Recombinant Acinetobacter baumannii Cysteine desulfurase (iscS)

How is the ISC pathway organized in bacteria, and what role does iscS play?

The enzyme functions through a multi-step process:

• Mobilizing sulfur from L-cysteine through PLP-dependent catalysis

• Generating a persulfide intermediate on a conserved cysteine residue

• Transferring this activated sulfur to scaffold proteins or directly to target proteins

• Facilitating the assembly of Fe-S clusters on proteins such as aconitase and succinate dehydrogenase

This process is fundamental for bacterial energy metabolism and numerous cellular functions dependent on Fe-S cluster-containing proteins.

What methods are used for producing recombinant A. baumannii iscS?

Recombinant A. baumannii iscS is typically produced using heterologous expression systems, primarily Escherichia coli. The methodological workflow includes:

• Gene acquisition: Either amplification from A. baumannii genomic DNA or gene synthesis based on the known sequence.

• Expression vector construction: Insertion of the iscS gene into a suitable expression vector (commonly pET-series) containing:

  • A strong promoter (typically T7)

  • Appropriate selection markers

  • Affinity tags for purification (frequently His-tag)

• Host transformation: Introduction of the recombinant vector into an E. coli expression strain, with BL21(DE3) being the standard host system .

• Expression optimization: Adjustment of conditions including:

  • Induction temperature (typically 16-37°C)

  • IPTG concentration (0.1-1.0 mM)

  • Duration of expression (3-24 hours)

  • Media composition (LB, TB, or defined media)

• Purification strategy: Isolation of the recombinant protein through:

  • Cell lysis (sonication or mechanical disruption)

  • Clarification by centrifugation

  • Affinity chromatography, typically nickel-based for His-tagged proteins

  • Optional additional purification steps (ion exchange, size exclusion)

The purified protein is typically assessed for quality using SDS-PAGE analysis, with >85% purity considered acceptable for most research applications .

What are the optimal storage conditions for recombinant iscS?

Maintaining the stability and activity of recombinant A. baumannii iscS requires careful attention to storage conditions. Based on standard protocols for similar enzymes and product documentation:

• Short-term storage (up to one week): 4°C in appropriate buffer.

• Long-term storage: -20°C to -80°C, with the latter recommended for extended periods .

• Cryoprotectant addition: Addition of 5-50% glycerol as a cryoprotectant is recommended to prevent freeze-thaw damage .

• Aliquoting: Division into single-use aliquots to avoid repeated freeze-thaw cycles, as "repeated freezing and thawing is not recommended" .

• Buffer composition: Typically includes:

  • Buffer component (often Tris-HCl, pH 7.5-8.0)

  • Salt (100-200 mM NaCl)

  • Reducing agent (DTT or β-mercaptoethanol) to protect thiol groups

  • Optional stabilizers or protease inhibitors

Following these guidelines helps maintain enzyme activity and structural integrity for experimental use.

How does iscS contribute to A. baumannii pathogenesis and antibiotic resistance?

A. baumannii has emerged as a significant hospital pathogen with multi-drug resistant (MDR) properties . The contribution of iscS to pathogenesis and antibiotic resistance occurs through several interconnected mechanisms:

• Support of essential metabolism: By facilitating Fe-S cluster assembly, iscS enables the function of numerous enzymes involved in:

  • Energy generation through respiratory chains

  • Central metabolic pathways

  • DNA replication and repair systems

• Oxidative stress resistance: Fe-S clusters are highly susceptible to oxidative damage. IscS-mediated repair and reassembly of these clusters is critical for bacterial survival during:

  • Host-generated oxidative burst

  • Antibiotic-induced oxidative stress

  • Environmental stress conditions

• Indirect contribution to antibiotic resistance: While not directly involved in resistance mechanisms like efflux pumps or drug-modifying enzymes, iscS supports cellular processes that contribute to:

  • Bacterial fitness and growth under antibiotic pressure

  • Metabolic adaptations necessary for persister cell formation

  • Energy-dependent resistance mechanisms

• Potential as a novel drug target: The essential nature of iscS makes it a promising target for novel antimicrobial development, particularly against MDR strains of A. baumannii that have become resistant to conventional antibiotics .

Understanding these connections provides valuable insights for developing strategies to combat A. baumannii infections, especially in the context of increasing antibiotic resistance.

What experimental approaches can assess iscS enzymatic activity in vitro?

Comprehensive characterization of recombinant A. baumannii iscS activity employs multiple complementary methodologies:

• Spectrophotometric assays:

  • Sulfide production: Quantification using colorimetric reagents (methylene blue method)

  • Alanine formation: Measurement through coupled enzyme assays

  • PLP cofactor absorbance: Monitoring changes at 420 nm during catalysis

• Fe-S cluster reconstitution assays:

  • Direct assembly: In vitro reconstitution of Fe-S clusters using iscS, iron source, reducing agent, and target apo-proteins

  • Activity restoration: Measurement of enzymatic activity in reconstituted Fe-S proteins (e.g., aconitase activity assay)

  • Spectroscopic monitoring: UV-visible absorption spectroscopy to track characteristic Fe-S cluster formation

• Enzyme kinetics analysis:

  • Steady-state kinetics: Determination of Km and Vmax values for cysteine desulfurization

  • Inhibition studies: Analysis of competitive, non-competitive, or uncompetitive inhibition patterns

  • pH and temperature profiles: Establishing optimal conditions for enzyme function

• Structural and interaction studies:

  • Thermal shift assays: Assessment of protein stability under various conditions

  • Circular dichroism: Analysis of secondary structure elements

  • Protein-protein interaction: Pull-down assays to identify binding partners

These methodologies provide complementary data about different aspects of iscS function, enabling comprehensive characterization of the enzyme's biochemical properties and interactions.

How does the structure of iscS relate to its function in A. baumannii?

While the specific crystal structure of A. baumannii iscS is not detailed in the provided search results, structural-functional relationships can be inferred from homologous enzymes and sequence analysis:

• Domain organization:

  • N-terminal domain: Contains the PLP binding site with a conserved lysine residue that forms a Schiff base with PLP

  • Central domain: Houses the active site cysteine that forms the catalytic persulfide

  • C-terminal domain: Involved in protein-protein interactions and substrate recognition

• Catalytic mechanism:

  • PLP-dependent activation of the cysteine substrate

  • Nucleophilic attack by the active site cysteine

  • Formation of a persulfide intermediate

  • Transfer of the activated sulfur to acceptor proteins

• Key structural features:

  • The enzyme typically exists as a homodimer

  • The active site lies at the interface between domains

  • Conformational changes occur during the catalytic cycle

  • Structural flexibility facilitates interactions with multiple protein partners

• Structure-based approaches for inhibitor design:

  • Targeting the PLP binding site

  • Designing competitive inhibitors for the substrate binding pocket

  • Developing compounds that interfere with protein-protein interactions

  • Creating covalent inhibitors for the active site cysteine

Understanding these structural aspects provides a foundation for rational drug design approaches targeting iscS in A. baumannii.

How can iscS be targeted for antimicrobial development against A. baumannii?

The essential nature of iscS in bacterial metabolism makes it an attractive target for novel antimicrobial strategies against multi-drug resistant A. baumannii:

• Direct enzyme inhibition strategies:

  • Active site targeting: Development of substrate analogs or transition state mimics

  • Allosteric inhibition: Compounds that disrupt enzyme dynamics or dimerization

  • Cofactor interference: Molecules that compete with or modify PLP binding

  • Covalent inhibitors: Compounds that form irreversible bonds with catalytic cysteine residues

• Pathway disruption approaches:

  • Targeting Fe-S cluster transfer: Inhibition of interactions between iscS and scaffold proteins

  • Synergistic combinations: Co-administration with conventional antibiotics to enhance efficacy

  • Multi-target strategies: Simultaneous inhibition of multiple components in Fe-S cluster assembly

• Challenges and considerations:

  • Selectivity: Ensuring specificity for bacterial iscS over human homologs

  • Penetration: Overcoming the Gram-negative outer membrane barrier

  • Resistance development: Designing inhibitors that minimize resistance emergence

  • Drug delivery: Developing appropriate formulations for effective delivery

• Experimental validation:

  • High-throughput screening assays for initial compound identification

  • Structure-guided optimization of lead compounds

  • In vitro and in vivo efficacy testing

  • Assessment of resistance development potential

Recent research has demonstrated the effectiveness of novel antimicrobial approaches against A. baumannii, such as recombinant antimicrobial peptides like Oncorhyncin II, which showed significant activity with a minimum inhibitory concentration (MIC) of 95.87 μg/ml . Similar innovative approaches targeting iscS could provide new therapeutic options for drug-resistant infections.

What is the role of iscS in bacterial adaptation to host environments?

A. baumannii iscS plays critical roles in bacterial adaptation to challenging host environments during infection:

• Adaptation to iron limitation:

  • Host iron sequestration is a primary defense mechanism

  • IscS enables efficient use of limited iron through Fe-S cluster assembly

  • This supports essential metabolic functions under iron-restricted conditions

• Response to oxidative stress:

  • Host immune cells generate reactive oxygen species (ROS)

  • Fe-S clusters are primary targets for oxidative damage

  • IscS-mediated repair and reassembly maintains bacterial viability

  • This contributes to bacterial persistence during inflammatory responses

• Metabolic adaptations:

  • Fe-S cluster-containing enzymes participate in metabolic rewiring

  • This allows utilization of alternative carbon and energy sources available in host tissues

  • Enables adaptation to nutrient-limiting conditions in infection sites

• Support for virulence factor expression:

  • Many virulence-associated processes depend on Fe-S proteins

  • IscS indirectly supports virulence factor production and function

  • This enhances bacterial capacity to establish and maintain infection

Understanding these adaptive mechanisms could inform new therapeutic approaches that target bacterial resilience during infection.

How can site-directed mutagenesis be used to study iscS function?

Site-directed mutagenesis provides powerful insights into structure-function relationships of A. baumannii iscS:

• Key residues for targeted mutagenesis:

  • Catalytic cysteine: Mutation abolishes persulfide formation and enzyme activity

  • PLP-binding lysine: Alteration prevents cofactor binding

  • Substrate binding pocket residues: Modifications affect substrate specificity and catalytic efficiency

  • Dimerization interface: Mutations can disrupt quaternary structure

• Experimental design approach:

  • Generate recombinant iscS variants using overlap extension PCR or commercial mutagenesis kits

  • Express and purify mutant proteins using established protocols for wild-type iscS

  • Compare biochemical properties (activity, stability, interactions) with wild-type enzyme

  • Assess functional consequences through in vitro activity assays

• Functional analysis:

  • Enzymatic activity: Measure sulfur transfer and desulfurase activity

  • Protein stability: Assess thermal stability and resistance to denaturation

  • Protein-protein interactions: Evaluate binding to partner proteins

  • Fe-S cluster assembly: Test ability to support cluster formation on target proteins

• Biological significance:

  • Complementation studies: Determine if mutants can rescue iscS-deficient phenotypes

  • Fitness assessment: Evaluate growth under various stress conditions

  • Virulence impact: Assess effects on pathogenicity in infection models

This approach provides mechanistic insights into iscS function while identifying critical features that could be targeted for inhibitor development.

What are the most effective purification strategies for obtaining high-quality recombinant iscS?

Obtaining high-purity, active recombinant A. baumannii iscS requires an optimized purification strategy:

• Expression optimization:

  • Host selection: E. coli BL21(DE3) is commonly used for iscS expression

  • Vector design: Incorporation of appropriate affinity tags (His-tag most common)

  • Induction conditions: Lower temperatures (16-25°C) often yield better solubility

  • Media supplementation: Addition of pyridoxine can improve PLP incorporation

• Multi-step purification protocol:

  • Initial capture: Nickel affinity chromatography (Ni-NTA) for His-tagged proteins

  • Intermediate purification: Ion exchange chromatography to remove contaminants

  • Polishing step: Size exclusion chromatography for homogeneity and buffer exchange

  • Tag removal: Optional protease cleavage of affinity tags if required for activity studies

• Critical parameters:

  • Buffer composition: Inclusion of reducing agents to protect thiol groups

  • pH optimization: Typically 7.5-8.0 for optimal stability

  • Salt concentration: Moderate levels (100-300 mM) to maintain solubility

  • Protease inhibitors: Addition during early purification stages

• Quality assessment:

  • Purity evaluation: SDS-PAGE analysis (target >85% purity)

  • Activity testing: Functional assays to confirm enzymatic activity

  • Oligomeric state analysis: Size exclusion chromatography or native PAGE

  • Spectroscopic analysis: UV-visible spectroscopy to assess PLP incorporation

This comprehensive approach yields enzyme preparations suitable for structural studies, enzymatic characterizations, and screening for potential inhibitors.

How can differential gene expression analysis be used to study iscS regulation?

Understanding the regulation of iscS expression provides insights into its role in A. baumannii physiology and pathogenesis:

• Experimental design for transcriptomic analysis:

  • Condition selection: Growth under varying iron availability, oxidative stress, antibiotic exposure

  • Time-course analysis: Sampling at multiple time points to capture dynamic responses

  • Comparative approach: Wild-type vs. regulatory mutants

  • In vitro vs. in vivo: Comparison between laboratory culture and infection models

• Methodological approaches:

  • RNA-Seq: Comprehensive transcriptome analysis

  • qRT-PCR: Targeted quantification of iscS transcript levels

  • Reporter gene assays: Fusion of iscS promoter to reporter genes

  • Chromatin immunoprecipitation (ChIP): Identification of transcription factors binding to iscS promoter

• Data analysis and interpretation:

  • Differential expression analysis: Identification of conditions affecting iscS expression

  • Co-expression networks: Finding genes regulated alongside iscS

  • Promoter motif analysis: Identifying potential regulatory elements

  • Pathway enrichment: Placing iscS regulation in broader cellular contexts

• Validation strategies:

  • Genetic manipulation: Deletion or mutation of potential regulatory elements

  • Protein-DNA interaction studies: Confirmation of transcription factor binding

  • Phenotypic assessment: Correlation of expression changes with physiological outcomes

This approach can reveal how A. baumannii modulates iscS expression in response to environmental challenges, providing insights into bacterial adaptation mechanisms.

How does iscS compare across different bacterial pathogens?

Comparative analysis of iscS across bacterial species reveals important evolutionary and functional insights:

Key comparative observations:

• Evolutionary adaptations:

  • Different bacteria have evolved varying degrees of complexity in their Fe-S cluster assembly pathways

  • Some pathogens like M. tuberculosis have remarkably streamlined systems with iscS as the sole component

  • These differences may reflect adaptation to specific ecological niches

• Functional conservation vs. specialization:

  • The core cysteine desulfurase activity is conserved across species

  • Substrate specificity and regulatory mechanisms show species-specific variations

  • Interaction networks with partner proteins vary in complexity

• Therapeutic implications:

  • Conserved features present broad-spectrum antimicrobial targets

  • Species-specific characteristics enable selective targeting

  • Understanding these differences guides rational drug design approaches

This comparative perspective informs both fundamental understanding of bacterial metabolism and targeted antimicrobial development strategies.

What is the potential for combining iscS inhibitors with existing antibiotics?

Combination therapy targeting iscS alongside conventional antibiotics presents promising opportunities for addressing multidrug-resistant A. baumannii infections:

• Synergistic mechanisms:

  • IscS inhibition would compromise multiple metabolic pathways simultaneously

  • This metabolic disruption could enhance antibiotic efficacy through:

    • Increased bacterial membrane permeability

    • Reduced energy availability for efflux pumps

    • Compromised DNA repair mechanisms

    • Disrupted stress response pathways

• Resistance mitigation:

  • Multi-target approach reduces likelihood of resistance development

  • Bacteria would need to evolve multiple simultaneous adaptations

  • Metabolic vulnerabilities created by iscS inhibition may prevent compensatory adaptations

• Dosing advantages:

  • Potential for lower effective doses of conventional antibiotics

  • Reduced side effects from high-dose antibiotic regimens

  • Extended useful lifespan of existing antimicrobials

• Experimental evidence from similar approaches:

  • Research with antimicrobial peptides like recombinant Oncorhyncin II has demonstrated efficacy against A. baumannii with MIC values of 95.87 μg/ml

  • When combined with another antimicrobial peptide (IB-AMP4), synergistic effects were observed

  • Similar principles could apply to iscS inhibitors in combination with conventional antibiotics

These combination approaches represent a promising strategy for addressing the urgent challenge of multidrug-resistant A. baumannii infections.

What technologies are emerging for studying iscS structural dynamics?

Advanced technologies are transforming our ability to understand the structural dynamics of enzymes like A. baumannii iscS:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of proteins in near-native states without crystallization

  • Can capture multiple conformational states

  • Particularly valuable for examining iscS interactions with partner proteins

  • Allows study of dynamic processes during catalysis

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein dynamics and conformational changes

  • Identifies regions with altered solvent accessibility during catalysis

  • Detects binding interfaces with substrates or protein partners

  • Provides insights into allosteric regulation mechanisms

• Single-molecule techniques:

  • Förster resonance energy transfer (FRET) to monitor conformational changes

  • Optical tweezers to study mechanical properties

  • Single-molecule fluorescence to track individual enzyme molecules

  • Reveals heterogeneity and rare conformational states

• Computational approaches:

  • Molecular dynamics simulations of enzyme flexibility

  • Machine learning for prediction of conformational changes

  • Virtual screening for potential inhibitors

  • Integration of experimental data with computational models

These technologies complement traditional structural biology approaches and provide unprecedented insights into the dynamic behavior of iscS during its catalytic cycle, informing both basic understanding and applied drug discovery efforts.

What are the major challenges and opportunities in iscS research?

Research on A. baumannii iscS faces several challenges while offering significant opportunities for advancement:

• Current challenges:

  • Limited structural information specific to A. baumannii iscS

  • Complexity of studying Fe-S cluster assembly in vitro

  • Difficulty in developing selective inhibitors that don't affect human homologs

  • Challenges in delivering compounds across the Gram-negative outer membrane

• Emerging opportunities:

  • Application of advanced structural biology techniques to elucidate iscS dynamics

  • Integration of systems biology approaches to understand pathway interactions

  • Development of targeted delivery systems for potential inhibitors

  • Exploration of combination therapies targeting multiple aspects of bacterial metabolism

• Future research directions:

  • Comprehensive characterization of the A. baumannii Fe-S cluster assembly network

  • Investigation of iscS regulation during infection and antibiotic exposure

  • High-throughput screening for novel iscS inhibitors

  • Development of in vivo models to assess iscS-targeting therapeutics

• Translational potential:

  • Novel antimicrobial agents against multidrug-resistant A. baumannii

  • Diagnostic tools based on iscS activity or expression

  • Biomarkers for monitoring treatment efficacy

  • Predictive models for antimicrobial resistance development

The critical role of iscS in bacterial metabolism combined with the urgent need for new approaches against multidrug-resistant pathogens ensures continued interest in this research area.

How can interdisciplinary approaches advance iscS research and applications?

Addressing the complex challenges of A. baumannii iscS research requires interdisciplinary collaboration:

• Integration of multiple disciplines:

  • Structural biology: Elucidation of enzyme architecture and dynamics

  • Biochemistry: Characterization of enzymatic mechanisms and regulation

  • Microbiology: Understanding the role in bacterial physiology and pathogenesis

  • Medicinal chemistry: Design of selective inhibitors and drug delivery systems

  • Computational biology: Modeling of enzyme dynamics and virtual screening

  • Clinical microbiology: Translating findings to therapeutic applications

• Technological synergies:

  • Combining high-resolution imaging with functional assays

  • Integrating genomic analyses with phenotypic studies

  • Coupling high-throughput screening with rational design approaches

  • Linking basic research findings with clinical observations

• Collaborative research frameworks:

  • Academic-industry partnerships for drug development

  • Multi-institutional research consortia

  • Cross-disciplinary training programs

  • Open data sharing platforms and resources

• Translational research pipeline:

  • Basic research → Target validation → Lead compound identification → Preclinical testing → Clinical development

The successful development of iscS-targeting therapeutics against multidrug-resistant A. baumannii will depend on effective collaboration across these diverse fields, combining scientific innovation with clinical application.