Recombinant Vibrio vulnificus Protein CyaY (cyaY)

What is the primary function of Vibrio vulnificus CyaY protein in bacterial metabolism?

CyaY functions primarily as an iron chaperone protein involved in iron-sulfur (Fe-S) cluster biosynthetic systems. It facilitates iron transport to various cellular components, playing a crucial role in iron homeostasis within Vibrio species. The protein serves as an iron donor for ferrochelatase, which catalyzes the insertion of Fe²⁺ into protoporphyrin IX during heme biosynthesis . This function is essential for various cellular processes that require iron-containing proteins, making CyaY a critical component in bacterial iron metabolism and utilization pathways.

How does V. vulnificus CyaY compare structurally to CyaY proteins in other Vibrio species?

While specific structural data comparing V. vulnificus CyaY to other Vibrio species is limited in the provided search results, research on V. cholerae CyaY provides valuable insights. The V. cholerae CyaY contains key functional residues including Tyr67 and Cys78, which serve as possible heme ligands . These residues likely create binding pockets that accommodate both iron and heme molecules. Structurally, V. vulnificus CyaY would be expected to share similar architecture to other bacterial frataxin-like proteins, particularly within the Vibrio genus, though species-specific variations may exist that affect binding affinities and functional properties.

What are the documented binding capabilities of CyaY protein?

Research demonstrates that CyaY proteins exhibit dual binding capabilities for both iron and heme molecules. Specifically, V. cholerae CyaY binds heme with high affinity, exhibiting an apparent dissociation constant of 21 ± 6 nM . Additionally, it binds iron with an apparent dissociation constant of 65.2 μM in its native state, which increases to 87.9 μM when heme is bound to the protein . Both ferric and ferrous forms of heme are accommodated by the protein, with binding occurring through anionic ligands such as tyrosine and/or cysteine residues . The protein's binding capabilities appear to be influenced by conformational changes, as circular dichroism spectra suggest heme binding induces rearrangement of aromatic residues .

What expression systems are most effective for producing recombinant V. vulnificus CyaY?

While the search results don't directly address expression systems for V. vulnificus CyaY, research on related iron-binding proteins suggests several effective approaches. For laboratory-scale production of functional recombinant CyaY, E. coli-based expression systems (particularly BL21(DE3) or similar strains) typically provide good yields when the protein is expressed with a His-tag for purification purposes. When designing expression systems, researchers should consider:

• Codon optimization based on V. vulnificus preferred codons

• Temperature control during induction (often lowered to 16-18°C)

• Addition of iron supplements to the growth medium

• Use of specialized vectors containing promoters that allow tight regulation

Successful expression typically requires careful optimization of induction parameters to prevent the formation of inclusion bodies, particularly given CyaY's metal-binding properties.

What purification strategies yield the highest quality recombinant CyaY for functional studies?

Purification of recombinant CyaY requires strategies that preserve both structure and function, particularly its ability to bind iron and heme. Based on studies of related proteins, an optimal purification protocol would likely include:

• Initial capture using immobilized metal affinity chromatography (IMAC) if the protein contains a His-tag

• Buffer optimization containing reducing agents to prevent oxidation of cysteine residues

• Size exclusion chromatography to separate monomeric from oligomeric forms, particularly since heme binding can mediate oligomerization of CyaY

• Quality control steps including circular dichroism to verify proper folding of aromatic residues, which are known to undergo rearrangement upon heme binding

• Activity verification through iron and heme binding assays

Researchers should monitor the oxidation state during purification, as both ferric and ferrous forms of CyaY-bound heme have been documented .

How can researchers accurately measure and characterize CyaY-heme interactions?

Characterizing CyaY-heme interactions requires multiple complementary techniques to establish binding parameters and structural changes. Based on methodologies described in the research literature, the following approaches are recommended:

• Absorption spectroscopy to monitor spectral shifts upon heme binding

• Resonance Raman spectroscopy to identify the nature of the iron-ligand interactions, which has previously revealed binding to anionic ligands such as tyrosine and/or cysteine

• Isothermal titration calorimetry (ITC) for accurate determination of binding constants (the Kd for heme binding to V. cholerae CyaY was determined to be 21 ± 6 nM)

• Circular dichroism to detect conformational changes in protein structure, particularly rearrangements of aromatic residues induced by heme binding

• Site-directed mutagenesis of potential binding residues (comparable to Tyr67 and Cys78 in V. cholerae) to confirm their roles in heme coordination

When conducting these analyses, researchers should account for the potential effect of oxidation states on binding properties, as both ferric and ferrous forms are biologically relevant.

How does CyaY contribute to virulence in Vibrio vulnificus infections?

Although direct evidence linking CyaY to V. vulnificus virulence is not presented in the provided search results, its role as an iron chaperone suggests potential contributions to pathogenesis through iron acquisition and utilization systems. V. vulnificus is a significant food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly from contaminated seafood consumption . Iron acquisition is critical for bacterial virulence, and CyaY's functions in iron transport for Fe-S cluster biosynthesis may support multiple virulence mechanisms by:

• Maintaining iron homeostasis during host colonization

• Supporting the synthesis of iron-containing virulence factors

• Contributing to bacterial survival under iron-restricted conditions in the host

Further research specifically investigating the relationship between CyaY and established virulence factors such as the MARTX toxin would help clarify its role in pathogenesis.

What is known about genetic variation in cyaY across different V. vulnificus strains?

Similar genetic diversity might exist in cyaY, potentially affecting iron acquisition capabilities among different strains. Research examining cyaY sequences across clinical and environmental isolates, similar to the approach used for rtxA1 analysis, would help identify whether strain-specific variations exist that might correlate with virulence potential or ecological adaptations.

Does CyaY interact with other virulence factors in Vibrio vulnificus?

While direct evidence of CyaY interaction with specific virulence factors is not presented in the search results, its role in iron metabolism suggests potential indirect relationships. V. vulnificus pathogenicity is linked to multiple virulence factors, including the multifunctional-autoprocessing RTX (MARTX Vv) toxin, which has been shown to be an important virulence factor in mouse models of infection .

Iron availability can regulate expression of virulence factors in many bacterial pathogens. As CyaY functions in iron transport for Fe-S cluster biosynthesis and transfers iron to ferrochelatase for heme synthesis , it may indirectly support virulence by:

• Contributing to iron homeostasis required for optimal expression of virulence genes

• Supporting biosynthesis of iron-containing enzymes involved in toxin production

• Potentially participating in iron-dependent regulatory networks that control virulence factor expression

Research examining protein-protein interactions or gene expression correlations between CyaY and established virulence factors would provide greater clarity on these potential relationships.

How do mutations in key binding residues affect CyaY's dual iron and heme binding capabilities?

Research on V. cholerae CyaY has identified Tyr67 and Cys78 as potential heme ligands . Mutations in these residues would likely have significant effects on binding capabilities. Based on biochemical principles and the available data, the following effects might be anticipated:

• Tyr67 mutations would likely diminish heme binding by eliminating a key anionic ligand, potentially affecting the spectroscopic properties observed in resonance Raman studies

• Cys78 alterations might impact both heme coordination and redox sensitivity of the protein

• Mutations that disrupt the binding pocket architecture would affect the dissociation constants for both iron (normally 65.2 μM) and heme (normally 21 ± 6 nM)

• Structural alterations might disrupt the heme-mediated oligomerization observed in size-exclusion chromatography

Systematic mutagenesis studies examining these residues would provide valuable insights into the molecular determinants of CyaY's binding properties and their relationship to protein function.

What is the molecular basis for the altered iron binding affinity observed when CyaY binds heme?

The binding of heme to CyaY increases the apparent dissociation constant for iron from 65.2 μM to 87.9 μM, indicating reduced affinity . This interesting finding suggests molecular crosstalk between binding sites. Several mechanisms could explain this phenomenon:

• Conformational changes induced by heme binding, supported by circular dichroism data showing rearrangement of aromatic residues , may alter the architecture of the iron-binding site

• Potential electrostatic interactions between the negatively charged heme propionate groups and positively charged residues involved in iron coordination

• Allosteric effects transmitted through the protein structure from the heme-binding site to the iron-binding site

• Competition for shared binding residues between iron and heme

This reduced iron affinity when heme is bound may represent a regulatory mechanism whereby heme binding modulates iron transport function, potentially serving as a feedback mechanism in iron utilization pathways.

How does heme-mediated oligomerization of CyaY affect its biological function?

Size-exclusion chromatography has demonstrated heme-mediated oligomerization of CyaY , which likely represents a significant regulatory mechanism. This oligomerization could affect biological function in several ways:

• Modulation of iron transport activity through assembly and disassembly of functional complexes

• Creation of higher-order structures with altered binding properties or specificities

• Protection of bound heme from oxidative damage or competitive binding

• Formation of a storage complex that sequesters excess heme or iron

• Establishment of multi-protein complexes that facilitate interactions with partner proteins in Fe-S cluster assembly

Understanding the structural basis and functional consequences of this oligomerization would provide significant insights into CyaY's biological roles and potential approaches for functional modulation.

How do the biochemical properties of V. vulnificus CyaY compare to frataxin-like proteins in other pathogenic bacteria?

While specific comparative data for V. vulnificus CyaY is limited in the search results, the information on V. cholerae CyaY provides a foundation for comparison with frataxin-like proteins in other systems. CyaY proteins belong to the frataxin family, characterized by their role in iron homeostasis. Key comparative aspects include:

• Binding affinities: V. cholerae CyaY exhibits a heme dissociation constant of 21 ± 6 nM and iron dissociation constants of 65.2-87.9 μM , which can be compared with other bacterial frataxins

• Dual binding capabilities: The ability to bind both iron and heme may not be universal among frataxin family proteins

• Ligand coordination: The use of anionic ligands (tyrosine and/or cysteine) for binding may represent a common or divergent feature

• Oligomerization properties: Heme-mediated oligomerization may be a specialized feature of Vibrio CyaY proteins or more broadly conserved

Comparative analysis across multiple pathogenic species would provide insights into evolutionary adaptation of these iron chaperones to different host environments and pathogenic lifestyles.

What role does CyaY play in iron competition between V. vulnificus and host organisms during infection?

Iron acquisition is a critical aspect of host-pathogen interactions, as hosts typically sequester iron as a defense mechanism. While the search results don't directly address CyaY's role in this competition, its function as an iron chaperone suggests several potential contributions:

• Facilitating efficient utilization of limited iron available during infection

• Supporting siderophore-based iron acquisition systems through downstream Fe-S cluster assembly

• Contributing to iron storage or mobilization depending on environmental conditions

• Potentially interfacing with host iron-containing proteins such as hemoglobin, transferrin, or lactoferrin

V. vulnificus is associated with severe food-borne infections through consumption of contaminated seafood , suggesting successful iron acquisition strategies within the host environment. Understanding CyaY's role in this process could provide insights into pathogenesis and potential therapeutic approaches targeting iron utilization pathways.

How have genetic recombination events shaped the evolution of iron utilization systems in Vibrio species?

The search results provide strong evidence that genetic recombination shapes virulence factor evolution in V. vulnificus, particularly for the rtxA1 gene encoding the MARTX toxin . Four distinct variants of rtxA1 have been identified, arising through recombination events with genes from plasmids or other marine pathogens like Vibrio anguillarum . This demonstrates the dynamic nature of genetic exchange in this pathogen.

Similar recombination processes may have influenced the evolution of iron utilization systems, including cyaY. Potential evolutionary mechanisms include:

• Horizontal gene transfer of iron acquisition components between Vibrio species

• Recombination events introducing novel binding capabilities, similar to how rtxA1 variants acquired different effector domains

• Selection pressures in different environments favoring specific variants with altered iron or heme binding properties

• Acquisition of regulatory elements affecting expression patterns

Genomic analysis across environmental and clinical isolates, similar to the approach used for rtxA1 , would reveal whether cyaY exhibits similar evolutionary dynamics and genetic diversity.

What are the critical controls needed when studying recombinant CyaY protein function in vitro?

Rigorous experimental design for studying recombinant CyaY requires several critical controls to ensure reliable results, particularly given its metal-binding properties. Based on biochemical principles and the available data, essential controls include:

• Metal contamination controls:

  • Metal-free buffers prepared with chelating agents and plastic labware

  • ICP-MS verification of metal content in protein preparations

  • Comparison of protein properties before and after metal removal treatments

• Protein quality controls:

  • Non-binding mutant versions (e.g., Tyr67 and Cys78 mutants based on V. cholerae data)

  • Heat-denatured protein samples

  • Circular dichroism to verify proper folding, especially of aromatic residues

• Binding specificity controls:

  • Competitive binding assays with known chelators

  • Testing binding of structurally related but non-physiological porphyrins

  • Evaluation of binding under varying pH and ionic strength conditions

• Functional validation controls:

  • Comparison with native (non-recombinant) protein when available

  • Assessment of oligomerization states by size-exclusion chromatography

  • Verification of expected spectroscopic properties for iron and heme binding

How can researchers effectively model the functional impact of CyaY in complex bacterial systems?

Understanding CyaY's role in complex bacterial systems requires approaches that bridge in vitro biochemistry with in vivo function. Effective modeling strategies include:

• Genetic approaches:

  • Construction of cyaY deletion mutants with complementation using wild-type and variant genes

  • Conditional expression systems to modulate CyaY levels

  • Reporter fusions to monitor iron-dependent processes

• Systems biology approaches:

  • Transcriptomic and proteomic analysis of wild-type versus cyaY mutant strains under varying iron conditions

  • Metabolomic profiling focusing on iron-dependent pathways

  • Protein interaction network mapping to identify functional partners

• Computational modeling:

  • Structural modeling of CyaY interactions with binding partners

  • Simulation of iron flux through bacterial systems with varying CyaY parameters

  • Evolutionary analysis comparing cyaY sequences across Vibrio isolates with different virulence properties

• Host-pathogen models:

  • Infection studies in iron-manipulated host environments

  • Competition assays between wild-type and cyaY mutant strains during infection

  • Tracking of iron distribution between host and pathogen compartments

These approaches would provide complementary perspectives on CyaY's functional impact within the complex context of bacterial physiology and pathogenesis.

What methods best address the challenges of studying CyaY function in iron-regulated virulence pathways?

Studying the intersection of CyaY function with iron-regulated virulence presents specific methodological challenges. Based on established approaches in bacterial pathogenesis research and the specific properties of CyaY, effective methods include:

• Iron-controlled experimental designs:

  • Precisely defined media with controlled iron availability

  • Use of iron chelators with varying specificities

  • Iron source diversity (heme, transferrin, ferric citrate) to model different host environments

• Virulence factor expression analysis:

  • Reporter constructs for iron-regulated virulence genes

  • Quantitative RT-PCR under varying iron conditions in wild-type versus cyaY mutants

  • Western blotting for key virulence factors like MARTX toxin components

• Functional virulence assays:

  • Cellular cytotoxicity assays with varying iron availability

  • Animal infection models with iron manipulation (similar to those used for rtxA1 studies)

  • Competition assays between wild-type and cyaY mutant strains

• Direct interaction studies:

  • Pull-down assays to identify CyaY-interacting proteins involved in virulence

  • Bacterial two-hybrid screening for protein-protein interactions

  • In situ proximity labeling to identify neighborhood proteins in the native environment

These methods would help address the challenge of connecting CyaY's biochemical functions to clinically relevant virulence outcomes while accounting for the complex regulatory effects of iron availability.

How should researchers reconcile conflicting data on CyaY function across different experimental systems?

Conflicting results in CyaY research may arise from methodological differences, strain variations, or environmental conditions. Researchers should approach data discrepancies through:

• Systematic comparison of experimental conditions:

  • Metal contamination levels in buffers and reagents

  • Protein preparation methods that may affect folding or post-translational modifications

  • Expression systems that might introduce strain-specific factors

• Strain-specific considerations:

  • Genetic background differences that might affect CyaY function

  • Presence of compensatory mechanisms in some strains

  • Potential strain-specific variations in cyaY sequence or regulation, similar to the variation observed in rtxA1

• Integrative analysis approaches:

  • Meta-analysis of published data with statistical evaluation of conflicting results

  • Development of testable hypotheses that could explain apparent contradictions

  • Collaborative cross-laboratory validation studies using standardized protocols

• Biological context assessment:

  • Evaluation of whether differences reflect biological variation rather than experimental artifacts

  • Consideration of whether CyaY might have context-dependent functions

  • Assessment of oligomerization states, which could affect function and are known to be influenced by heme binding

What are the most significant technical challenges in studying CyaY-mediated iron transport?

Iron transport studies present unique technical challenges due to iron's chemical properties and biological handling. For CyaY research, significant challenges include:

• Maintaining defined metal states:

  • Preventing oxidation of ferrous iron during experiments

  • Controlling adventitious metal contamination in buffers and protein preparations

  • Distinguishing specific from non-specific binding events

• Tracking iron movement:

  • Developing assays that can monitor iron transfer between CyaY and recipient proteins

  • Distinguishing CyaY-mediated transport from passive diffusion or other transport systems

  • Creating appropriate fluorescent or radioactive tracers without disrupting normal function

• Reconstituting physiologically relevant conditions:

  • Establishing appropriate redox conditions that mimic the bacterial cytoplasm

  • Including all relevant components of Fe-S cluster assembly machinery

  • Accounting for potential regulatory factors present in vivo but absent in vitro

• Addressing CyaY's dual binding capabilities:

  • Separating the effects of iron binding from heme binding

  • Understanding how the increase in iron dissociation constant from 65.2 to 87.9 μM when heme is bound affects transport function

  • Determining the biological significance of heme-induced conformational changes and oligomerization

How can researchers distinguish between direct and indirect effects of CyaY on bacterial virulence?

Establishing causal relationships between CyaY function and virulence outcomes requires careful experimental design to separate direct from indirect effects. Effective approaches include:

• Genetic complementation strategies:

  • Wild-type complementation to confirm phenotype restoration

  • Complementation with binding-deficient mutants (e.g., mutations in residues equivalent to Tyr67 and Cys78)

  • Domain-swap experiments with homologous proteins from non-pathogenic species

• Temporal analysis approaches:

  • Time-course studies to establish order of events following cyaY perturbation

  • Inducible expression systems to initiate CyaY function at defined timepoints

  • Real-time monitoring of iron-dependent processes following CyaY manipulation

• Direct interaction verification:

  • Targeted analysis of CyaY interaction with specific virulence factors

  • In vivo crosslinking to capture transient interactions

  • Proximity-dependent labeling to identify the CyaY interaction network during infection

• Pathway dissection strategies:

  • Epistasis analysis using multiple deletion/complementation combinations

  • Specific inhibitors of downstream pathways to block indirect effects

  • Systems biology approaches to model network effects and identify key nodes

These approaches would help establish whether CyaY affects virulence directly through interaction with virulence factors, or indirectly by supporting general iron homeostasis required for pathogen function.

What emerging technologies will advance our understanding of CyaY's role in bacterial iron homeostasis?

Several cutting-edge technologies show promise for deepening our understanding of CyaY function:

• Advanced structural biology approaches:

  • Cryo-electron microscopy to visualize CyaY-partner protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon binding

  • Time-resolved X-ray crystallography to capture dynamic binding events

• Single-molecule techniques:

  • FRET-based sensors to monitor CyaY-substrate interactions in real-time

  • Optical tweezers to measure binding forces and kinetics

  • Single-molecule tracking in live bacteria to observe CyaY localization and movement

• Advanced genetic tools:

  • CRISPR interference for precise temporal control of cyaY expression

  • Deep mutational scanning to comprehensively map structure-function relationships

  • Ribosome profiling to assess translational regulation of CyaY and related proteins

• Imaging innovations:

  • Element-specific imaging to track iron distribution in bacteria with subcellular resolution

  • Correlative light and electron microscopy to connect CyaY localization with ultrastructural features

  • Super-resolution microscopy to visualize CyaY oligomerization in vivo

These technologies would provide unprecedented insights into the molecular mechanisms and cellular contexts of CyaY function.

How might CyaY research contribute to novel antimicrobial strategies?

Given V. vulnificus's status as a significant food-borne pathogen associated with high mortality , targeting iron acquisition systems could lead to novel therapeutic approaches:

• Direct CyaY inhibition strategies:

  • Small molecule inhibitors targeting the iron or heme binding sites

  • Peptide-based inhibitors that disrupt CyaY-partner protein interactions

  • Compounds that lock CyaY in non-functional oligomeric states

• Iron homeostasis disruption approaches:

  • Exploitation of the reduced iron affinity when heme is bound (dissociation constant increase from 65.2 to 87.9 μM)

  • Molecules that artificially induce conformational changes similar to those observed in circular dichroism studies

  • Targeted iron sequestration strategies that overwhelm CyaY-dependent acquisition systems

• Virulence modulation strategies:

  • Compounds that disrupt potential interactions between CyaY and virulence factors

  • Agents that enhance host iron sequestration mechanisms to counter bacterial acquisition

  • Targeted vaccine approaches against iron transport systems

• Diagnostic applications:

  • Detection of CyaY variants associated with heightened virulence

  • Biosensors based on CyaY binding properties for environmental monitoring

  • Identification of genetic signatures in cyaY that predict strain virulence potential

These approaches would capitalize on the essential nature of iron acquisition for pathogen survival while leveraging the specific properties of CyaY.

What interdisciplinary approaches could resolve outstanding questions about CyaY evolution and adaptation?

Understanding the evolutionary trajectory and adaptive significance of CyaY requires integrative approaches spanning multiple disciplines:

• Comparative genomics and evolutionary biology:

  • Phylogenetic analysis of cyaY across Vibrio species and related pathogens

  • Identification of selection signatures indicating adaptive evolution

  • Investigation of genetic recombination patterns similar to those observed for rtxA1

• Environmental microbiology:

  • Sampling of V. vulnificus strains across diverse marine environments

  • Correlation of cyaY variants with environmental iron availability

  • Assessment of cyaY expression under different ecological conditions

• Structural biology and biochemistry:

  • Comparative analysis of binding properties across evolutionary diverse CyaY proteins

  • Structure-guided reconstruction of ancestral CyaY proteins

  • Biochemical characterization of CyaY from multiple Vibrio species

• Host-pathogen systems biology:

  • Transcriptomic analysis of host and pathogen during infection

  • Metabolic modeling of iron flux between host and pathogen compartments

  • Mathematical modeling of evolutionary dynamics under changing selective pressures