcB homolog from F. psychrophilum requires a multi-faceted approach combining structural and functional analyses. Given that CrcB is predicted to function as a fluoride ion channel, the following methodological framework is recommended:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure content

  • Thermal denaturation assays to assess protein stability under various conditions

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

  • Limited proteolysis to probe for well-folded domains resistant to digestion

• Fluoride transport assays:

  • Liposome-based fluoride transport assays using fluoride-sensitive probes

  • Whole-cell assays comparing fluoride sensitivity in expression systems with and without recombinant CrcB

  • Patch-clamp electrophysiology for direct measurement of channel activity (for advanced studies)

• Binding assays:

  • Microscale thermophoresis to measure fluoride binding affinity

  • Isothermal titration calorimetry for quantitative binding parameters

  • Fluorescence-based ligand binding assays using intrinsic tryptophan fluorescence

• Comparative analysis with known functional CrcB proteins:

  • Side-by-side functional assays with well-characterized CrcB proteins from other bacteria

  • Complementation assays in CrcB-deficient bacterial strains

The experimental design should include appropriate positive and negative controls. For instance, site-directed mutagenesis of predicted key residues in the ion channel pathway would be expected to abolish function if the protein is properly folded but render it non-functional. This approach parallels methodologies used to characterize other functional proteins from F. psychrophilum, such as the analysis of extracellular enzymes and hemolytic activity that revealed similarities in mode of action across strains .

How does CrcB potentially contribute to F. psychrophilum pathogenicity and virulence?

While the direct role of CrcB homolog in F. psychrophilum pathogenicity has not been explicitly characterized in the provided literature, several methodological approaches can be employed to investigate its potential contributions to virulence:

• Comparative genomic analysis: Examining the presence and conservation of crcB across virulent and less virulent F. psychrophilum strains can provide initial insights. The comparative genome analysis approach that revealed equal distribution of virulence factors across isolates could be applied specifically to crcB to determine if it follows similar patterns of conservation.

• Gene knockout/knockdown studies: Creating crcB deletion mutants and assessing changes in:

  • Survival under environmental stress conditions

  • Biofilm formation capabilities (shown to be similar across F. psychrophilum strains )

  • Hemolytic activity

  • Secretion of extracellular enzymes

  • Adherence to host cells

  • Fluoride resistance

• Transcriptomic analysis: Comparing crcB expression levels between:

  • Growth in standard media versus in vivo conditions

  • Different stages of infection

  • Response to host immune factors

  • Different environmental conditions (temperature, pH, salt concentration)

• Protein-protein interaction studies:

  • Identifying potential interactions between CrcB and known virulence factors

  • Examining if CrcB associates with other membrane proteins involved in pathogenicity

• In vivo infection models:

  • Comparing virulence of wild-type and crcB mutant strains in fish infection models

  • Assessing bacterial burden and tissue distribution

The methodological framework should incorporate the knowledge that F. psychrophilum isolates show similar modes of action on adhesion, colonization, and destruction of fish tissues across large spatial and temporal scales , suggesting conserved virulence mechanisms that might involve membrane proteins like CrcB.

What approaches can be used to study potential protein-protein interactions involving CrcB?

Investigating protein-protein interactions involving the CrcB homolog in F. psychrophilum requires a comprehensive approach combining complementary methodologies. The following research framework is recommended:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against CrcB or use epitope-tagged recombinant CrcB

  • Perform Co-IP from membrane fractions under conditions that preserve native interactions

  • Identify interaction partners using mass spectrometry

  • Validate interactions with reverse Co-IP experiments

• Bacterial two-hybrid (B2H) system:

  • Clone the crcB gene into appropriate bacterial two-hybrid vectors

  • Screen against a genomic library of F. psychrophilum to identify interaction partners

  • Validate positive interactions through focused B2H assays

  • Consider specialized membrane protein-compatible B2H systems

• Proximity-based labeling methods:

  • Express CrcB fused to enzymes like BioID or APEX2 in F. psychrophilum

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures both stable and transient interactions in the native environment

• Microscopy-based approaches:

  • Fluorescence resonance energy transfer (FRET) between fluorescently labeled proteins

  • Bimolecular fluorescence complementation (BiFC) for visualizing interactions in vivo

  • Super-resolution microscopy to examine co-localization of proteins

• Chemical cross-linking coupled with mass spectrometry:

  • Use membrane-permeable cross-linkers to stabilize protein complexes

  • Purify CrcB and cross-linked partners

  • Identify interaction sites through cross-link-specific mass spectrometry

This multi-method approach provides complementary data to build a comprehensive interaction network. The immunoproteomic analysis methodology that successfully identified immunogenic proteins in F. psychrophilum demonstrates that protein-specific analytical techniques can be effectively applied to this bacterium, and similar approaches could be adapted for interaction studies involving CrcB.

How can researchers investigate the role of CrcB in fluoride resistance in F. psychrophilum?

To systematically investigate the role of CrcB homolog in fluoride resistance in F. psychrophilum, researchers should implement a comprehensive experimental approach:

• Growth inhibition assays:

  • Determine minimum inhibitory concentrations (MICs) of fluoride for wild-type F. psychrophilum strains

  • Compare growth curves in media with various fluoride concentrations

  • Assess strain-specific variations in fluoride tolerance across the 11 genetically characterized isolates

  • Create dose-response curves to quantify susceptibility

• Gene expression analysis:

  • Quantify crcB expression using RT-qPCR under varying fluoride concentrations

  • Perform RNA-seq to identify genome-wide transcriptional responses to fluoride exposure

  • Compare crcB expression across different environmental conditions

• Genetic manipulation approaches:

  • Generate crcB knockout mutants using targeted gene deletion

  • Create crcB overexpression strains

  • Perform complementation studies to confirm phenotypes

  • Conduct competitive growth assays between wild-type and mutant strains

• Fluoride transport measurements:

  • Use fluoride-selective electrodes to measure intracellular fluoride accumulation

  • Compare fluoride uptake/export kinetics between wild-type and crcB-mutant strains

  • Employ fluorescent probes to visualize fluoride distribution within cells

• Structural and functional analysis:

  • Identify conserved residues in CrcB through sequence alignment with characterized fluoride channels

  • Perform site-directed mutagenesis of key residues predicted to be involved in fluoride transport

  • Assess changes in fluoride resistance resulting from these mutations

This methodological framework draws on approaches used to characterize other functional aspects of F. psychrophilum and adapts them specifically to investigate fluoride resistance mechanisms.

Could recombinant CrcB be a potential vaccine candidate against F. psychrophilum infections?

Evaluating the potential of recombinant CrcB homolog as a vaccine candidate against F. psychrophilum infections requires a systematic assessment of several critical factors. Based on previous successful approaches in identifying immunogenic proteins in F. psychrophilum, the following methodological framework is recommended:

• Immunogenicity assessment:

  • Determine if CrcB is naturally immunogenic during infection using sera from recovered fish

  • Perform immunoproteomic analysis similar to the approach that identified 15 immunogenic proteins in F. psychrophilum

  • Evaluate if CrcB belongs to any of the protective molecular mass fractions previously identified

• Conservation analysis:

  • Assess sequence conservation of CrcB across diverse F. psychrophilum strains

  • Determine if CrcB is part of the core genome (1743 genes)

  • Identify conserved epitopes that could provide broad protection

  • Compare with the distribution pattern of known protective antigens

• Accessibility evaluation:

  • Determine cellular localization and surface exposure of CrcB

  • Assess if antibodies against CrcB can bind to intact bacteria

  • Evaluate accessibility for immune system recognition

• Immunization trials:

  • Formulate purified recombinant CrcB with appropriate adjuvants

  • Conduct dose-response studies to determine optimal antigen concentration

  • Perform challenge experiments with vaccinated fish

  • Measure antibody titers, cell-mediated immune responses, and survival rates

• Comparative analysis:

  • Compare protection levels with other known protective antigens such as OmpA (P60), ClpB, and gliding motility protein GldN

  • Consider combination approaches with multiple antigens

The precedent for identifying protective immunogenic proteins in F. psychrophilum has been established through previous research that correlated specific antibodies with protection . The outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, and gliding motility protein GldN were identified as potentially important for protective immunity , providing a comparative framework for evaluating CrcB.

What methods are most effective for evaluating the immunogenicity of recombinant F. psychrophilum proteins?

Evaluating the immunogenicity of recombinant F. psychrophilum proteins, including CrcB homolog, requires a multi-faceted approach combining in vitro and in vivo methodologies. Based on successful immunogenicity studies of F. psychrophilum proteins, the following comprehensive methodology is recommended:

• In vitro antigenicity assessment:

  • Western blotting using sera from naturally infected or vaccinated fish

  • Enzyme-linked immunosorbent assay (ELISA) to quantify antibody binding

  • Epitope mapping to identify immunodominant regions

  • T-cell proliferation assays to assess cellular immune recognition

• Animal immunization studies:

  • Immunize fish with purified recombinant protein using appropriate adjuvants

  • Collect sera at different time points to monitor antibody development

  • Measure antibody titers using ELISA

  • Assess antibody avidity and isotype distribution

  • Evaluate duration of antibody response

• Immunoproteomic approaches:

  • Two-dimensional polyacrylamide gel electrophoresis followed by Western blotting with immune sera, as previously employed for F. psychrophilum

  • Mass spectrometry identification of immunoreactive spots

  • Comparison with known immunogenic proteins like OmpA (P60), trigger factor, and ClpB

• Functional antibody assays:

  • Bacterial agglutination tests

  • Opsonophagocytosis assays with fish macrophages or neutrophils

  • Complement-mediated killing assays

  • Neutralization of specific bacterial functions

• Challenge studies:

  • Vaccinate fish with candidate proteins

  • Challenge with virulent F. psychrophilum strains

  • Compare survival rates between vaccinated and control groups

  • Assess correlates of protection

The methodological approach should build upon the documented success in identifying 15 immunogenic proteins through immunoproteomic analysis of F. psychrophilum . This study demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection .

How can researchers assess cross-protection against different F. psychrophilum serotypes using recombinant proteins?

Assessing cross-protection against different F. psychrophilum serotypes using recombinant proteins like CrcB requires a methodical approach considering the serological diversity of this pathogen. Based on the research documenting three main serotypes (Fp T, Th, and Fd) , the following comprehensive methodology is recommended:

• Serotype-specific challenge panel development:

  • Assemble a panel of well-characterized F. psychrophilum isolates representing all three serotypes (Fp T, Th, and Fd)

  • Include multiple strains from each serotype to account for within-serotype variation

  • Characterize challenge strains using the multiplex PCR-based serotyping scheme

  • Standardize challenge doses across serotypes based on preliminary virulence testing

• Recombinant protein conservation analysis:

  • Compare the sequence conservation of CrcB across strains representing different serotypes

  • Identify conserved epitopes that may confer cross-protection

  • Assess if CrcB belongs to the core genome (like the 1743 core genes identified) or is associated with serotype-specific genomic regions

• Cross-reactivity assessment:

  • Develop antisera against recombinant CrcB

  • Test reactivity against whole cells of different serotypes using ELISA, immunoblotting, or flow cytometry

  • Evaluate if antibodies recognize native CrcB across serotypes

• Vaccination and challenge studies:

  • Immunize separate groups of fish with recombinant CrcB

  • Challenge immunized fish with representative strains from each serotype

  • Calculate relative percent survival for each challenge strain

  • Compare protection levels across serotypes

• Immune response analysis:

  • Measure antibody titers against each serotype

  • Assess cellular immune responses

  • Identify correlates of protection that predict cross-protection

This framework builds upon the striking correlation between PCR-serotype and fish host species documented in F. psychrophilum and adapts established methodologies for assessing cross-protection. The molecular determinants of serotypes identified through genomic analysis provide critical context for evaluating whether CrcB-based immunity would transcend serotype boundaries.

How can CRISPR-Cas9 technology be applied to study CrcB function in F. psychrophilum?

CRISPR-Cas9 technology offers powerful approaches for investigating CrcB function in F. psychrophilum through precise genetic manipulation. Building on the knowledge that F. psychrophilum naturally possesses CRISPR systems , the following methodological framework for applying CRISPR-Cas9 to study CrcB is recommended:

• Gene knockout studies:

  • Design guide RNAs (gRNAs) targeting the crcB gene with high specificity

  • Construct CRISPR-Cas9 delivery systems adapted for F. psychrophilum

  • Generate precise gene deletions without polar effects on adjacent genes

  • Create conditional knockouts using inducible promoters to study essential genes

  • Verify knockouts through sequencing and expression analysis

• Gene tagging and visualization:

  • Use CRISPR-Cas9 to introduce fluorescent protein tags at the C-terminus of CrcB

  • Employ epitope tags for protein detection and purification

  • Create translational fusions that maintain protein function

  • Visualize protein localization under various environmental conditions

• Base editing and point mutations:

  • Apply CRISPR base editors to introduce specific mutations in crcB

  • Create amino acid substitutions in predicted functional domains

  • Generate mutations that mimic naturally occurring variants

  • Assess the impact of specific residues on fluoride channel function

• CRISPRi for gene repression:

  • Implement CRISPR interference (CRISPRi) using catalytically inactive Cas9 (dCas9)

  • Achieve tunable repression of crcB expression

  • Study phenotypes under partial loss of function

  • Investigate gene essentiality under various conditions

• CRISPR screening approaches:

  • Develop CRISPR libraries targeting genes potentially interacting with crcB

  • Perform screens for synthetic lethality or genetic interactions

  • Identify compensatory mechanisms for fluoride resistance

Implementation considerations must include optimization for the low-temperature growth conditions preferred by F. psychrophilum and adaptation of transformation methods for this specific bacterium. The analysis of CRISPR arrays in F. psychrophilum revealed two different loci with dissimilar spacer content , suggesting that understanding the native CRISPR biology of this organism may inform the development of optimized CRISPR-Cas9 tools for genetic manipulation.

What are the challenges in structural characterization of CrcB and how can they be overcome?

Structural characterization of the F. psychrophilum CrcB homolog presents several significant challenges typical of membrane proteins. The following methodological framework addresses these challenges with modern approaches:

• Challenges in protein production:

  • Membrane proteins often express poorly in heterologous systems

  • Proper folding and insertion into membranes is critical

  • Detergent selection impacts structural integrity

  • Stability issues during purification and crystallization

• X-ray crystallography approaches:

  • Fusion protein strategy: Incorporate stable, crystallizable protein domains (e.g., T4 lysozyme, BRIL) to facilitate crystal contacts

  • Antibody fragment co-crystallization: Generate and purify Fab or nanobody fragments that stabilize specific conformations

  • Lipidic cubic phase crystallization: Employ lipidic mesophases that better mimic the membrane environment

  • Detergent screening: Systematically test different detergents and additives to identify optimal crystallization conditions

• Cryo-electron microscopy (cryo-EM) strategies:

  • Amphipol reconstitution: Transfer protein from detergent to amphipathic polymers that better preserve native structure

  • Nanodiscs: Incorporate CrcB into nanodiscs with defined lipid composition for single-particle analysis

  • Focused refinement: Apply computational approaches to improve resolution of flexible regions

  • Direct electron detectors: Utilize latest-generation detectors to enhance signal-to-noise ratio

• Nuclear magnetic resonance (NMR) approaches:

  • Selective isotope labeling: Incorporate NMR-active isotopes into specific residues to reduce spectral complexity

  • Fragment-based analysis: Study individual domains or segments separately

  • Solid-state NMR: Apply to study CrcB in a more native-like lipid environment

• Integrative structural biology:

  • Combine low-resolution techniques (SAXS, cross-linking mass spectrometry) with computational modeling

  • Utilize evolutionary coupling analysis to predict structural contacts

  • Apply molecular dynamics simulations to refine structures and study conformational dynamics

By employing complementary approaches and adapting methods to the specific challenges posed by CrcB, researchers can overcome the significant hurdles in membrane protein structural biology. The success in identifying and characterizing other F. psychrophilum proteins using advanced methodologies suggests that similar technical innovations could be applied to structural studies of CrcB.

How can systems biology approaches integrate CrcB function into broader F. psychrophilum pathogenicity networks?

Systems biology approaches offer powerful frameworks for integrating CrcB function into comprehensive F. psychrophilum pathogenicity networks. Building on the genomic and phenotypic characterization of F. psychrophilum , the following methodological strategies are recommended:

• Multi-omics integration:

  • Genomics: Analyze crcB presence, conservation, and genetic context across the pan-genome of F. psychrophilum (3373 genes)

  • Transcriptomics: Profile genome-wide expression changes in wild-type vs. crcB mutants under various conditions

  • Proteomics: Quantify protein abundance changes, with special attention to virulence factors

  • Metabolomics: Measure metabolic shifts associated with crcB function

  • Integrate datasets using computational tools to identify correlations and causative relationships

• Network analysis approaches:

  • Construct protein-protein interaction networks centered on CrcB

  • Develop gene regulatory networks showing transcriptional connections

  • Create metabolic networks highlighting potential bottlenecks

  • Identify network motifs and regulatory hubs connected to CrcB function

  • Perform network perturbation analysis through targeted mutations

• Host-pathogen interaction mapping:

  • Profile host transcriptional responses to wild-type vs. crcB-mutant bacteria

  • Characterize immune signaling networks activated during infection

  • Develop interactome maps between bacterial and host proteins

  • Measure dynamic changes in host-pathogen interfaces during infection progression

• Predictive modeling:

  • Develop mathematical models of CrcB function in fluoride homeostasis

  • Create genome-scale metabolic models incorporating CrcB-associated pathways

  • Build machine learning models to predict virulence based on genomic features

  • Simulate in silico gene knockouts to predict phenotypic outcomes

• Experimental validation pipeline:

  • Test model predictions through targeted experiments

  • Refine models based on experimental outcomes

  • Develop high-throughput validation approaches for network connections

This systems biology framework builds upon the previous findings that F. psychrophilum isolates have a similar mode of action on adhesion, colonization, and destruction of fish tissues across large spatial and temporal scales . Understanding how CrcB functions within this conserved virulence network could reveal new insights into F. psychrophilum pathogenicity and potentially identify novel intervention targets.