Recombinant Salmonella paratyphi C Cysteine desulfurase (iscS)

What is the role of cysteine desulfurase (IscS) in Salmonella paratyphi C?

Cysteine desulfurase (IscS) in Salmonella species, including S. paratyphi C, functions as a key enzyme in sulfur mobilization pathways. It catalyzes the removal of sulfur from L-cysteine, which is subsequently incorporated into various biomolecules. The primary function of IscS in S. paratyphi C is the formation of thiolated nucleosides in tRNA, which occurs through two distinct biosynthetic pathways . These modifications are crucial for proper tRNA function and translation fidelity, ultimately affecting bacterial metabolism and virulence. Additionally, IscS plays a central role in the biosynthesis of iron-sulfur clusters, which are essential cofactors for numerous proteins involved in diverse cellular processes, including electron transport and enzyme catalysis.

How does IscS contribute to tRNA modification in Salmonella species?

IscS is required for the formation of all four thiolated nucleosides in tRNA in Salmonella: 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-thiocytidine (s2C), and N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) . The synthesis of these modified nucleosides occurs via two principally different biosynthetic pathways:

• Iron-sulfur cluster protein-independent pathway: Responsible for s4U and mnm5s2U synthesis. This pathway involves the direct transfer of sulfur from IscS via various proteins to the target nucleoside in tRNA .

• Iron-sulfur cluster protein-dependent pathway: Required for s2C and ms2io6A synthesis. This pathway depends on iron-sulfur cluster proteins, whose formation and maintenance are dependent on IscS .

The following table summarizes the thiolated nucleosides in tRNA and their respective biosynthetic pathways:

What are the genetic characteristics of S. paratyphi C in relation to other Salmonella species?

S. paratyphi C is a human-adapted typhoid agent with distinct genetic characteristics. Its genome consists of a chromosome of 4,833,080 bp and a plasmid of 55,414 bp, containing 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid) .

Genetic comparison reveals that S. paratyphi C shares 4,346 genes with S. choleraesuis (a swine pathogen), covering 96.66% of the S. paratyphi C genome and 98.23% of the S. choleraesuis genome . In contrast, S. paratyphi C shares only 4,008 genes with S. typhi (another human-adapted typhoid agent), accounting for 89.78% of the S. paratyphi C genome and 90.23% of the S. typhi genome . This genetic comparison suggests that S. paratyphi C is more closely related to S. choleraesuis than to S. typhi, despite both S. paratyphi C and S. typhi being human-adapted typhoid agents.

What experimental approaches are most effective for studying structural alterations of IscS in S. paratyphi C?

Investigating structural alterations of IscS in S. paratyphi C requires a multi-faceted approach combining molecular genetics, biochemistry, and structural biology techniques. Based on previous successful methodologies, the following experimental approaches are recommended:

• Site-directed mutagenesis: Generate specific mutations in the iscS gene, particularly in the C-terminal region which has been shown to specifically affect certain thiolated nucleoside synthesis pathways . This approach allows researchers to investigate structure-function relationships of IscS.

• Complementation studies: Express wild-type and mutant iscS genes in an iscS-deficient background to assess the ability of different IscS variants to restore thiolation activity. This method has successfully identified altered forms of IscS that influence the synthesis of specific subsets of thiolated nucleosides .

• Biochemical characterization: Purify recombinant wild-type and mutant IscS proteins for in vitro assays measuring cysteine desulfurase activity, protein-protein interactions, and sulfur transfer capabilities. Differential effects on specific pathways can be quantitatively assessed through these biochemical approaches.

• Structural biology techniques: Employ X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy to determine the three-dimensional structures of wild-type and mutant IscS proteins, providing insights into how specific alterations affect protein conformation and function.

How do mutations in the C-terminal region of IscS specifically affect thiolated nucleoside synthesis?

Research has demonstrated that alterations in the C-terminal region of IscS have differential effects on thiolated nucleoside synthesis pathways. Mutations in this region preferentially reduced the level of ms2io6A, suggesting that the synthesis of this nucleoside is particularly sensitive to minor aberrations in iron-sulfur cluster transfer activity .

The C-terminal region of IscS is believed to be involved in protein-protein interactions that are critical for the iron-sulfur cluster assembly pathway. When mutations occur in this region, they likely disrupt specific interactions required for efficient iron-sulfur cluster formation and transfer to target proteins, while having less impact on direct sulfur transfer reactions. This explains why ms2io6A synthesis, which depends on iron-sulfur cluster proteins, is more severely affected than s4U or mnm5s2U synthesis, which occur via direct sulfur transfer .

To investigate this phenomenon, researchers should:

• Generate a series of systematic mutations throughout the C-terminal domain

• Analyze the impact on all four thiolated nucleosides quantitatively

• Correlate structural changes with functional effects using integrated structural biology approaches

• Examine protein-protein interactions between IscS variants and other components of the iron-sulfur cluster assembly machinery

What methodologies can be used to investigate the intrinsic substrate specificity of IscS in S. paratyphi C?

IscS demonstrates intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation required for tRNA thiolation . To investigate this specificity, researchers should employ the following methodologies:

• Comparative enzyme kinetics: Measure the kinetic parameters (Km, kcat, kcat/Km) of purified recombinant IscS with various substrate analogs to quantify preferences for different cysteine derivatives or reaction partners.

• Protein-protein interaction studies:

  • Pull-down assays to identify binding partners of IscS

  • Surface plasmon resonance or isothermal titration calorimetry to quantify binding affinities

  • Crosslinking followed by mass spectrometry to map interaction interfaces

• In vivo sulfur tracking: Utilize radioactive sulfur (35S) incorporation studies to trace the flow of sulfur from IscS to different target molecules, allowing quantification of the relative efficiency of different pathways.

• Structural modeling and docking: Employ computational approaches to predict and visualize how IscS interacts with different protein partners and substrates, generating testable hypotheses about the molecular basis of specificity.

How can researchers distinguish between iron-sulfur cluster protein-dependent and independent pathways in IscS function?

Distinguishing between the two pathways through which IscS mediates tRNA thiolation requires sophisticated experimental approaches that selectively perturb one pathway while preserving the other. The following methodological approaches are recommended:

• Genetic dissection: Create mutants that specifically affect either:

  • Iron-sulfur cluster formation machinery (targeting genes like iscU, iscA)

  • Direct sulfur transfer pathway components (targeting genes like tusA, mnmA)

• Biochemical assays: Develop in vitro reconstitution systems that allow:

  • Quantification of direct sulfur transfer from IscS to acceptor proteins

  • Monitoring of iron-sulfur cluster assembly on scaffold proteins

  • Analysis of subsequent transfer to target apoproteins

• Analytical techniques for tRNA modification analysis:

  • HPLC or LC-MS/MS analysis to quantify the levels of each thiolated nucleoside

  • Next-generation sequencing-based methods to profile tRNA modifications at single-nucleotide resolution

  • Radioactive labeling to track the incorporation of sulfur into specific tRNA positions

• Drug-based approach: Utilize small molecules that specifically inhibit either:

  • Iron-sulfur cluster assembly (iron chelators at sub-inhibitory concentrations)

  • Direct sulfur transfer (thiol-reactive compounds at carefully titrated doses)

What are optimal conditions for expressing and purifying recombinant S. paratyphi C IscS?

Successful expression and purification of recombinant S. paratyphi C IscS requires careful optimization of conditions to maintain protein stability and activity. Based on established protocols for similar enzymes, the following approach is recommended:

• Expression system:

  • E. coli BL21(DE3) strain is preferred due to its reduced protease activity

  • pET vector systems with T7 promoter provide high-level expression

  • Consider using a fusion tag (His6, GST, or MBP) to facilitate purification

• Culture conditions:

  • Initial growth at 37°C to OD600 of 0.6-0.8

  • Induction with 0.1-0.5 mM IPTG

  • Post-induction temperature reduction to 16-25°C for 4-16 hours to enhance solubility

• Buffer optimization:

  • Include 5-10% glycerol to enhance stability

  • Add 1-5 mM DTT or β-mercaptoethanol to maintain reduced cysteines

  • Consider including pyridoxal 5'-phosphate (PLP, 0.1-0.2 mM) as IscS is a PLP-dependent enzyme

• Purification strategy:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

  • Intermediate purification using ion exchange chromatography

  • Final polishing step using size exclusion chromatography

The following table summarizes optimal buffer conditions for different purification stages:

How can researchers design experiments to analyze IscS function in the context of S. paratyphi C pathogenesis?

Investigating the role of IscS in S. paratyphi C pathogenesis requires integration of molecular genetics, biochemistry, and infection models. The following experimental design considerations are recommended:

• Generation of iscS mutant strains:

  • Create precise point mutations using CRISPR-Cas9 or lambda Red recombination

  • Develop conditional expression systems (tetracycline-regulated promoters) for essential genes

  • Engineer complementation constructs with wild-type and mutant alleles

• In vitro characterization:

  • Assess growth kinetics under various stress conditions (oxidative stress, iron limitation)

  • Analyze tRNA modification profiles using LC-MS/MS

  • Measure activities of Fe-S cluster-dependent enzymes (aconitase, fumarase)

• Cellular infection models:

  • Human intestinal epithelial cell invasion assays

  • Macrophage survival and replication studies

  • Cytokine induction profiles

• In vivo infection studies (if ethically approved):

  • Mouse infection models to assess colonization, dissemination, and persistence

  • Competition assays between wild-type and iscS mutant strains

  • Analysis of virulence gene expression in vivo

• Systems biology approaches:

  • Transcriptomics to identify genes differentially regulated in iscS mutants

  • Proteomics to detect changes in protein abundance and post-translational modifications

  • Metabolomics to characterize metabolic perturbations resulting from altered IscS function

What are the challenges in resolving contradictory data regarding IscS function across different Salmonella species?

Researchers investigating IscS function across Salmonella species may encounter contradictory data due to several factors. Addressing these challenges requires careful experimental design and data interpretation:

• Phylogenetic considerations:

  • Despite being human-adapted typhoid agents, S. paratyphi C and S. typhi do not share a common ancestor but have evolved by convergent processes

  • S. paratyphi C is more closely related to S. choleraesuis (a swine pathogen) than to S. typhi

  • Researchers should account for these evolutionary relationships when comparing IscS function

• Genetic context effects:

  • The genetic background in which iscS operates differs between species

  • S. paratyphi C shares 4,346 genes with S. choleraesuis but only 4,008 genes with S. typhi

  • These differences may affect IscS substrate availability, interaction partners, and regulatory mechanisms

• Methodological approaches to resolve contradictions:

  • Cross-species complementation studies: Express iscS from different Salmonella species in a common genetic background

  • Domain swapping experiments: Create chimeric IscS proteins to identify species-specific functional domains

  • Standardized assay conditions: Develop uniform protocols for measuring IscS activity across species

  • Mathematical modeling: Develop quantitative models that account for species-specific genetic contexts

• Systematic documentation and reporting:

  • Maintain detailed records of experimental conditions

  • Perform statistical analyses appropriate for the experimental design

  • Consider publishing negative or contradictory results to advance the field

  • Develop a community database for standardized storage and sharing of IscS functional data

How does genomic divergence between S. paratyphi C and other Salmonella species impact IscS structure and function?

S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to humans . This divergence likely influences IscS structure and function through several mechanisms:

• Sequence variations: Although the core functional domains of IscS are conserved across Salmonella species, subtle amino acid differences may alter:

  • Substrate binding affinities

  • Catalytic efficiency

  • Interaction with partner proteins

  • Regulation by post-translational modifications

• Genomic context: The genomic environment of the iscS gene differs between Salmonella species, potentially affecting:

  • Expression levels through altered promoter sequences

  • Operon structure and co-regulation with neighboring genes

  • Responses to environmental signals relevant to host adaptation

• Research approaches to investigate impact of genomic divergence:

  • Comparative genomics to identify species-specific variations in iscS and related genes

  • Heterologous expression studies to assess functional differences

  • Structural biology approaches to characterize species-specific conformational features

  • Systems-level analyses to map species-specific interaction networks

What role might IscS play in the adaptation of S. paratyphi C to the human host?

S. paratyphi C is a human-adapted typhoid agent that has evolved from a common ancestor with the swine pathogen S. choleraesuis . IscS may contribute to this host adaptation through several mechanisms:

• Modulation of tRNA modification profiles:

  • Different hosts present distinct physiological environments (temperature, pH, nutrient availability)

  • Adaptations in IscS function may optimize tRNA modification patterns for translation efficiency under human host conditions

  • Species-specific modifications may regulate expression of virulence factors needed for human infection

• Iron-sulfur cluster homeostasis:

  • The human host restricts iron availability as an innate immune defense

  • Adaptations in IscS function may enhance iron-sulfur cluster assembly under iron-limiting conditions

  • Efficient iron-sulfur cluster formation supports growth and virulence in the human environment

• Stress response regulation:

  • Human host defenses include oxidative burst and nitrosative stress

  • IscS-mediated processes may contribute to redox homeostasis and stress resistance

  • Specialized adaptations may protect iron-sulfur clusters from oxidative damage during infection

• Metabolic adaptations:

  • Different hosts offer distinct nutrient profiles

  • IscS-dependent enzymes support metabolic pathways needed for growth in the human environment

  • Host-specific adaptations in IscS may optimize these pathways for human infection