Recombinant Salmonella choleraesuis Cobalt transport protein CbiN (cbiN)

How does CbiN interact with other components of the cobalt transport system?

CbiN functions within the CbiMNQO transporter complex, where it appears to play a coupling role between CbiM and CbiQ components. Based on structural studies, CbiN is positioned near the interface between CbiM and CbiQ, facilitating conformational changes between these components during the transport cycle .

The current model suggests that CbiN functions in transmitting conformational changes induced by ATP binding and hydrolysis (occurring at the CbiO component) to the substrate-binding CbiM component. This coordination is essential for the transport mechanism where rotation or toppling of both CbiQ and CbiM components is required for efficient cobalt transport across the membrane .

What is the evolutionary significance of CbiN across different bacterial species?

CbiN is part of the Energy-coupling factor (ECF) transporters, which represent a large family of ATP-binding cassette transporters identified in various microorganisms. The protein is specifically found in group-I ECF transporters, which are dedicated to specific substrates like cobalt. The conservation of CbiN across different Salmonella species suggests its essential role in cobalt acquisition, which is critical for various metabolic processes .

Comparative analysis between Salmonella choleraesuis and other species like Salmonella arizonae shows conservation of key functional domains, highlighting the evolutionary importance of this transport system for bacterial survival in diverse environments .

What are the optimal conditions for recombinant expression of Salmonella CbiN protein?

For recombinant expression of CbiN, the following methodology has proven effective:

• Expression System: E. coli is the preferred heterologous host for CbiN expression, as demonstrated with Salmonella arizonae CbiN .

• Protein Tagging: N-terminal His-tagging facilitates purification while maintaining protein functionality. The full-length protein (amino acids 1-93) with the His-tag demonstrates good expression levels .

• Expression Conditions: While specific optimization parameters were not detailed in the available research, standard induction protocols for membrane proteins (lower temperatures of 16-25°C, reduced IPTG concentrations) are likely beneficial for proper folding.

• Purification: Standard nickel affinity chromatography followed by size exclusion chromatography can yield >90% purity as determined by SDS-PAGE .

What storage conditions maintain the stability of purified recombinant CbiN protein?

Optimal storage conditions for recombinant CbiN protein include:

• Storage Temperature: -20°C/-80°C for long-term storage .

• Buffer Composition: Tris/PBS-based buffer containing 6% Trehalose, pH 8.0 has been demonstrated to maintain stability .

• Recommended Handling:

  • Lyophilization is an effective preservation method

  • Brief centrifugation prior to opening vials ensures content retrieval

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of glycerol (5-50% final concentration, with 50% being optimal) for storage at -20°C/-80°C

  • Aliquoting is necessary to prevent protein degradation from freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

What analytical methods are most effective for confirming the structural integrity of recombinant CbiN?

For structural integrity confirmation, a multi-method approach is recommended:

• SDS-PAGE: Primary method for assessing purity (>90%) and confirming the expected molecular weight .

• Western Blotting: Utilizing anti-His antibodies or specific anti-CbiN antibodies to confirm identity.

• Circular Dichroism (CD): While not explicitly mentioned in the search results, CD spectroscopy would be valuable for assessing secondary structure integrity of the purified protein.

• Size Exclusion Chromatography: To assess aggregation state and homogeneity.

• Functional Assays: Reconstitution into liposomes or proteoliposomes followed by cobalt transport assays to confirm functional integrity.

How can CbiN be utilized in recombinant attenuated Salmonella vaccine development?

The methodology for utilizing CbiN in vaccine development can be extrapolated from successful approaches with other Salmonella surface proteins:

• Vector Construction: Based on the attenuated Salmonella Choleraesuis vector system (similar to rSC0016), CbiN can be incorporated into expression plasmids with appropriate regulatory elements to ensure efficient in vivo expression .

• Delivery System Design: The protein can be expressed either surface-displayed (using autotransporters like MisL) or secreted, depending on the desired immune response. Surface display leverages the natural immunogenicity of bacterial surface components .

• Attenuation Strategies: Implementing regulated delayed attenuation and delayed exogenous synthesis systems ensures bacterial vector survival long enough to deliver the antigen effectively while maintaining safety .

• Immune Response Assessment: Evaluation should include measurements of:

  • Antigen-specific mucosal IgA

  • Serum IgG titers

  • Cellular immune responses (Th1/Th2 cytokine profiles)

  • Protection efficacy through challenge studies

What experimental approaches are effective for studying CbiN's role in the CbiMNQO transport mechanism?

To investigate CbiN's functional role in the cobalt transport mechanism:

• Structural Studies:

  • X-ray crystallography of the complete CbiMNQO complex

  • Cryo-electron microscopy to capture different conformational states during the transport cycle

  • Molecular modeling based on the interaction between CbiM and CbiQ

• Functional Assays:

  • Reconstitution of purified components into liposomes for transport assays

  • ATPase activity measurements to assess how CbiN affects the ATP hydrolysis by CbiO

  • Isothermal titration calorimetry to quantify binding interactions between components

• Mutagenesis Studies:

  • Site-directed mutagenesis of key residues in CbiN

  • Construction of chimeric proteins to identify functional domains

  • Deletion mutants to assess the minimal functional unit

• In vivo Tracking:

  • Fluorescently tagged CbiN to track localization and dynamics

  • Co-immunoprecipitation to identify interaction partners

  • Crosslinking studies to capture transient interactions during the transport cycle

How does ATP binding and hydrolysis in CbiO affect the conformational changes in CbiN within the transport complex?

The coupling mechanism between ATP hydrolysis and CbiN conformational changes involves a complex series of events:

• Energy Transduction Pathway:

  • ATP binding to CbiO induces a closed conformation of the nucleotide-binding domains

  • This conformational change is transmitted to CbiQ, the scaffold component

  • CbiQ then undergoes rotation or toppling that alters its interaction with CbiN

  • CbiN transmits these changes to CbiM, facilitating substrate transport

• Structural Transitions:

  • The complex transitions between inward-open and outward-open conformations

  • CbiN appears to function as a conformational coupler in this process

  • The L1 loop of CbiM has been identified as having a substrate-gating function that may be influenced by CbiN's positioning

• ATP Hydrolysis Cycle:

  • β, γ-methyleneadenosine 5′-triphosphate binding induces the closed conformation of CbiO

  • Product release triggers conformational reset

  • These cycles drive the conformational changes throughout the complex, including CbiN

What are the challenges in distinguishing CbiN's specific functions from other components of the CbiMNQO complex?

Several methodological challenges exist in isolating CbiN's specific contributions:

• Functional Redundancy:

  • Components of transport complexes often have overlapping functions

  • Knockout studies may be compensated by alternate pathways

• Complex Stability Issues:

  • CbiN may be unstable or improperly folded when expressed alone

  • The complex may require all components for structural integrity

• Experimental Approaches to Address These Challenges:

  • Complementation studies with chimeric proteins

  • Conditional expression systems to regulate component levels

  • Cross-species functional complementation to identify conserved mechanisms

  • Single-molecule studies to observe real-time dynamics of individual components

How can researchers optimize recombinant Salmonella strains expressing CbiN for targeted delivery to specific tissues?

Optimization strategies for targeted delivery include:

• Promoter Selection and Regulation:

  • In vivo-inducible promoters for tissue-specific expression

  • Oxygen-regulated or pH-dependent promoters for environmental sensing

• Tissue-Targeting Modifications:

  • Based on successful approaches with other recombinant Salmonella, tumor-targeting can be enhanced through:

    • Auxotrophic mutations that promote preferential replication in target tissues

    • Surface modifications to enhance tissue tropism

    • Co-expression of tissue-specific adhesins

• Quantification of Targeting Efficiency:

  • Tissue colonization can be quantified as CFU/g of tissue

  • Immunohistochemical detection using anti-Salmonella antibodies

• In vivo Administration Protocol:

  • Based on tumor-targeting studies:

    • Administration of 1×10⁷ CFU in 100 μl PBS via tail vein

    • Multiple doses (four doses at 7-day intervals)

    • Concurrent antibiotic (ampicillin 0.6 mg/ml) in drinking water to maintain plasmid selection

How do differences in experimental models affect the interpretation of CbiN function in different Salmonella species?

When comparing CbiN research across different experimental systems:

• Species-Specific Differences:

  • Data from Salmonella arizonae CbiN (93 amino acids) may not directly translate to Salmonella choleraesuis

  • Functional conservation needs to be experimentally verified across species

• Model System Limitations:

  • Mouse models provide valuable insights but have limitations for translating to other hosts

  • As noted in vaccine research: "it is crucial to note that the outcomes observed in mice cannot be extrapolated to pigs" - requiring species-specific validation

• Experimental Context Considerations:

  • In vitro studies of purified components may not capture the complexity of in vivo interactions

  • Different expression systems (E. coli vs. native Salmonella) may affect protein folding and function

  • Recombinant tags (His-tag) may influence protein behavior in some experimental contexts

What are the current contradictions in the literature regarding CbiN's role in cobalt transport and virulence?

The available research presents several areas where further clarification is needed:

• Transport Mechanism Discrepancies:

  • The exact positioning of CbiN within the complex structure

  • Whether CbiN directly interacts with the substrate or solely functions in conformational coupling

• Virulence Connections:

  • While cobalt acquisition is linked to virulence in several bacterial pathogens, the specific contribution of CbiN to Salmonella pathogenesis remains to be fully characterized

  • Potential dual roles in both essential metabolism and virulence require further investigation

• Research Gaps to Address:

  • Comparative studies across Salmonella serovars

  • Direct assessment of how CbiN mutations affect colonization and virulence

  • Integration of structural insights with in vivo function

How might CbiN be exploited as a target for novel antimicrobial development?

Targeting CbiN for antimicrobial development presents several strategic approaches:

• Rationale for Targeting:

  • Essential role in cobalt acquisition

  • Surface accessibility

  • Significant divergence from human transporters

• Potential Approaches:

  • Small molecule inhibitors targeting the CbiN-CbiM interface

  • Peptide inhibitors mimicking key interaction domains

  • Antibodies or antibody fragments targeting surface-exposed regions

  • CRISPR-Cas delivery systems targeting cbiN genes

• Screening Methodologies:

  • High-throughput screening using reconstituted transport systems

  • Structure-based virtual screening leveraging CbiMQO complex structures

  • Phenotypic screens under cobalt-limited conditions

What technical innovations could improve the stability and immunogenicity of CbiN-based vaccine constructs?

Based on advances with other Salmonella-based vaccines, several approaches could enhance CbiN-based vaccines:

• Stability Enhancements:

  • Codon optimization for improved expression

  • Fusion partners to increase protein stability

  • Directed evolution to select for stable variants

• Immunogenicity Improvements:

  • Strategic epitope mapping and enhancement

  • Co-expression with molecular adjuvants

  • Display scaffolds optimizing epitope presentation

• Delivery Optimization:

  • Regulated delayed lysis systems for controlled antigen release

  • Dual plasmid systems with differential regulation

  • Bacterial ghost technology for non-living delivery vehicles

• Assessment Methodology:

  • Comprehensive immune profiling including:

    • Antigen-specific mucosal, humoral, and cellular immune responses

    • T-cell subset analysis (Th1/Th2 balance)

    • Memory B and T cell generation

    • Challenge studies with relevant pathogens

How might systems biology approaches advance our understanding of CbiN's role in bacterial metabolism and pathogenesis?

Integrative systems approaches offer powerful tools for contextualizing CbiN function:

• Multi-omics Integration:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to map interaction networks

  • Metabolomics to track cobalt-dependent pathways

  • Correlation of these datasets to construct predictive models

• Network Analysis:

  • Identification of condition-specific regulatory networks

  • Flux balance analysis to quantify metabolic impacts

  • Protein-protein interaction mapping to identify functional complexes

• In vivo Dynamics:

  • Single-cell analysis of gene expression in different microenvironments

  • In vivo imaging to track bacterial metabolism in real-time

  • Host-pathogen interaction profiling

• Computational Modeling:

  • Molecular dynamics simulations of the transport cycle

  • Machine learning approaches to predict functional partners

  • Evolutionary analysis to identify selection pressures