Recombinant Salmonella paratyphi B Cobalt transport protein CbiN (cbiN)

What is the Cobalt transport protein CbiN and what is its primary function in Salmonella paratyphi?

Cobalt transport protein CbiN (cbiN) is a membrane protein that functions as part of the Energy-coupling factor (ECF) transporter system in Salmonella paratyphi. It serves as a probable substrate-capture protein component (also called the S component) within this system, specifically facilitating the transport of cobalt ions across the bacterial membrane . The protein consists of 93 amino acids and plays a critical role in cobalt homeostasis, which is essential for various metabolic processes within the bacterium, particularly cobalamin (vitamin B12) biosynthesis. This transport function is vital for bacterial survival and virulence, as cobalt serves as a cofactor for several enzymes involved in critical cellular processes.

How does the amino acid sequence of CbiN differ between Salmonella paratyphi A and B strains?

The CbiN protein sequences from Salmonella paratyphi A and B strains show high similarity but contain distinct differences:

The key difference appears at position 8, where S. paratyphi A has alanine while S. paratyphi B has valine . This amino acid substitution, while subtle, may influence protein structure and potentially affect substrate binding characteristics, though functional studies would be required to determine the significance of this variation.

What role does the CbiN protein play in the pathogenesis of Salmonella paratyphi?

While the search results don't directly address the specific role of CbiN in pathogenesis, we can infer its importance based on the function of cobalt transport systems in bacterial virulence. Cobalt acquisition is essential for bacterial metabolic processes, particularly in the nutrient-limited environment of a host. The CbiN protein, as part of the ECF transport system for cobalt, likely contributes to Salmonella paratyphi's ability to survive and replicate within the host.

Transport proteins are often evaluated as potential vaccine candidates because they are typically exposed on the bacterial surface. Similar outer membrane proteins from Salmonella have shown immunogenicity and protective efficacy. For instance, studies have identified several outer membrane proteins of S. paratyphi A with significant immunoprotection rates, including LamB, PagC, TolC, NmpC, and FadL . While CbiN was not specifically mentioned in these studies, its role as a transport protein suggests it might have similar potential for vaccine development.

What experimental approaches are optimal for studying CbiN protein function and structure?

To comprehensively investigate CbiN protein function and structure, researchers should employ a multi-faceted experimental approach:

• Protein Expression and Purification:

  • Recombinant expression using E. coli or other suitable expression systems

  • Purification using affinity chromatography with appropriate tags (determined during production process)

  • Storage in optimized buffer (Tris-based buffer with 50% glycerol) at -20°C or -80°C for extended storage

• Structural Analysis:

  • X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure

  • Circular dichroism spectroscopy to analyze secondary structure elements

  • Nuclear magnetic resonance (NMR) spectroscopy for dynamic studies of the protein

• Functional Assays:

  • Metal binding assays using isothermal titration calorimetry

  • Radiolabeled cobalt transport assays in reconstituted membrane vesicles

  • Site-directed mutagenesis to identify key residues involved in cobalt binding and transport

• Interaction Studies:

  • Pull-down assays to identify interaction partners within the ECF transport complex

  • Surface plasmon resonance to quantify binding kinetics with other components

  • Crosslinking studies to capture transient protein-protein interactions

How might recombinant CbiN be utilized in the development of vaccines against paratyphoid fever?

Recombinant CbiN protein could be investigated as a potential component in vaccine development against Salmonella paratyphi through several approaches:

• Subunit Vaccine Development:
  Similar to other membrane proteins studied in Salmonella, CbiN could be evaluated as a subunit vaccine component. Studies have shown that outer membrane proteins of S. paratyphi A, when intraperitoneally immunized at a dose of 100 μg, have demonstrated significant immunoprotection with protection rates ranging from 70-95% .

• Adjuvant Combination Studies:
  Researchers should test various adjuvant formulations with recombinant CbiN to enhance immunogenicity, following approaches used with other Salmonella membrane proteins.

• Outer Membrane Vesicle (OMV) Incorporation:
  CbiN could be incorporated into OMV-based vaccine platforms, which have shown promise for enteric fever vaccines. Studies have demonstrated that bivalent OMV-based immunogens derived from S. Typhi and S. Paratyphi A can induce significant humoral responses and protect against heterologous Salmonella strains .

• Attenuated Live Vaccine Vector:
  The protein could be overexpressed in attenuated Salmonella strains, similar to approaches used with other immunogenic proteins. Several attenuated S. Paratyphi A strains have been developed by deleting virulence-associated genes like aroC/yncD, guaBA/clpX, and sptP, which could potentially serve as vectors for increased CbiN expression .

What methods can be used to assess the immunogenicity of CbiN protein?

To evaluate the immunogenicity of CbiN protein, researchers should implement a comprehensive assessment strategy:

• In vitro Assays:

  • Bactericidal assays using antisera raised against recombinant CbiN

  • Serum IgG antibody titer determination using ELISA

  • B-cell epitope mapping using peptide arrays

• Animal Model Studies:

  • Measure humoral immune response by quantifying serum IgG and mucosal IgA levels

  • Assess cellular immunity by analyzing CD4, CD8, and CD19 cell populations in immunized mice spleen

  • Evaluate Th1 and Th17-cell mediated immunity through cytokine profiling

  • Conduct challenge studies with heterologous Salmonella strains to assess protective efficacy

• Functional Antibody Assessment:

  • Investigate protective mechanisms of anti-CbiN antibodies by evaluating inhibition of bacterial motility

  • Assess prevention of mucin penetration ability

  • Determine opsonization capacity and complement-mediated killing

How does CbiN interact with other components of the Energy-coupling factor transport system?

The CbiN protein functions as the substrate-capture component (S-component) of the Energy-coupling factor (ECF) transport system . Based on general knowledge of ECF transporters:

• Component Interaction:

  • The S-component (CbiN) typically interacts with the energizing module consisting of an ATPase (A-component) and a transmembrane protein (T-component)

  • This interaction is critical for coupling ATP hydrolysis to substrate transport

• Experimental Approaches to Study Interactions:

  • Co-immunoprecipitation to identify protein-protein interactions

  • Bacterial two-hybrid systems to verify direct interactions

  • Blue native PAGE to analyze intact complexes

  • Crosslinking mass spectrometry to map interaction interfaces

• Functional Coupling Assays:

  • Reconstitution of the complete ECF transport complex in proteoliposomes

  • Measurement of ATP hydrolysis coupled to cobalt transport

  • Mutational analysis of key residues at predicted interfaces

What protocols are recommended for characterizing the metal-binding properties of recombinant CbiN?

To characterize the metal-binding properties of recombinant CbiN protein, researchers should employ multiple complementary techniques:

• Isothermal Titration Calorimetry (ITC):

  • Measures heat changes during binding events

  • Provides binding affinity (Kd), stoichiometry, and thermodynamic parameters

  • Sample preparation: Purified CbiN (10-20 μM) in buffer without metal chelators

• Differential Scanning Fluorimetry (DSF):

  • Assesses protein thermal stability changes upon metal binding

  • Method: Incubate protein with varying concentrations of cobalt and other divalent metals

  • Analysis: Compare melting temperatures (Tm) in presence/absence of metals

• Spectroscopic Methods:

  • UV-Visible spectroscopy to detect spectral shifts upon metal binding

  • Circular dichroism to assess conformational changes

  • Fluorescence spectroscopy with intrinsic tryptophan or external probes

• Metal Quantification:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to determine metal content

  • Atomic absorption spectroscopy for quantitative analysis of bound metals

  • Colorimetric assays with metal-specific chelators

• Structural Analysis of Metal Binding:

  • X-ray absorption spectroscopy (XAS) to determine metal coordination environment

  • Nuclear magnetic resonance (NMR) for binding site mapping

  • Mutational analysis of predicted metal-coordinating residues

How can researchers optimize the expression and purification of recombinant Salmonella paratyphi CbiN protein?

Optimizing expression and purification of membrane proteins like CbiN requires addressing several challenges:

• Expression System Selection:

  • E. coli BL21(DE3) for high-yield expression

  • C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression

  • Consider cell-free expression systems for toxic proteins

• Vector Design Considerations:

  • Incorporate appropriate fusion tags (His, GST, MBP) for purification and solubility

  • Include TEV or PreScission protease sites for tag removal

  • Consider codon optimization for the expression host

• Expression Condition Optimization:

  • Test various induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Evaluate different inducer concentrations (IPTG: 0.1-1.0 mM)

  • Explore extended expression times (5-24 hours) at lower temperatures

• Membrane Protein Solubilization:

  • Screen detergents (DDM, LDAO, Triton X-100) for efficient extraction

  • Test mild solubilization conditions to maintain native structure

  • Consider nanodiscs or amphipols for stabilization

• Purification Strategy:

  • Multi-step approach: affinity chromatography followed by size exclusion

  • Buffer optimization: include glycerol (50%) and appropriate pH for stability

  • Storage considerations: avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

What analytical techniques should be employed to validate the integrity and functionality of purified CbiN protein?

To ensure the quality and functionality of purified CbiN protein, researchers should employ a comprehensive validation workflow:

• Purity Assessment:

  • SDS-PAGE with Coomassie or silver staining

  • Western blotting with specific antibodies

  • Mass spectrometry for protein identification and integrity verification

• Structural Integrity Analysis:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal shift assays to assess stability

  • Dynamic light scattering to evaluate homogeneity and aggregation state

• Functional Validation:

  • Metal binding assays using fluorescent probes or ITC

  • Reconstituted transport assays in liposomes

  • ATPase activity assays when co-purified with other ECF components

• Biophysical Characterization:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation to determine oligomeric state

  • Microscale thermophoresis for interaction studies

How might CbiN be utilized as a target for antimicrobial development?

The essential role of CbiN in cobalt transport suggests several strategies for antimicrobial development:

• Small Molecule Inhibitor Development:

  • Target the metal-binding site of CbiN to prevent cobalt acquisition

  • Screen chemical libraries for compounds that disrupt CbiN-substrate interactions

  • Design peptidomimetics that interfere with CbiN assembly into the ECF complex

• Antibody-Based Approaches:

  • Develop antibodies that specifically bind and neutralize CbiN function

  • Construct bispecific antibodies targeting CbiN and immune effector cells

  • Consider antibody-antibiotic conjugates for targeted delivery

• Screening Methodologies:

  • Develop high-throughput assays based on cobalt transport or protein-protein interactions

  • Implement virtual screening using computational models of CbiN structure

  • Design phenotypic screens based on bacterial growth under cobalt-limited conditions

• Validation Studies:

  • Assess effects of CbiN inhibition on bacterial growth and virulence

  • Evaluate potential for resistance development

  • Conduct mechanistic studies to confirm target engagement

What role could CbiN play in developing bivalent vaccines against both Salmonella Typhi and Paratyphi?

The development of bivalent vaccines targeting both S. Typhi and S. Paratyphi represents a significant research priority, and CbiN could potentially contribute to this effort:

• Comparative Immunogenicity Assessment:

  • Evaluate sequence conservation of CbiN across Salmonella serovars

  • Assess cross-reactivity of anti-CbiN antibodies between S. Typhi and S. Paratyphi

  • Identify conserved epitopes that could provide cross-protection

• OMV-Based Vaccine Approaches:

  • Incorporate CbiN into bivalent Outer Membrane Vesicle (OMV) formulations

  • Build upon successful OMV-based immunogens that have shown protection against heterologous Salmonella strains

  • Consider GMMA (Generalized Modules for Membrane Antigens) technology, which has been used to engineer S. Paratyphi A strains that display S. Typhi antigens

• Combination Strategies:

  • Evaluate CbiN in combination with other immunogenic outer membrane proteins

  • Consider incorporating CbiN with Vi-polysaccharide, which has been engineered for expression in S. Paratyphi A and shown to provide protection against both serovars

  • Test co-administration with other established vaccine components

• Evaluation Framework:

  • Assess humoral and cell-mediated immune responses against both serovars

  • Measure protective efficacy in appropriate animal models

  • Conduct comparative studies with existing vaccine candidates

How can genetic manipulation of the cbiN gene contribute to attenuated vaccine strain development?

Genetic modification of the cbiN gene could be explored as a strategy for developing attenuated vaccine strains:

• Attenuation Approaches:

  • Generate conditional cbiN mutants with reduced expression under in vivo conditions

  • Create cobalt transport-deficient strains with altered cbiN function

  • Develop strains with modified CbiN that allow sufficient growth for immunogenicity while reducing virulence

• Contextual Considerations:

  • Combine cbiN modification with established attenuation strategies, such as aroC/yncD deletion or guaBA/clpX deletion, which have shown success in S. Paratyphi A vaccine development

  • Evaluate attenuation in comparison with existing vaccine strains like SPADD01, CVD 1902, and sptP deletion mutants

• Assessment Protocol:

  • Determine the 50% lethal dose (LD50) compared to wild-type and established attenuated strains

  • Evaluate bacterial persistence and colonization in mucosal tissues

  • Measure immunogenicity through antibody titers and T-cell responses

  • Assess protection against challenge with virulent strains

• Safety and Stability Evaluation:

  • Monitor genetic stability through multiple passages

  • Evaluate potential for reversion to virulence

  • Assess shedding patterns and environmental persistence

What emerging technologies could advance our understanding of CbiN structure and function?

Several cutting-edge technologies offer promise for deeper insights into CbiN biology:

• Structural Biology Advances:

  • Cryo-electron microscopy for high-resolution structure determination of membrane protein complexes

  • Integrative structural biology combining multiple techniques (X-ray, NMR, SAXS, crosslinking MS)

  • Computational approaches like AlphaFold2 for structure prediction and molecular dynamics simulations

• Single-Molecule Techniques:

  • Single-molecule FRET to observe conformational changes during transport

  • Atomic force microscopy for topological studies of CbiN in membranes

  • Nanopore recording to observe individual transport events

• Functional Genomics Approaches:

  • CRISPR-Cas9 screening to identify genetic interactions with cbiN

  • Transposon sequencing to map fitness contributions under various conditions

  • RNA-seq to characterize transcriptional responses to CbiN manipulation

• Advanced Imaging Methods:

  • Super-resolution microscopy to visualize CbiN localization and dynamics in bacterial cells

  • Correlative light and electron microscopy for contextualized structural insights

  • Live-cell imaging with fluorescent metal sensors to observe transport in real-time

How might systems biology approaches enhance our understanding of CbiN's role in Salmonella pathogenesis?

Systems biology offers powerful frameworks for understanding CbiN in the broader context of pathogenesis:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data to map CbiN's influence on cellular networks

  • Analyze cobalt-dependent metabolic pathways affected by CbiN function

  • Identify condition-specific regulation of cbiN expression

• Network Analysis Approaches:

  • Construct protein-protein interaction networks centered on CbiN

  • Develop metabolic models incorporating cobalt-dependent processes

  • Map signaling pathways influenced by cobalt availability

• Host-Pathogen Interaction Studies:

  • Dual RNA-seq to simultaneously profile host and bacterial responses

  • Spatial transcriptomics to map expression patterns during infection

  • Proteomics of the Salmonella-containing vacuole to assess CbiN's role

• Computational Modeling:

  • Develop mathematical models of cobalt transport kinetics

  • Simulate effects of CbiN inhibition on bacterial fitness

  • Model evolutionary trajectories under selective pressure