Recombinant Salmonella choleraesuis Disulfide bond formation protein B (dsbB)

What is DsbB and what is its basic function in Salmonella choleraesuis?

DsbB is an inner membrane protein with four transmembrane segments that functions as an oxidoreductase in the bacterial periplasm. It plays a critical role in the oxidative protein folding pathway by reoxidizing the periplasmic disulfide bond-forming protein DsbA. This creates a catalytic cycle essential for proper folding of numerous periplasmic and secreted proteins containing disulfide bonds, many of which are virulence factors .

The DsbB protein contains two essential periplasmic loops, each with a pair of cysteine residues that participate in disulfide exchange reactions. DsbB oxidizes DsbA through disulfide exchange with one cysteine pair, then transfers electrons via the second periplasmic loop to quinones in the cytoplasmic membrane, which serve as the ultimate electron acceptors .

How does the DsbB/DsbA system differ from the paralogous DsbL/DsbI system in Salmonella?

Salmonella enterica possesses both the DsbA/DsbB system and a paralogous DsbL/DsbI system. Key differences include:

While both systems contribute to periplasmic disulfide bond formation, they differ in their substrate specificity and evolutionary conservation, suggesting complementary but distinct roles in bacterial physiology .

How is DsbB involved in Salmonella pathogenicity?

DsbB contributes significantly to Salmonella pathogenicity through several mechanisms:

• It ensures proper folding of virulence factors containing disulfide bonds

• It affects transcription of the Salmonella pathogenicity island 1 (SPI1) type three secretion system genes

• It influences activation of the RcsCDB regulatory system, which controls various virulence determinants

• It plays a role in bacterial transmission networks, as Salmonella Choleraesuis (including its properly folded virulence factors) is transmitted between food, environment, humans, and livestock, with swine playing a significant role

The importance of DsbB is demonstrated by the fact that disruption of the disulfide bond formation pathway significantly reduces bacterial virulence and survival in host environments.

What are the optimized methods for expression and purification of recombinant DsbB?

Based on established protocols for DsbB purification from E. coli, which can be adapted for S. choleraesuis:

• Expression System:

  • Clone the dsbB gene with a C-terminal His-tag into an appropriate expression vector

  • Transform into a suitable E. coli strain lacking endogenous DsbB to prevent interference

  • Induce expression under controlled conditions (typically IPTG induction at mid-log phase)

• Membrane Extraction:

  • Harvest cells and disrupt by sonication or French press

  • Isolate membrane fraction by ultracentrifugation (typically >100,000 × g)

  • Solubilize membrane proteins with appropriate detergents (0.1% dodecyl-β-d-maltoside is effective)

• Purification Steps:

  • Apply solubilized membrane proteins to Ni-NTA column

  • Wash extensively with high-salt buffer containing detergent

  • Include washing step with lauroylsarcosine to remove contaminants

  • Elute with imidazole gradient (0-500 mM)

  • Further purify using hydroxyapatite chromatography with sodium phosphate gradient

  • Dialyze against appropriate buffer to remove excess salt

This approach typically yields approximately 4 mg of purified DsbB per liter of bacterial culture .

How can researchers accurately assess DsbB activity in vitro?

Several complementary approaches can be used to assess DsbB activity:

• Redox State Analysis:

  • Monitor the oxidation state of DsbB's cysteine residues using thiol-specific reagents (e.g., Ellman's reagent)

  • Use non-reducing SDS-PAGE to distinguish oxidized from reduced forms

• Coupled Enzyme Assays:

  • Measure DsbB activity through its ability to oxidize reduced DsbA

  • Monitor DsbA oxidation state changes using fluorescence-based assays

  • Quantify quinone reduction spectrophotometrically as electrons flow from DsbA through DsbB to quinones

• Substrate Protein Folding:

  • Use model substrates like alkaline phosphatase (PhoA) whose activity depends on disulfide bond formation

  • Compare PhoA activity and protein stability in reconstituted systems with purified DsbB vs. controls

• Electron Transfer Kinetics:

  • Determine electron transfer rates between DsbA and DsbB using stopped-flow spectroscopy

  • Measure kinetic parameters (Km, kcat) under varying conditions to assess catalytic efficiency

What genetic approaches are most effective for studying DsbB function in vivo?

Effective genetic approaches include:

• Gene Deletion and Complementation:

  • Create clean deletion mutants (ΔdsbB) using lambda Red recombination or CRISPR-Cas9

  • Complement with wild-type and mutant variants of dsbB on plasmids or chromosomally integrated constructs

  • Create double mutants (e.g., ΔdsbA ΔdsbB, ΔdsbB ΔdsbI) to assess functional redundancy

• Reporter Systems:

  • Use transcriptional fusions (e.g., hilA-lacZ) to monitor effects on virulence gene expression

  • Employ enzymatic reporters requiring disulfide bonds (e.g., PhoA-6×His) to assess pathway functionality

  • Monitor reporter protein levels by Western blot to differentiate effects on expression versus stability

• Cysteine Mutagenesis:

  • Create point mutations in catalytic cysteine residues (based on E. coli model: Cys41-Cys44 and Cys104-Cys130)

  • Replace non-essential cysteines (e.g., Cys8, Cys49) with serine or valine to simplify analysis

  • Generate variants with different combinations of cysteine mutations (CCSS, SSCC, SSSS) to dissect reaction mechanisms

• Conditional Expression Systems:

  • Use inducible promoters to control DsbB levels and timing of expression

  • Employ depletion strategies to study effects of acute loss of DsbB function

How do interactions between DsbB and quinones affect disulfide bond formation efficiency?

The interaction between DsbB and quinones represents a critical node in the electron transfer pathway:

Research strategies include spectroscopic analysis of quinone-DsbB interactions, site-directed mutagenesis of quinone binding residues, and comparative analysis of DsbB function under varying oxygen tensions.

What is the relationship between DsbB function and antimicrobial resistance in Salmonella choleraesuis?

The relationship between DsbB and antimicrobial resistance involves several interconnected mechanisms:

• Proper Folding of Resistance Determinants:

  • Many antimicrobial resistance proteins require disulfide bonds for proper folding

  • DsbB dysfunction may compromise the stability and function of resistance determinants

  • This creates an indirect relationship between DsbB activity and resistance phenotypes

• Mobile Genetic Elements:

  • Antimicrobial resistance genes often reside on mobile genetic elements

  • Horizontal gene transfer facilitates spread of resistance via plasmids and transposons

  • The proper folding of proteins involved in conjugation and transposition may depend on the DsbB pathway

• Specific Resistance Mechanisms:

  • Colistin resistance gene mcr-3, commonly detected in swine, has been found in Salmonella Choleraesuis strains from human infections

  • Proper expression and function of resistance elements like efflux pumps may require functional disulfide bond formation

• Stress Responses and Adaptation:

  • Antimicrobial exposure triggers bacterial stress responses

  • DsbB function may be modulated as part of these adaptive responses

  • Compromised disulfide bond formation may trigger compensatory mechanisms affecting resistance

Research approaches should include comparative genomic analysis of resistant isolates, assessment of resistance determinant stability in dsbB mutants, and evaluation of antimicrobial susceptibility profiles under conditions affecting DsbB function.

How do DsbB sequence variations across Salmonella serovars correlate with host specificity and virulence?

Analysis of DsbB sequence variations across Salmonella serovars reveals important relationships with host adaptation:

• Sequence Conservation Patterns:

  • Core catalytic regions show high conservation across serovars

  • Variable regions may correlate with host range or tissue tropism

  • Specific polymorphisms might reflect adaptation to host-specific environments

• Co-evolution with Partner Proteins:

  • DsbB variations may co-evolve with corresponding changes in DsbA

  • Sequence changes affecting interaction interfaces would be particularly significant

  • Co-evolution patterns can identify functional constraints and adaptations

• Host-Specific Adaptations:

  • Salmonella Choleraesuis shows host adaptation to swine and can cause severe infections in humans

  • Transmission patterns between swine, humans, and the environment may exert selective pressure on DsbB

  • International pork trade has influenced the global transmission of Salmonella serovars, potentially selecting for specific DsbB variants

• Virulence Correlations:

  • DsbB variants may correlate with differences in virulence phenotypes

  • Effects on SPI1 expression vary depending on genetic background

  • Combination with mutations in other disulfide bond formation proteins (DsbA, DsbL, DsbI, SrgA) produces synergistic effects on virulence

Research approaches should include phylogenetic analysis of DsbB sequences, correlation with host range and virulence phenotypes, and functional complementation studies across serovars.

How should researchers design experiments to distinguish between DsbB-dependent and DsbB-independent disulfide bond formation pathways?

Designing experiments to distinguish these pathways requires a systematic approach:

• Genetic Dissection Strategy:

  • Create single and combinatorial mutations in disulfide bond formation genes

  • Test ΔdsbB alone, ΔdsbI alone, and ΔdsbB ΔdsbI double mutants

  • Include additional mutations in DsbA and SrgA to fully map pathway interdependencies

  • Complement with plasmid-encoded genes to confirm specificity of phenotypes

• Substrate-Specific Analysis:

  • Identify and test multiple substrate proteins with different dependencies

  • PhoA serves as a model substrate dependent on disulfide bond formation

  • Flagellar assembly specifically requires DsbA/DsbB but not DsbL/DsbI

  • SPI1 expression shows complex dependencies on multiple disulfide bond formation systems

• Biochemical Approaches:

  • Examine redox states of periplasmic proteins in various mutant backgrounds

  • Use alkylating agents to trap proteins with free thiols

  • Perform in vitro reconstitution with purified components

  • Employ redox potential measurements to characterize electron flow through different pathways

• Environmental Modulation:

  • Test pathway dependencies under different growth conditions

  • Examine effects of oxidative stress, reducing agents, and pH changes

  • Monitor pathway switching in response to environmental signals

This experimental design should be coupled with appropriate controls and statistical analysis to robustly distinguish between the pathways.

What are the key considerations for analyzing conflicting data regarding DsbB function across experimental systems?

When confronted with conflicting data regarding DsbB function, researchers should systematically evaluate:

• Strain and Serovar Differences:

  • Confirm the exact Salmonella strain and serovar used in each study

  • Consider genetic background effects (e.g., presence of prophages, plasmids)

  • Sequence DsbB and interacting proteins to identify potential polymorphisms

  • Directly compare multiple reference strains under identical conditions

• Methodological Variations:

  • Evaluate differences in expression systems (native vs. recombinant)

  • Compare protein purification methods and their effects on activity

  • Assess assay conditions (temperature, pH, redox environment)

  • Consider timing of measurements and growth phases used

• Functional Redundancy:

  • Account for compensatory effects from DsbL/DsbI system

  • Consider contributions from SrgA and other redox proteins

  • Examine potential upregulation of alternative pathways in mutants

  • Use double or triple mutants to overcome redundancy issues

• Substrate Specificity Differences:

  • Different substrates may show variable dependence on DsbB

  • Consider substrate availability and expression levels

  • Examine potential substrate-specific cofactors or chaperones

  • Test multiple substrates in parallel to identify patterns

When interpreting conflicting results, researchers should clearly define experimental contexts and develop testable hypotheses to resolve discrepancies.

How can researchers effectively measure the impact of DsbB on global protein folding in Salmonella choleraesuis?

To comprehensively assess DsbB's impact on global protein folding:

• Proteome-Wide Approaches:

  • Compare proteomes of wild-type and ΔdsbB strains using 2D gel electrophoresis

  • Employ diagonal redox 2D-PAGE to identify proteins with altered disulfide bonding

  • Use quantitative proteomics to measure protein abundance changes

  • Combine with pulse-chase labeling to distinguish folding effects from expression changes

• Thiol-Trapping Methods:

  • Use thiol-reactive fluorescent dyes to label proteins with free thiols

  • Employ isotope-coded affinity tags specific for thiols

  • Analyze by mass spectrometry to identify affected proteins

  • Quantify the ratio of oxidized to reduced forms of specific proteins

• Functional Genomics:

  • Perform transposon mutagenesis screens in wild-type and ΔdsbB backgrounds

  • Identify genetic interactions using synthetic lethal/sick screens

  • Use transcriptome analysis to identify compensatory responses

  • Apply gene ontology enrichment analysis to identify affected pathways

• In Vivo Folding Sensors:

  • Develop fluorescent reporters that indicate disulfide bond formation status

  • Use split-protein complementation based on disulfide-dependent interactions

  • Measure protein stability using targeted degradation tags in different genetic backgrounds

  • Monitor activity of reporter enzymes requiring disulfide bonds (e.g., PhoA)

These approaches should be combined with appropriate controls and statistical analysis to generate a comprehensive picture of DsbB's impact on the proteome.

What are the most promising therapeutic approaches targeting DsbB in Salmonella choleraesuis infections?

Several therapeutic approaches targeting DsbB show promise:

• Small Molecule Inhibitors:

  • Design competitive inhibitors of DsbB-DsbA interaction

  • Develop compounds that interfere with quinone binding

  • Create allosteric modulators affecting DsbB conformation

  • Target the cysteine residues involved in electron transfer

• Peptide-Based Approaches:

  • Design peptide mimics of DsbA interaction interfaces

  • Develop cell-penetrating peptides targeting DsbB

  • Create cyclic peptides with enhanced stability and specificity

  • Combine with antimicrobial peptides for dual-action therapy

• Combination Therapies:

  • Pair DsbB inhibitors with conventional antibiotics

  • Target multiple disulfide bond formation pathways simultaneously

  • Combine with inhibitors of virulence pathways dependent on DsbB

  • Develop adjuvants enhancing host immunity against misfolded bacterial proteins

• Vaccine Approaches:

  • Use attenuated strains with modified DsbB activity as live vaccines

  • Develop subunit vaccines including DsbB epitopes

  • Create outer membrane vesicle (OMV) vaccines from dsbB-modified strains

  • Design DNA vaccines encoding modified DsbB variants

These approaches require careful consideration of specificity, given the conservation of disulfide bond formation pathways across many bacterial species.

How might advanced structural biology techniques advance our understanding of DsbB function?

Advanced structural biology techniques can provide crucial insights:

• Cryo-Electron Microscopy (Cryo-EM):

  • Determine high-resolution structures of DsbB in native membrane environments

  • Visualize DsbB-DsbA complexes during electron transfer

  • Capture different conformational states in the reaction cycle

  • Examine interactions with quinones and membrane lipids

• Integrative Structural Biology:

  • Combine X-ray crystallography, NMR, and molecular dynamics simulations

  • Map conformational changes during the catalytic cycle

  • Identify allosteric networks within the protein structure

  • Predict effects of mutations on structure and function

• Time-Resolved Structural Methods:

  • Use time-resolved X-ray crystallography to capture reaction intermediates

  • Apply temperature-jump techniques with rapid structural analysis

  • Employ hydrogen-deuterium exchange mass spectrometry to track conformational dynamics

  • Develop FRET-based sensors to monitor distance changes during catalysis

• In-Cell Structural Biology:

  • Apply in-cell NMR to study DsbB conformations in living bacteria

  • Use genetic code expansion to introduce structural probes at specific positions

  • Employ cross-linking mass spectrometry to map interaction networks

  • Develop cellular cryo-electron tomography approaches to visualize DsbB in situ

These approaches will provide unprecedented insights into DsbB structure-function relationships and catalytic mechanisms.

What role might DsbB play in emerging Salmonella choleraesuis transmission patterns and epidemiology?

DsbB's role in emerging transmission patterns involves several important aspects:

• Global Trade Influences:

  • International pork trade appears concordant with global transmission of Salmonella enterica serotypes

  • Euramerica-centralized sourcing has shaped Salmonella transmission patterns

  • DsbB function may influence bacterial survival during food processing and transport

• Host Adaptation Mechanisms:

  • Swine play a significant role in Salmonella Choleraesuis transmission between food, environment, humans, and other livestock

  • DsbB's role in virulence factor folding affects host range and adaptation

  • Proper protein folding influences bacterial fitness across diverse host environments

• Antimicrobial Resistance Connections:

  • Colistin-resistant gene mcr-3, commonly detected in swine, has been found in human Salmonella Choleraesuis isolates in China and the UK

  • Antimicrobial use in agriculture may select for variants with altered DsbB function

  • DsbB activity influences the proper folding of proteins involved in resistance mechanisms

• Surveillance Implications:

  • Monitoring DsbB sequence variants could provide epidemiological insights

  • Tracking disulfide bond-dependent virulence factors may help predict emerging threats

  • Understanding transmission networks requires assessing bacterial protein folding capacity

Research in this area should combine molecular epidemiology, whole genome sequencing, and functional analysis of DsbB variants to understand evolving transmission dynamics.