Recombinant Shigella boydii serotype 18 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is ubiquinone biosynthesis protein UbiB and what is its function in Shigella boydii?

Ubiquinone biosynthesis protein UbiB is a key component in the biosynthetic pathway of ubiquinone (coenzyme Q) in Shigella boydii. UbiB belongs to a family of proteins that participate in the assembly of the ubiquinone molecule, which is critical for bacterial respiratory electron transport chains. In the context of Shigella boydii serotype 18, UbiB plays a role in energy metabolism by contributing to the production of ubiquinone, which serves as an electron carrier in the respiratory chain and as a membrane antioxidant .

Unlike some other ubiquinone biosynthesis proteins (such as UbiJ and UbiK) that form distinct complexes, UbiB operates within the metabolon – a multienzyme complex that facilitates the coordinated synthesis of ubiquinone. The protein likely functions in association with the bacterial inner membrane, where ubiquinone is ultimately incorporated to perform its biological functions .

How conserved is UbiB across different Shigella serotypes and related bacterial species?

UbiB is highly conserved across different Shigella serotypes and closely related Enterobacteriaceae, particularly between Shigella and Escherichia coli. This conservation reflects the essential nature of ubiquinone in bacterial energy metabolism. Sequence analysis shows >98% homology in UbiB across different Shigella species, similar to the high conservation observed in other proteins like IpaB (invasion plasmid antigen B) .

This high degree of conservation makes UbiB a potential target for broad-spectrum research applications. Unlike surface antigens like O-polysaccharides that vary significantly between serotypes (hence why Shigella has multiple serotypes including 15 for S. dysenteriae, 14 for S. flexneri, 20 for S. boydii, and 1 for S. sonnei), proteins involved in core metabolic functions like UbiB tend to show much less variation . This conservation is particularly relevant when considering potential cross-protective research applications.

What are the optimal expression systems for producing recombinant Shigella boydii UbiB for research purposes?

For efficient expression of recombinant Shigella boydii UbiB, several expression systems have proven effective, with E. coli-based systems being the most commonly utilized due to the genetic similarity between E. coli and Shigella. The following expression systems and methodologies are recommended:

E. coli Expression Systems:

• BL21(DE3) strain with pET vector systems provides high-yield expression when UbiB is fused with tags like His6 or GST for purification

• ArcticExpress or Rosetta strains can improve proper folding when expression issues arise

• For membrane-associated proteins like UbiB, C41(DE3) or C43(DE3) strains (derivatives of BL21) often yield better results

Expression Conditions:

• Induction with 0.1-0.5 mM IPTG at lower temperatures (16-25°C)

• Extended expression times (16-24 hours) at reduced temperatures can improve proper folding

• Supplementation with specific cofactors may enhance protein stability

Similar to approaches used for the UbiJ-UbiK complex, cell-free protein synthesis systems may be considered for UbiB expression when traditional methods yield poor results. These systems have been successfully employed for expressing membrane-associated proteins involved in ubiquinone biosynthesis .

What purification strategies are most effective for isolating Shigella boydii UbiB while maintaining protein stability and function?

Purifying Shigella boydii UbiB requires careful consideration of its potential membrane association and interaction with other ubiquinone biosynthesis proteins. The following purification strategy is recommended:

Step 1: Membrane Fraction Preparation

• Gentle lysis using French press or sonication in buffer containing protease inhibitors

• Differential centrifugation to separate membrane fractions

• Solubilization using mild detergents (0.5-1% n-dodecyl-β-D-maltoside or CHAPS)

Step 2: Affinity Chromatography

• For His-tagged UbiB: Ni-NTA affinity chromatography with imidazole gradient elution

• For GST-tagged UbiB: Glutathione Sepharose with reduced glutathione elution

Step 3: Secondary Purification

• Size exclusion chromatography using Superdex 200 columns to separate monomeric UbiB from aggregates or other complexes

• Ion exchange chromatography as an alternative or additional step

Critical Factors for Maintaining Stability:

• Inclusion of stabilizing agents like glycerol (10-15%)

• Maintaining pH between 7.0-8.0

• Addition of reducing agents (1-5 mM DTT or 2-mercaptoethanol)

• Consider purifying entire protein complexes if UbiB tends to co-purify with other metabolon components

The purification should be verified using SDS-PAGE, Western blotting, and activity assays to confirm both purity and retention of functional properties .

How can researchers effectively measure the enzymatic activity of recombinant UbiB in vitro?

Measuring the enzymatic activity of recombinant UbiB presents challenges due to its operation within the ubiquinone biosynthesis metabolon. Nevertheless, several approaches can be employed:

In Vitro Reconstitution Assays:

• Prepare liposomes or nanodiscs containing purified UbiB and other essential metabolon components

• Add ubiquinone precursors and cofactors (ATP, electron donors)

• Monitor conversion of precursors to intermediates or final ubiquinone product using:

  • HPLC with UV detection

  • LC-MS/MS for precise identification of intermediates

  • Radioisotope-labeled precursors for tracking conversion rates

Complementation Assays:

• Express recombinant UbiB in UbiB-deficient strains

• Measure restoration of ubiquinone synthesis by analyzing cellular ubiquinone content

• Quantify growth recovery under conditions requiring functional respiratory chains

Binding Assays:

• Assess interaction with other metabolon components using:

  • Surface plasmon resonance

  • Microscale thermophoresis

  • Isothermal titration calorimetry

• Evaluate membrane interaction capabilities through liposome binding assays

When measuring UbiB activity, researchers should consider the complexity of the ubiquinone biosynthesis pathway and the potential requirement for additional proteins within the metabolon. Similar to the approach used for studying the UbiJ-UbiK complex, combining multiple methods provides the most comprehensive assessment of UbiB function .

How does UbiB interact with other proteins in the ubiquinone biosynthesis metabolon?

UbiB functions as part of a larger metabolon complex that coordinates ubiquinone biosynthesis. Based on studies of related ubiquinone biosynthesis proteins, we can infer UbiB's interactions:

• Metabolon Assembly Interactions:

  • UbiB likely interacts with other Ubi proteins (potentially UbiA, UbiD, UbiX) to form the biosynthetic complex

  • These interactions help channel intermediates between enzymatic components, increasing pathway efficiency

  • Protein-protein interaction domains within UbiB mediate these associations

• Membrane Interface Interactions:

  • Similar to the UbiJ-UbiK complex that serves as an interface between soluble enzymes and the membrane, UbiB may play a role in membrane association

  • UbiB might contribute to the release of newly synthesized ubiquinone into the membrane, similar to how the UbiJ-UbiK complex functions

• Substrate Channeling:

  • UbiB potentially creates microenvironments that protect reactive intermediates from degradation

  • Such protein-protein interactions prevent side reactions and increase biosynthetic efficiency

By analogy with the extensively studied UbiJ-UbiK complex, which forms a UbiJ-UbiK₂ heterotrimer that mediates interactions between the metabolon and the membrane, UbiB likely participates in similar spatial organization of the ubiquinone biosynthesis machinery .

What is the proposed mechanism by which UbiB contributes to ubiquinone biosynthesis?

The mechanistic contribution of UbiB to ubiquinone biosynthesis involves several proposed functions:

• Potential Enzymatic Role:

  • UbiB may possess enzymatic activity facilitating specific steps in the conversion of precursors to ubiquinone

  • It potentially participates in the hydroxylation or methylation reactions of the ubiquinone head group

• Structural Support:

  • UbiB likely provides structural integrity to the metabolon complex

  • This structural role helps maintain the spatial organization required for efficient biosynthesis

• Substrate Delivery and Release:

  • Similar to the UbiJ-UbiK complex, UbiB may facilitate the delivery of intermediates to catalytic sites

  • It potentially assists in the release of completed ubiquinone molecules into the membrane

  • Molecular modeling suggests an energy barrier of approximately 3 kcal/mol for ubiquinone release, a process in which UbiB might participate

• Membrane Association:

  • UbiB likely contains membrane-interacting domains similar to those identified in UbiJ and UbiK

  • These domains could include amphipathic helices that anchor the metabolon to the membrane surface

While direct experimental evidence for UbiB's precise mechanism is still emerging, its classification as a "probable ubiquinone biosynthesis protein" reflects its putative involvement in these processes based on homology to better-characterized components of the ubiquinone biosynthetic machinery .

How does UbiB function compare with UbiJ-UbiK complex in ubiquinone biosynthesis?

The UbiB protein and the UbiJ-UbiK complex represent distinct components of the ubiquinone biosynthesis pathway, with both similarities and differences in their functions:

How might UbiB function differ between various Shigella serotypes, particularly in the context of metabolic adaptation?

While UbiB is highly conserved across Shigella serotypes, subtle variations in its sequence or expression levels may contribute to metabolic adaptations specific to different ecological niches or pathogenic behaviors:

• Serotype-Specific Metabolic Requirements:

  • S. boydii serotype 18 may have specific metabolic adaptations related to its epidemiological distribution

  • Different serotypes might exhibit varying levels of respiratory versus fermentative metabolism, potentially influencing UbiB function or expression

  • These differences could manifest in subtle sequence variations affecting protein-protein interactions within the metabolon

• Regulatory Variations:

  • Expression patterns of UbiB may differ between serotypes in response to environmental stresses

  • Oxygen tension, nutrient availability, and host environments might differentially regulate UbiB expression

• Integration with Virulence Mechanisms:

  • Metabolic adaptation through UbiB might interface with serotype-specific virulence factors

  • Energy production via ubiquinone-dependent respiration could support different invasion strategies

Research approaches to investigate these differences should include comparative genomics across serotypes, transcriptomic analysis under various conditions, and biochemical characterization of UbiB from multiple Shigella serotypes to identify functional differences that might contribute to their distinct ecological adaptations and pathogenic behaviors .

What techniques can be employed to study the structure-function relationship of UbiB within the context of the ubiquinone biosynthesis metabolon?

Elucidating the structure-function relationship of UbiB within the ubiquinone biosynthesis metabolon requires integrating multiple advanced techniques:

A particularly promising approach would combine multiscale molecular modeling (as applied to the UbiJ-UbiK complex) with experimental validation through mutagenesis of key residues identified in silico. This strategy could reveal how UbiB interacts with both the membrane and other components of the metabolon to facilitate ubiquinone biosynthesis .

How might genetic variations in UbiB impact antibiotic resistance mechanisms in Shigella boydii?

Genetic variations in UbiB could significantly impact antibiotic resistance mechanisms in Shigella boydii through several interconnected pathways:

• Respiratory Chain Modulation:

  • Variations affecting UbiB function could alter ubiquinone levels, affecting electron transport chain efficiency

  • Modified respiratory capacity might influence susceptibility to antibiotics targeting energy metabolism

  • Altered membrane potential resulting from respiratory changes could affect uptake of aminoglycosides and other membrane potential-dependent antibiotics

• Membrane Composition Effects:

  • UbiB's role in ubiquinone biosynthesis impacts membrane composition

  • Changes in membrane ubiquinone content could alter membrane permeability and fluidity

  • These alterations potentially affect the penetration of hydrophobic antibiotics

• Oxidative Stress Response:

  • Ubiquinone functions as an antioxidant in bacterial membranes

  • UbiB variations might alter oxidative stress tolerance

  • This could impact susceptibility to antibiotics that generate reactive oxygen species as part of their killing mechanism

• Metabolic State Influence:

  • Changes in energy production efficiency due to UbiB variations might shift bacterial metabolic state

  • Metabolically dormant or altered states often exhibit reduced antibiotic sensitivity

  • This represents a form of non-genetic adaptive resistance

Research approaches to investigate these connections should include comparative analysis of UbiB sequences from resistant isolates, generation of UbiB variants with altered function, and assessment of their impact on antibiotic susceptibility profiles. Understanding these relationships could potentially identify UbiB as a target for adjuvant therapies designed to enhance antibiotic efficacy against resistant Shigella strains .

Could UbiB serve as a potential target for novel Shigella vaccine development approaches?

Evaluating UbiB as a potential target for Shigella vaccine development requires careful consideration of several factors:

• Advantages as a Vaccine Target:

  • High conservation across Shigella serotypes (>98% homology), potentially providing broad protection similar to IpaB

  • Essential metabolic function, making escape mutations less likely

  • Potential to generate cross-protective immunity against multiple Shigella serotypes

• Challenges and Limitations:

  • Limited surface exposure may reduce accessibility to antibodies

  • As an intracellular protein, UbiB might not be readily accessible during initial infection stages

  • Potential homology with human proteins could raise safety concerns

• Potential Vaccine Strategies:

  • Subunit vaccines using recombinant UbiB or immunogenic epitopes

  • Conjugate approaches similar to OPS-IpaB vaccines, potentially creating UbiB-carrier conjugates

  • Attenuated live vaccines with modified UbiB to enhance immunogenicity while maintaining safety

• Combination Approaches:

  • UbiB could serve as a carrier protein for Shigella O-polysaccharide (OPS) similar to how IpaB has been used

  • This approach could potentially address the limitations of current vaccine candidates that rely exclusively on O-antigen specificity

  • Such conjugates might provide both serotype-specific immunity (via OPS) and broader protection (via UbiB)

Current Shigella vaccine development has focused heavily on O-antigen specificity, but this necessitates multivalent formulations to cover prevalent serotypes. The high conservation of UbiB across Shigella species could potentially address this limitation if immunogenic epitopes can be identified and effectively presented to the immune system .

How does current research on UbiB compare with established Shigella vaccine approaches targeting O-polysaccharides and invasion plasmid antigens?

Current research on UbiB exists in a different context compared to established Shigella vaccine approaches:

The most advanced Shigella vaccine candidates currently rely on O-polysaccharide conjugates, which face the limitation of serotype specificity. Novel approaches using invasion plasmid antigens like IpaB have shown promise by offering broader protection. UbiB research represents a less explored avenue that could potentially contribute to future vaccine strategies, particularly if integrated with existing approaches .

For example, the novel OPS-IpaB conjugate vaccine approach demonstrated robust protection against lethal challenge with both S. flexneri 2a and S. sonnei in mouse models. This approach uses the conserved IpaB protein as both a carrier and protective antigen, offering broader protection than traditional conjugates. A similar conceptual approach might be possible with UbiB if it proves sufficiently immunogenic .

What role might UbiB play in developing attenuated live Shigella vaccine strains with modified metabolism?

UbiB modifications could serve as a sophisticated attenuation strategy for developing live Shigella vaccine strains:

• Metabolic Attenuation Approach:

  • Controlled modification of UbiB could create strains with reduced virulence while maintaining immunogenicity

  • Partial reduction in ubiquinone biosynthesis through UbiB modulation could limit bacterial replication without preventing antigen presentation

  • Such metabolically attenuated strains might achieve the balance between safety and immunogenicity that has challenged previous live vaccine candidates

• Advantages Over Current Attenuation Strategies:

  • More precise metabolic control compared to traditional auxotrophic mutations (e.g., aroA deletions)

  • Potentially more stable than plasmid-based attenuation methods

  • Allows for fine-tuning of attenuation level through specific UbiB modifications

• Integration with Existing Approaches:

  • UbiB modifications could complement other attenuation strategies

  • Combined with deletions in virulence genes (e.g., virG), a UbiB-modified strain might achieve optimal safety and efficacy

  • Similar to the approach used in developing attenuated S. flexneri 2a strains like CVD 1203, which harbors deletions in aroA and virG

• Implementation Strategy:

  • Identification of UbiB modifications that reduce function without eliminating it

  • Integration of these modifications into candidate vaccine strains

  • Assessment of attenuation, stability, immunogenicity, and protective efficacy

Clinical trials with previous attenuated Shigella vaccines have demonstrated challenges in balancing reactogenicity with immunogenicity. For example, CVD 1203 (S. flexneri 2a with aroA and virG deletions) was well-tolerated at lower doses but caused unacceptable reactogenicity at higher doses needed for strong immune responses . UbiB-based attenuation strategies might provide an alternative approach to achieve this critical balance by more precisely controlling bacterial metabolism and replication during immunization.