Recombinant Salmonella paratyphi A Cobalt transport protein CbiN (cbiN)

What is the CbiN protein and what role does it play in Salmonella paratyphi A?

CbiN is a membrane component of the CbiMNQO complex, which functions as a group-I cobalt energy coupling factor (ECF) transporter in Salmonella paratyphi A. This transporter is responsible for micronutrient uptake from the environment, specifically facilitating cobalt ion transport across the bacterial membrane . Within the CbiMNQO complex, CbiN corresponds to part of the EcfS component found in group-II ECF transporters, working alongside CbiM to form the substrate-binding module of the transporter complex . The protein plays a crucial role in coupling conformational changes between the CbiQ and CbiM subunits during the transport cycle, which is essential for the proper functioning of the cobalt transport mechanism in S. paratyphi A.

How does the CbiN protein structurally interact with other components of the CbiMNQO complex?

The CbiN protein functions as part of an integrated complex where it mediates interactions between CbiM and CbiQ components. Based on experimental results, CbiN is positioned at the interface between these two proteins, serving as a coupling element . Structurally, CbiN helps coordinate the conformational changes necessary for transport activity by facilitating the rotation or toppling movements of both CbiQ and CbiM during the transport process . The precise positioning of CbiN is critical, as it transmits the energy derived from ATP hydrolysis (performed by CbiO) to the substrate-binding component (CbiM), enabling the conformational changes required for cobalt transport across the membrane.

What genetic factors influence cbiN expression in S. paratyphi A?

The cbiN gene in S. paratyphi A is part of the cobalt transport genetic system that responds to environmental cobalt availability. Regulation occurs primarily through metal-dependent transcriptional regulators that sense intracellular cobalt concentrations. During infection, expression of cbiN may be modulated by environmental conditions encountered within the host, particularly as S. paratyphi A migrates from the intestinal epithelium through Peyer's patches and into systemic circulation . The genomic context of cbiN can vary between different S. paratyphi A strains, as evidenced by comparative genomic analyses of isolates from outbreaks such as the one documented in Vadodara, India . These genomic variations may influence the expression levels and functional characteristics of the CbiN protein across different clinical isolates.

What expression systems are optimal for producing recombinant CbiN protein?

For recombinant expression of CbiN from S. paratyphi A, several expression systems should be considered based on the protein's membrane-associated nature. E. coli BL21(DE3) with pET vector systems provides a strong starting point, particularly when modified with rare codon supplementation for S. paratyphi A codons. Expression optimization requires careful temperature control (typically 16-25°C) to prevent inclusion body formation. For functional studies, membrane-protein specialized systems such as C41/C43(DE3) E. coli strains or cell-free expression systems supplemented with nanodiscs or liposomes may preserve native structural conformations.

Expression yields can be significantly improved by incorporating an N-terminal signal sequence and a C-terminal purification tag (His6 or Strep-tag II) separated by a TEV protease cleavage site. This approach facilitates both proper membrane insertion during expression and subsequent purification while allowing tag removal for structural studies. Empirical optimization of induction conditions (IPTG concentration: 0.1-0.5 mM; induction duration: 4-16 hours) is essential for achieving the balance between yield and proper folding.

How can site-directed mutagenesis be applied to study functional domains in CbiN?

Site-directed mutagenesis provides critical insights into CbiN's structure-function relationships. Based on structural data of the CbiMNQO complex, key residues at the interfaces between CbiN and its partner proteins (CbiM and CbiQ) should be targeted . The QuikChange mutagenesis protocol or Gibson Assembly methods are most appropriate for generating these mutations.

A systematic approach should target:

• Conserved residues at protein-protein interfaces (alanine scanning)

• Residues implicated in conformational changes during transport cycles

• Potential metal coordination sites if CbiN participates in cobalt binding

Following mutagenesis, a comprehensive functional assessment workflow includes:

• Expression level analysis by Western blotting

• Membrane localization verification by fractionation

• Complex assembly assessment via pull-down assays

• Transport activity measurements using radioisotope (57Co) uptake assays

• Conformational change analysis through intrinsic fluorescence or FRET

This systematic mutation analysis will map the functional topology of CbiN and clarify its precise role in the cobalt transport mechanism.

What purification strategies maintain native CbiN structure?

Purification of recombinant CbiN requires specialized approaches due to its membrane association and interaction partners. A multi-step strategy is recommended:

• Membrane Extraction: Solubilization using mild detergents (n-dodecyl-β-D-maltoside or digitonin at 1-2%) in phosphate buffer with 100-300 mM NaCl preserves protein-protein interactions within the CbiMNQO complex.

• Affinity Chromatography: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or TALON resin with gradual imidazole elution (20-250 mM) minimizes non-specific binding.

• Size Exclusion Chromatography: Critical for separating monomeric CbiN from aggregates or partial complexes, using Superdex 200 columns equilibrated with purification buffer containing detergent at concentrations slightly above critical micelle concentration.

• Detergent Exchange or Removal: For structural studies, consider exchanging harsh detergents for milder ones using successive dialysis, or reconstitution into nanodiscs or proteoliposomes for functional assays.

Throughout purification, maintain physiologically relevant concentrations of cobalt (1-10 μM) in buffers to stabilize the transport-competent conformation. Verify protein integrity at each step using SDS-PAGE and Western blotting, while confirming structural integrity through circular dichroism spectroscopy.

What are the challenges in crystallizing the CbiMNQO complex containing CbiN?

Crystallizing the complete CbiMNQO complex presents several challenges due to its membrane-embedded nature and dynamic conformational states. The primary obstacles include:

• Conformational Heterogeneity: The transport cycle involves multiple conformational states of the complex, creating a mixture that complicates crystal formation. Stabilizing specific conformations using ATP analogs (AMP-PNP or ADP-AlF4) can help overcome this issue by locking the complex in defined states .

• Detergent Micelle Interference: The detergent micelle surrounding the hydrophobic regions of the complex can hinder crystal contact formation. Screening different detergents (maltosides, glucosides, and newer amphipols) or using lipidic cubic phase crystallization may mitigate this problem.

• Complex Stability: The CbiMNQO complex may dissociate during purification and crystallization. Co-expression of all components with appropriate tags, followed by tandem affinity purification, helps maintain complex integrity.

• Component Flexibility: The coupling mechanism between CbiN and other components involves flexible regions that can impede crystallization. Limited proteolysis or surface entropy reduction through mutation of flexible surface residues can improve crystallizability.

How does the structure of CbiN in S. paratyphi A compare to homologous proteins?

CbiN from S. paratyphi A shares structural and functional homology with equivalent components in other bacterial ECF transporters, though with distinct characteristics. Comparative structural analysis reveals:

Understanding these structural similarities and differences provides insights into the evolution of cobalt transport mechanisms and may guide the development of targeted interventions against pathogenic bacteria like S. paratyphi A.

What methods are most effective for assessing CbiN function in vitro?

Several complementary approaches provide robust assessment of CbiN function in vitro:

• Radioisotope Transport Assays: Using 57Co2+ to directly measure cobalt uptake in reconstituted proteoliposomes containing purified CbiMNQO complex or in whole-cell systems. This approach quantifies transport kinetics (Km and Vmax) and can evaluate the impact of mutations or inhibitors.

• ATPase Activity Coupling: Measuring ATP hydrolysis rates using colorimetric phosphate release assays or coupled enzyme systems. CbiM has been found to stimulate CbiQO's basal ATPase activity, and this stimulation can serve as a proxy for proper complex assembly and function .

• Conformational Change Monitoring: Using intrinsic tryptophan fluorescence, FRET with strategically placed fluorophores, or EPR spectroscopy with spin labels to detect the conformational changes in CbiN during the transport cycle.

• Binding Assays: Isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to characterize interactions between CbiN and other complex components, as well as potential direct interactions with cobalt ions.

• Crosslinking Mass Spectrometry: Chemical crosslinking followed by mass spectrometry analysis to map the interaction interfaces between CbiN and its partner proteins in different conformational states.

These methods should be performed using purified components reconstituted in appropriate membrane mimetics (nanodiscs or proteoliposomes) to maintain the native-like environment necessary for proper function.

How can isotope labeling be applied to study CbiN interactions?

Isotope labeling techniques provide powerful tools for investigating CbiN's interactions with partner proteins and its structural dynamics:

• NMR Spectroscopy Applications:

  • Selective 15N/13C labeling of CbiN enables monitoring of chemical shift perturbations upon complex formation

  • TROSY-based experiments for detecting residue-specific interactions in the membrane-embedded state

  • Methyl-TROSY approaches focusing on Ile, Leu, Val residues for monitoring conformational changes in large complexes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Mapping solvent-accessible regions of CbiN to identify protected interfaces upon complex formation

  • Detecting conformational changes induced by cobalt binding or ATP hydrolysis

  • Time-resolved measurements to capture transient states during the transport cycle

• Crosslinking with Isotope-Coded Reagents:

  • Deuterium-labeled crosslinkers combined with mass spectrometry to quantify dynamic changes in protein-protein interfaces

  • Identification of transient interactions that may be missed by traditional structural methods

• Neutron Scattering with Deuterium Contrast Variation:

  • Selective deuteration of CbiN or partner proteins to highlight specific components within the complex

  • Low-resolution structural information complementary to X-ray crystallography or cryo-EM

These approaches should be integrated with functional assays to correlate structural insights with transport activity, providing a comprehensive understanding of CbiN's role in the cobalt transport mechanism.

How might CbiN function contribute to S. paratyphi A virulence?

CbiN's role in cobalt transport has significant implications for S. paratyphi A virulence and pathogenesis:

• Nutrient Acquisition: As part of the CbiMNQO complex, CbiN facilitates cobalt uptake, which is essential for vitamin B12 (cobalamin) biosynthesis. Cobalamin is a critical cofactor for several metabolic enzymes, making cobalt acquisition important for bacterial survival in the nutrient-limited host environment .

• Adaptive Metabolism: During different stages of infection—from intestinal colonization to systemic spread—S. paratyphi A encounters varying nutrient landscapes. The efficient cobalt transport system helps the pathogen adapt to these changing conditions, potentially enhancing its fitness during infection .

• Immune Evasion Connection: Metal homeostasis systems, including cobalt transporters, can influence bacterial resistance to oxidative stress generated by host immune responses. Proper functioning of CbiN and the entire cobalt transport system may contribute to S. paratyphi A's ability to withstand host defense mechanisms.

• Potential Target for Attenuation: Given its importance for bacterial metabolism, the CbiN protein and the broader cobalt transport system could represent targets for developing attenuated strains. Current vaccine development efforts for S. paratyphi A have focused on mutations in genes like guaBA, clpX, and sptP , and incorporating mutations in cobalt transport genes might offer additional attenuation strategies.

Understanding CbiN's contribution to virulence could inform new approaches to vaccine development and therapeutic intervention against paratyphoid fever A.

Could recombinant CbiN be utilized in vaccine development against S. paratyphi A?

Recombinant CbiN protein could potentially contribute to S. paratyphi A vaccine development through several strategies:

• Subunit Vaccine Component: As a membrane-associated protein involved in essential nutrient acquisition, CbiN could serve as an antigenic target in subunit vaccine formulations. This approach would be conceptually similar to utilizing other S. paratyphi A outer membrane proteins that have demonstrated immunoprotective properties in mouse models, such as LamB, PagC, TolC, NmpC, and FadL, which showed protection rates of 70-95% when used in immunization studies .

• Live Attenuated Vaccine Enhancement: Current attenuated S. paratyphi A vaccine candidates, such as CVD 1902 (with guaBA and clpX deletions), demonstrate attenuation and immunogenicity in animal and human studies . Incorporating additional mutations in the cbiN gene could potentially enhance attenuation while preserving immunogenicity, creating a more balanced safety-immunogenicity profile.

• Diagnostic Marker Application: Recombinant CbiN could serve as a diagnostic antigen for developing serological tests to distinguish between different enteric fever infections, particularly given the increasing proportion of paratyphoid fever A cases in regions where S. Typhi vaccines are deployed .

• Challenges to Consider:

  • Expression level variations of CbiN during different infection stages might affect its suitability as a vaccine target

  • Potential cross-reactivity with homologous proteins in commensal bacteria could reduce specificity

  • The membrane-embedded nature of CbiN presents challenges for recombinant production while maintaining native conformation

Research exploring CbiN as a vaccine component would require thorough immunogenicity studies and protection assays in appropriate animal models, followed by careful safety assessment in human trials similar to those conducted for other S. paratyphi A vaccine candidates .

What bioinformatic approaches can identify CbiN homologs and predict functional conservation?

Several bioinformatic strategies can effectively identify CbiN homologs across bacterial species and predict functional conservation:

• Profile-Based Sequence Searches:

  • Position-Specific Scoring Matrix (PSSM) searches using PSI-BLAST

  • Hidden Markov Model (HMM) profiles generated from aligned CbiN sequences

  • Sensitive detection methods like HMMER or JACKHMMER to identify distant homologs

• Structural Homology Assessment:

  • Threading approaches (I-TASSER, Phyre2) to identify proteins with similar fold despite low sequence identity

  • Contact prediction methods (AlphaFold2, RoseTTAFold) to compare predicted structural features

• Genomic Context Analysis:

  • Examination of gene neighborhood conservation across species

  • Identification of conserved operonic structures containing cbiN

  • Correlation with other components of the cobalt transport system (cbiM, cbiQ, cbiO)

• Evolutionary Analysis:

  • Calculation of selection pressures (dN/dS ratios) across different domains

  • Identification of co-evolving residues using methods like direct coupling analysis

  • Reconstruction of evolutionary history to identify adaptation events

• Integrated Functional Prediction:

  • Gene expression correlation network analysis

  • Protein-protein interaction predictions

  • Integration of structural data with sequence conservation patterns

These approaches should be applied systematically across diverse bacterial genomes, with particular attention to pathogenic species and those with established cobalt transport systems. The resulting data can guide experimental validation of predicted functional sites and inform targeted mutagenesis studies.

How can contradictory results in CbiN functional studies be reconciled?

Reconciling contradictory results in CbiN functional studies requires a systematic approach to identify sources of variability and establish consensus findings:

• Experimental System Variations:

  • Different expression systems (E. coli, yeast, cell-free) may produce proteins with varying post-translational modifications or folding properties

  • Membrane mimetic environments (detergents, nanodiscs, liposomes) significantly impact membrane protein function

  • Presence or absence of partner proteins (CbiM, CbiQ, CbiO) may alter CbiN behavior

• Methodological Standardization:

  • Develop standard operating procedures for CbiN purification and functional assays

  • Establish reference preparations with defined activity metrics

  • Create positive and negative controls for each assay type

• Meta-analysis Approaches:

  • Systematic review of published data with statistical assessment of effect sizes

  • Identification of moderator variables that explain inconsistencies

  • Bayesian integration of diverse data types to generate consensus models

• Collaborative Validation Studies:

  • Ring trials involving multiple laboratories using standardized protocols

  • Blind testing of key hypotheses across different experimental platforms

  • Open data sharing to enable comprehensive reanalysis

• Computational Reconciliation:

  • Develop mechanistic models that can accommodate seemingly contradictory results

  • Use machine learning to identify patterns in experimental conditions that predict outcomes

  • Simulate experimental variations to predict their impact on results

By addressing methodological inconsistencies and integrating diverse data types, researchers can develop a more coherent understanding of CbiN function and resolve apparent contradictions in the literature.

What emerging technologies could advance CbiN structural and functional studies?

Several cutting-edge technologies show promise for advancing CbiN research:

• Cryo-Electron Microscopy Innovations:

  • Time-resolved cryo-EM to capture transport intermediates

  • Microcrystal electron diffraction (MicroED) for structural determination from nano-sized crystals

  • In situ structural studies within native-like membrane environments

• Advanced Spectroscopic Methods:

  • Single-molecule FRET to track conformational dynamics during transport

  • Vibrational spectroscopy (FTIR, Raman) to detect subtle structural changes

  • Ultrafast X-ray free electron laser (XFEL) studies for time-resolved structural changes

• Genetic Engineering Approaches:

  • CRISPR-based genetic screens to identify genetic interactions with cbiN

  • In vivo proximity labeling (BioID, APEX) to map protein interaction networks

  • Synthetic biology approaches to engineer novel transport properties

• Computational Advances:

  • Molecular dynamics simulations across biologically relevant timescales

  • Machine learning for predicting functional impacts of mutations

  • Quantum mechanical/molecular mechanical (QM/MM) modeling of metal coordination and transport

• Imaging Technologies:

  • Super-resolution microscopy to visualize CbiN distribution in bacterial cells

  • Correlative light and electron microscopy to connect function with ultrastructure

  • Nanoscale secondary ion mass spectrometry (NanoSIMS) to track cobalt distribution

These emerging technologies can be integrated to create a comprehensive understanding of CbiN's role in cobalt transport, potentially revealing new therapeutic targets against S. paratyphi A infection.

How might CbiN research contribute to broader understanding of metal transport systems?

Research on the CbiN protein has significant implications for our understanding of bacterial metal transport systems:

• Evolutionary Insights:

  • CbiN represents a component of group-I ECF transporters, which differ structurally from group-II transporters while maintaining similar functions

  • Comparative studies between CbiN and analogous components in other transport systems can illuminate evolutionary pathways of metal acquisition mechanisms

• Mechanistic Principles:

  • The coupling function of CbiN between CbiQ and CbiM provides a model for understanding energy transduction in membrane transporters

  • Structure-function studies of CbiN contribute to general principles of conformational coupling in transport proteins

• Bacterial Adaptation:

  • Understanding how CbiN contributes to cobalt transport efficiency in different environmental conditions can reveal mechanisms of bacterial adaptation to metal-limited environments

  • This knowledge extends to how pathogens like S. paratyphi A compete for essential nutrients during infection

• Therapeutic Applications:

  • Insights from CbiN research may inform the development of inhibitors targeting metal transport systems in various pathogens

  • The approach could lead to novel antibacterial strategies that exploit the essential nature of metal acquisition

• Synthetic Biology Potential:

  • Understanding the modular nature of ECF transporters, including CbiN's role, could enable engineering of synthetic transporters with novel specificities

  • Such applications could extend to bioremediation, biosensing, or controlled metal accumulation

By positioning CbiN research within this broader context, investigators can contribute not only to understanding S. paratyphi A pathogenesis but also to fundamental knowledge of bacterial metal homeostasis mechanisms.

What are the key knowledge gaps in CbiN research that need addressing?

Despite progress in understanding the CbiMNQO complex structure and function, several critical knowledge gaps remain in CbiN research:

• Precise Transport Mechanism: While structural data suggests CbiN functions in coupling conformational changes between CbiQ and CbiM during transport , the exact molecular details of how energy is transferred through the complex remain poorly defined.

• Regulatory Mechanisms: The transcriptional and post-translational regulation of cbiN expression in response to environmental signals, particularly during infection, needs further characterization.

• Species-Specific Variations: Comparative analysis of CbiN across different Salmonella serovars and closely related bacteria would clarify adaptation mechanisms and potential functional differences.

• In Vivo Significance: Direct evidence linking CbiN function to S. paratyphi A virulence, persistence, or antibiotic resistance is lacking, particularly in relevant infection models.

• Structural Dynamics: Time-resolved structural studies capturing the conformational changes in CbiN during the transport cycle would provide crucial insights into its coupling function.

Addressing these knowledge gaps requires interdisciplinary approaches combining structural biology, biochemistry, microbial genetics, and infection biology. Progress in these areas will enhance our understanding of bacterial metal transport systems and potentially reveal new therapeutic targets against S. paratyphi A.

How can CbiN research inform therapeutic strategies against paratyphoid fever?

Research on S. paratyphi A CbiN protein could inform multiple therapeutic strategies against paratyphoid fever:

• Target-Based Drug Discovery: The structural details of CbiN and its interfaces with other components of the CbiMNQO complex provide potential binding sites for small molecule inhibitors . Disrupting cobalt transport could attenuate bacterial growth in the metal-limited host environment.

• Vaccine Development Support: Understanding CbiN's expression patterns, immunogenicity, and essentiality contributes to rational vaccine design approaches. This could complement existing vaccine candidates that target other components of S. paratyphi A .

• Diagnostic Applications: Knowledge of CbiN conservation and expression could support the development of improved diagnostics that distinguish between typhoid and paratyphoid fever, addressing the increasing proportion of paratyphoid fever A cases in regions where S. Typhi vaccines are deployed .

• Combination Therapy Approaches: Insights into CbiN function could reveal synergistic targets that, when inhibited together with conventional antibiotics, might enhance treatment efficacy or prevent resistance development.

• Host-Directed Therapies: Understanding how host factors interact with bacterial metal transport systems could suggest interventions that modify the host environment to restrict pathogen access to essential metals.