Recombinant Salmonella paratyphi A Cysteine desulfurase (iscS)

What is the role of cysteine desulfurase (iscS) in Salmonella paratyphi A?

Cysteine desulfurase (iscS) plays a critical role in Salmonella paratyphi A as an enzyme responsible for the production of hydrogen sulfide (H₂S). This gasotransmitter is particularly important for bacterial survival under anaerobic conditions when the pathogen faces oxidative stress. Research demonstrates that iscS functions as the primary cysteine desulfurase in anaerobic Salmonella, converting cysteine to alanine while releasing sulfur, which is subsequently incorporated into the production of H₂S. This biochemical pathway represents a crucial defense mechanism that enhances bacterial resistance to peroxide stress, allowing Salmonella to survive hostile host environments .

How does Salmonella paratyphi A differ from Salmonella Typhi in terms of pathogenesis?

While Salmonella paratyphi A and Salmonella Typhi both cause enteric fever with clinically indistinguishable symptoms, they exhibit significant genetic and pathogenic differences. Unlike S. Typhi, S. paratyphi A naturally lacks the Vi capsular polysaccharide, which serves as a virulence factor in S. Typhi. Both pathogens are human-restricted, which has limited comprehensive understanding of their host-pathogen interactions. Metabolomic analysis using two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) has revealed distinct metabolite profiles in plasma samples from patients infected with either pathogen, indicating serovar-specific systemic biomarkers that can be detected during enteric fever . These metabolomic differences suggest that the pathogens employ different strategies to manipulate host cellular processes despite causing similar clinical presentations.

What experimental methods are typically used to study iscS function in Salmonella?

The study of iscS function in Salmonella typically employs several complementary experimental approaches:

• Gene deletion studies: Creating ΔiscS knockout strains through targeted mutagenesis to observe phenotypic changes

• Complementation assays: Expressing iscS from plasmids (such as pWSK29) in knockout strains to confirm functional roles

• H₂S production measurement: Quantitative assessment of H₂S production in wild-type versus mutant strains under various conditions

• Oxidative stress resistance assays: Evaluating bacterial survival in the presence of H₂O₂ or other oxidative stressors

• Transcriptomic analyses: RNA sequencing to identify genes whose expression is affected by iscS deletion

• Biochemical enzyme assays: In vitro assessment of cysteine desulfurase activity

Research has shown that deletion of iscS prevents H₂S production in anaerobic Salmonella exposed to H₂O₂, while complementation with iscS on an expression vector restores this protective mechanism . These methodologies collectively provide insights into the physiological roles and regulatory networks involving iscS.

What are the optimal parameters for creating stable recombinant S. paratyphi A strains expressing modified iscS?

Creating stable recombinant S. paratyphi A strains with modified iscS requires careful optimization of several experimental parameters. The process typically involves:

• Vector selection: Low-copy plasmids like pWSK29 have shown effectiveness for iscS complementation studies

• Promoter choice: Native promoters maintain physiological expression levels, while inducible promoters enable controlled expression

• Integration site selection: Chromosomal integration provides greater stability than plasmid-based expression

• Selection markers: Kanamycin resistance markers are commonly used for initial selection

• Counter-selection strategies: Sucrose sensitivity (via sacB) allows for marker removal and selection of clean recombinants

For chromosomal integration, techniques similar to those used for the viaB locus integration in S. paratyphi A can be applied. This involves constructing recombination cassettes with homologous flanking regions, electroporation into the target strain, selection with appropriate antibiotics, and counter-selection on sucrose-containing media . For stable chromosomal modifications, maintaining the recombinant strain through numerous passages (>200) and performing routine PCR verification ensures genetic stability of the integrated construct.

How can researchers differentiate between the effects of iscS-dependent H₂S production and other stress response mechanisms in Salmonella?

Differentiating between iscS-dependent H₂S production and other stress response mechanisms requires a multi-faceted experimental approach:

• Specific inhibitor studies: Comparing the effects of iscS deletion to chemical inhibitors of H₂S production

• H₂S donor complementation: Using chemical H₂S donors like GYY4137 to rescue ΔiscS phenotypes

• Comparative deletion analysis: Creating knockout strains of other cysteine desulfurases (cadA, sufS) and H₂S-producing enzymes

• Double/triple knockout studies: Creating combinatorial deletions to assess redundancy and crosstalk

• Transcriptome/proteome analysis: Comparing global expression changes between single and multiple knockouts

• Temporal dynamics assessment: Measuring the kinetics of H₂S production relative to other stress responses

Research data indicates that while multiple cysteine desulfurases exist in Salmonella (iscS, cadA, sufS), deletion of iscS specifically prevents H₂S production in response to peroxide stress under anaerobic conditions . The addition of H₂S donor GYY4137 enhances resistance to H₂O₂ in ΔdmsABC Salmonella, providing a methodological approach to distinguish the specific protective effects of H₂S from other mechanisms.

What methodological approaches can be used to study the regulatory network controlling iscS expression in different microenvironments?

The regulatory network controlling iscS expression across different microenvironments can be studied through these methodological approaches:

• Reporter gene fusions: Constructing transcriptional/translational fusions of iscS promoter to reporters like GFP or luciferase

• Chromatin immunoprecipitation (ChIP-seq): Identifying transcription factors binding to the iscS promoter

• RNA-seq under various conditions: Oxygen levels, nutrient availability, host-derived signals

• In vivo expression technology (IVET): Assessing iscS expression during infection of animal models

• Single-cell analysis: Using flow cytometry or microscopy to assess cell-to-cell variation in expression

• Metabolic flux analysis: Quantifying how changes in metabolic pathways affect iscS expression

• Computational modeling: Integrating transcriptomic, proteomic, and metabolomic data to predict regulatory interactions

Research suggests that iscS expression is likely affected by oxygen availability and oxidative stress conditions, as its role in H₂S production is particularly important under anaerobic conditions when bacteria face peroxide stress . Implementation of these approaches would provide comprehensive insights into how S. paratyphi A modulates iscS expression across the diverse microenvironments encountered during infection.

What are the most effective methods for purifying and assessing the enzymatic activity of recombinant iscS from S. paratyphi A?

The purification and enzymatic characterization of recombinant iscS from S. paratyphi A typically follows this methodological workflow:

• Expression system optimization:

  • E. coli BL21(DE3) with pET vector systems for high-yield expression

  • Optimization of induction parameters (IPTG concentration, temperature, duration)

  • Addition of PLP (pyridoxal 5'-phosphate) as a cofactor during expression

• Purification protocol:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography for final polishing and buffer exchange

• Activity assay methods:

  • Methylene blue assay for H₂S production quantification

  • Cysteine desulfurase activity measurement using dithiothreitol-dependent reduction of cysteine

  • Coupled enzyme assays monitoring alanine production

  • Lead acetate precipitation assays for sulfide detection

• Kinetic parameter determination:

  • Measurement of Km, Vmax, and kcat using varying substrate concentrations

  • Inhibition studies to identify regulatory molecules

The purified enzyme requires careful handling under anaerobic or low-oxygen conditions to maintain activity, as oxidation of the catalytic cysteine residue can impair function. Including stabilizing agents such as DTT or β-mercaptoethanol in purification buffers helps maintain enzymatic activity during the isolation process.

How can researchers quantitatively assess H₂S production by iscS in vitro and in bacterial cultures?

Quantitative assessment of H₂S production by iscS can be performed through several complementary methods:

• For in vitro enzymatic assays:

  • Methylene blue method: Quantifies H₂S through formation of methylene blue, measured spectrophotometrically at 670 nm

  • Fluorescent probes: H₂S-specific probes that increase fluorescence upon reaction with H₂S

  • Gas chromatography: Direct measurement of headspace H₂S

  • Polarographic H₂S sensors: Real-time monitoring of H₂S production

• For bacterial cultures:

  • Lead acetate paper assays: Qualitative detection of H₂S production

  • Bismuth sulfite precipitation: Formation of black precipitate in media containing bismuth

  • Fluorescent probe permeabilization: Treatment of cultures with fluorescent H₂S probes

  • Zinc acetate trapping followed by spectrophotometric quantification

• Data analysis considerations:

  • Calibration with Na₂S standards at physiologically relevant concentrations

  • Accounting for abiotic H₂S loss through volatilization or oxidation

  • Normalization to protein concentration or cell density

  • Kinetic measurements to determine rates of H₂S production

When comparing wild-type and ΔiscS strains, results indicate that deletion of iscS prevents H₂S production in anaerobic Salmonella exposed to H₂O₂ . This quantitative difference provides a useful readout for assessing the functional importance of iscS in H₂S biosynthesis under specific environmental conditions.

What is the methodology for studying the relationship between iron-sulfur cluster biosynthesis and iscS function in S. paratyphi A?

Studying the relationship between iron-sulfur (Fe-S) cluster biosynthesis and iscS function requires specialized techniques:

• Fe-S cluster protein activity assays:

  • Measuring activities of Fe-S-dependent enzymes (aconitase, fumarase, etc.)

  • EPR spectroscopy to directly detect Fe-S clusters

  • 55Fe radiolabeling to track incorporation into Fe-S proteins

  • Mössbauer spectroscopy for Fe-S cluster type characterization

• Protein-protein interaction studies:

  • Bacterial two-hybrid assays to identify interactions between iscS and Fe-S assembly proteins

  • Co-immunoprecipitation to confirm interactions

  • FRET-based approaches to detect dynamic interactions in vivo

  • Crosslinking mass spectrometry to map interaction domains

• Transcriptional analysis:

  • RNA-seq comparing iscS mutants and wild-type to identify effects on the Fe-S regulon

  • qRT-PCR targeting specific genes in the iscSUA-hscBA-fdx operon

  • Chromatin immunoprecipitation to identify regulatory factors

• Metabolic labeling:

  • 35S-cysteine tracing to follow sulfur transfer from iscS to Fe-S clusters

  • Pulse-chase experiments to determine Fe-S cluster assembly rates

The iscS protein functions within the ISC (Iron-Sulfur Cluster) system, the primary machinery for Fe-S cluster assembly in Salmonella. By acting as a sulfur donor, iscS converts cysteine to alanine, liberating sulfur that is transferred to scaffold proteins for Fe-S cluster formation. These clusters are crucial for anaerobic respiration pathways, potentially linking iscS function to both H₂S production and respiratory flexibility in varying host environments.

What experimental approaches can best evaluate how iscS-dependent H₂S production affects S. paratyphi A virulence in infection models?

Evaluating how iscS-dependent H₂S production affects S. paratyphi A virulence requires a combination of in vitro and in vivo approaches:

• Cellular infection models:

  • Macrophage infection assays comparing survival of wild-type vs. ΔiscS strains

  • Gentamicin protection assays to assess intracellular survival

  • Cell culture systems mimicking intestinal epithelium (Caco-2, T84 cells)

  • Co-culture systems with immune cells to assess inflammatory responses

• Animal infection models:

  • Mouse models using genetically susceptible strains

  • Competitive index assays (wild-type vs. ΔiscS in the same animal)

  • Bacterial burden quantification in tissues

  • Survival studies and histopathological assessments

• Mechanistic investigations:

  • ROS/RNS measurement in infected cells exposed to wild-type vs. ΔiscS strains

  • Cytokine profiling to assess host immune response differences

  • Transcriptomics of host cells infected with different strains

  • H₂S donors/inhibitors to rescue/mimic phenotypes in vivo

• Human organoid models:

  • Intestinal organoids to model host-specific interactions

  • Microfluidic organ-on-chip technology for dynamic infection studies

Research data indicates that H₂S production by iscS provides protection against oxidative stress, suggesting it may enhance bacterial survival when facing host-derived reactive oxygen species during infection . Complementation studies using both genetic (iscS expression vectors) and chemical (H₂S donors like GYY4137) approaches would help establish causality between H₂S production and virulence-associated phenotypes.

How can recombinant S. paratyphi A strains with modified iscS be designed for potential vaccine development?

Designing recombinant S. paratyphi A strains with modified iscS for vaccine development requires a systematic approach:

• Rational attenuation strategy:

  • Mutation design for iscS: Partial activity variants vs. complete deletion

  • Combination with other attenuating mutations (e.g., htrA, phoPQ) as demonstrated in Vi-producing S. paratyphi A vaccine candidates

  • Analysis of single vs. double/triple attenuated strains for safety profiles

• Immunogenicity assessment:

  • Measuring serum IgG responses to S. paratyphi A antigens

  • Secretory IgA quantification in intestinal contents

  • T-cell responses evaluation (Th1/Th17 polarization)

  • Cytokine profiling after immunization

• Protection studies:

  • Challenge experiments with wild-type S. paratyphi A

  • Cross-protection assessment against S. Typhi

  • Long-term immunity studies and memory response evaluation

• Safety evaluation:

  • Genetic stability assessment through multiple passages (>200 passages)

  • Reversion frequency measurements

  • Distribution and persistence in tissues

  • Shedding patterns post-immunization

A promising approach would be similar to that used for developing Vi-producing attenuated S. paratyphi A, where deletion of virulence loci (htrA and phoPQ) successfully attenuated the strain while maintaining protective immunogenicity . For iscS-modified strains, careful balance between attenuation (reducing virulence) and immunogenicity (maintaining sufficient in vivo persistence to stimulate immunity) would be crucial.

What are the methodological considerations for investigating the impact of iscS-mediated H₂S production on antibiotic susceptibility in S. paratyphi A?

Investigating the impact of iscS-mediated H₂S production on antibiotic susceptibility requires careful experimental design:

• Susceptibility testing approaches:

  • Minimum inhibitory concentration (MIC) determination comparing wild-type and ΔiscS strains

  • Time-kill assays to assess killing kinetics under different oxygen tensions

  • Post-antibiotic effect studies with and without H₂S donors

  • Biofilm susceptibility assays to model therapeutic challenges

• Mechanistic investigations:

  • ROS measurement during antibiotic exposure with/without functional iscS

  • Membrane potential and permeability assessment

  • Gene expression profiling during antibiotic stress

  • Metabolomic analysis to identify metabolic adaptations

• Experimental conditions to consider:

  • Aerobic vs. anaerobic testing environments

  • pH variations to model different infection sites

  • Growth phase considerations (log vs. stationary)

  • Pre-conditioning with sub-inhibitory oxidative stress

• Data analysis and interpretation:

  • Fold-change in MIC between wild-type and mutant strains

  • Area under the kill curve comparisons

  • Statistical analysis adjusting for growth rate differences

  • Synergy/antagonism assessment with antioxidants

Research suggests that H₂S production via iscS may influence antibiotic susceptibility through its impact on bacterial redox homeostasis and stress resistance . Since many antibiotics exert bactericidal effects partly through inducing oxidative stress, iscS-dependent H₂S production might modulate these effects. Complementation studies using both genetic approaches (iscS expression vectors) and chemical approaches (H₂S donors) would help establish causality between H₂S production and changes in antibiotic susceptibility.

What are the experimental controls required when studying iscS function in recombinant S. paratyphi A strains?

Robust experimental design for studying iscS function requires these essential controls:

• Genetic controls:

  • Wild-type S. paratyphi A (positive control for normal iscS function)

  • ΔiscS knockout strain (negative control for iscS-dependent phenotypes)

  • Complemented strain (ΔiscS + plasmid-expressed iscS) to confirm phenotypes are due to iscS

  • Empty vector control in the ΔiscS background

  • Mutants of other cysteine desulfurases (ΔcadA, ΔsufS) to assess specificity

• Chemical controls:

  • H₂S donor compounds (e.g., GYY4137) to mimic iscS function

  • H₂S scavengers to neutralize H₂S effects

  • Specific enzyme inhibitors with appropriate vehicle controls

  • Antioxidants to distinguish between H₂S-specific and general redox effects

• Experimental condition controls:

  • Strict anaerobic vs. aerobic conditions with appropriate monitoring

  • pH and temperature controls relevant to host environments

  • Growth phase standardization across experiments

  • Media composition controls (minimal vs. rich media)

• Technical validation controls:

  • PCR confirmation of genetic modifications

  • Western blot verification of protein expression

  • Enzyme activity assays confirming functional differences

  • Growth curve analysis to account for growth rate effects

Research has demonstrated that complementation with the iscS gene expressed from pWSK29 improves H₂S synthesis in ΔdmsABC Salmonella and protects against H₂O₂ cytotoxicity, confirming the specific role of iscS . These controls help distinguish iscS-specific effects from artifacts or secondary consequences of genetic manipulation.

What statistical approaches are most appropriate for analyzing complex datasets from experiments involving recombinant S. paratyphi A iscS variants?

Analyzing complex datasets from experiments with recombinant S. paratyphi A iscS variants requires sophisticated statistical approaches:

• For hypothesis testing:

  • ANOVA with appropriate post-hoc tests for multiple strain comparisons

  • Mixed-effects models for repeated measures or nested experimental designs

  • Non-parametric alternatives when normality assumptions are violated

  • Survival analysis techniques for time-to-event data

• For high-dimensional data:

  • Principal Component Analysis (PCA) for dimensionality reduction

  • Hierarchical clustering to identify patterns across experimental conditions

  • Partial Least Squares Discriminant Analysis (PLS-DA) for metabolomic data

  • Gene Set Enrichment Analysis (GSEA) for transcriptomic data

• Experimental design considerations:

  • Power analysis to determine appropriate sample sizes

  • Randomization and blocking strategies to control for batch effects

  • Factorial designs to efficiently assess interaction effects

  • Latin square designs for complex multi-factor experiments

• Advanced analytical approaches:

  • Bayesian statistical frameworks for incorporating prior knowledge

  • Machine learning techniques for predictive modeling

  • Network analysis for protein-protein interaction data

  • Time-series analysis for dynamic phenotypes

When applying these methods to iscS research, it's important to account for potential confounding variables such as growth rate differences between strains and the influence of oxygen availability on experimental outcomes. For metabolomic data, approaches similar to those used in distinguishing S. Typhi and S. Paratyphi A infections based on host metabolites could be adapted to analyze bacterial metabolic profiles .

How can understanding iscS function in S. paratyphi A contribute to new diagnostic approaches for enteric fever?

Understanding iscS function can contribute to novel diagnostic approaches for enteric fever through several research applications:

• Metabolite biomarker development:

  • Identification of iscS-dependent metabolites in bacterial culture supernatants

  • Detection of H₂S-modified host proteins or metabolites in patient samples

  • Integration with existing metabolomic approaches that have successfully distinguished S. Typhi and S. Paratyphi A infections

  • Development of field-deployable tests targeting these biomarkers

• Immunodiagnostic approaches:

  • Identification of iscS-dependent antigens expressed during infection

  • Development of serological assays targeting these antigens

  • Engineering of aptamer-based detection systems for pathogen-specific signatures

  • Creation of rapid lateral flow assays for point-of-care diagnosis

• Molecular diagnostic strategies:

  • Nucleic acid amplification tests targeting the iscS gene or iscS-regulated genes

  • Design of primers recognizing serovar-specific regions of the iscS gene

  • CRISPR-Cas-based detection systems for rapid, specific diagnosis

  • Multiplexed PCR panels including iscS and other serovar-specific targets

• Systems biology integration:

  • Combining metabolomic, proteomic, and transcriptomic data to identify robust biomarker panels

  • Machine learning approaches to distinguish infection based on multi-parameter signatures

  • Development of diagnostic algorithms incorporating host and pathogen biomarkers

Research has demonstrated that reproducible and serovar-specific systemic biomarkers can be detected during enteric fever, with a combination of just six metabolites accurately defining the etiological agent . Integrating knowledge of iscS function with these approaches could further refine diagnostic specificity and potentially lead to tests that not only identify the pathogen but also provide information about antibiotic susceptibility or virulence potential.

What research directions could explore the therapeutic targeting of iscS or H₂S signaling pathways in Salmonella infections?

Therapeutic targeting of iscS or H₂S signaling pathways represents a promising research direction with several potential approaches:

• Direct iscS inhibitor development:

  • Structure-based design of specific inhibitors targeting the catalytic site

  • High-throughput screening of compound libraries against purified iscS

  • Fragment-based drug discovery approaches

  • Peptidomimetic inhibitors blocking protein-protein interactions in the Fe-S cluster assembly machinery

• H₂S signaling modulation strategies:

  • Compounds that scavenge bacterial H₂S without affecting host H₂S signaling

  • Inhibitors targeting bacterial persulfidation of specific proteins

  • Molecules disrupting H₂S-mediated protection against oxidative stress

  • Combination therapies pairing H₂S inhibitors with conventional antibiotics

• Host-directed therapeutic approaches:

  • Immunomodulatory compounds that enhance oxidative burst in phagocytes

  • Agents that modify the intracellular redox environment to counter H₂S effects

  • Drugs targeting host pathways exploited by bacterial H₂S

  • Precision probiotics engineered to compete with pathogens in H₂S-rich environments

• Novel antibiotic development strategies:

  • Antibiotic conjugates targeting iscS-dependent metabolic vulnerabilities

  • Compounds with enhanced activity under conditions where iscS function is critical

  • Antibiotics specifically active against anaerobic, H₂S-producing bacteria

  • Narrow-spectrum agents targeting unique features of S. paratyphi A metabolism

Research has shown that H₂S production via the cysteine desulfurase iscS protects anaerobic Salmonella from peroxide stress, suggesting that inhibiting this pathway could potentiate the effects of oxidative stress-inducing antibiotics or host immune responses . The addition of H₂S donors like GYY4137 enhances bacterial resistance to H₂O₂, further supporting the therapeutic potential of targeting this pathway .

How might comparative genomics and evolution studies of iscS across Salmonella serovars inform our understanding of pathogen adaptation?

Comparative genomics and evolutionary studies of iscS across Salmonella serovars can provide valuable insights into pathogen adaptation:

• Sequence-function relationship analysis:

  • Identification of conserved vs. variable regions in iscS across serovars

  • Correlation of sequence variations with host range or virulence differences

  • Structural modeling to predict functional consequences of sequence polymorphisms

  • Experimental validation of predictions through chimeric proteins or site-directed mutagenesis

• Evolutionary pressure analysis:

  • Calculation of dN/dS ratios to identify signatures of selection

  • Bayesian evolutionary analysis to reconstruct ancestral sequences

  • Identification of horizontally transferred elements affecting iscS function

  • Dating of evolutionary events in relation to host adaptation

• Regulatory network evolution:

  • Comparative analysis of iscS promoter regions across serovars

  • Identification of serovar-specific transcription factor binding sites

  • Experimental validation of regulatory differences using reporter constructs

  • Systems biology approaches to model regulatory network evolution

• Host adaptation correlation:

  • Comparison of iscS from human-restricted serovars (S. Typhi, S. Paratyphi A) vs. broad-host-range serovars

  • Analysis of iscS from closely related species with different host preferences

  • Experimental assessment of iscS function under conditions mimicking different host environments

  • Correlating iscS variants with epidemiological or clinical outcome data