Recombinant Bartonella henselae NADH-quinone oxidoreductase subunit I (nuoI)

What is the biological function of NADH-quinone oxidoreductase in Bartonella henselae?

NADH-quinone oxidoreductase serves as the entry point for electrons into the respiratory chain in many pathogenic bacteria, including Bartonella species. While detailed studies have been conducted in other bacteria such as Vibrio cholerae (where it functions as the main sodium pump), in Bartonella henselae this enzyme complex is critical for energy metabolism . The enzyme catalyzes the oxidation of NADH and the reduction of quinones while simultaneously translocating ions across the bacterial membrane, contributing to the establishment of electrochemical gradients essential for ATP synthesis and various cellular processes. The subunit I (nuoI) specifically contributes to the structural integrity and functional capacity of this multi-subunit enzyme complex.

How is the nuoI subunit structurally characterized within the NADH-quinone oxidoreductase complex?

The nuoI subunit represents one component of the multi-subunit NADH-quinone oxidoreductase complex. While specific structural data for B. henselae nuoI is limited, comparative analyses with homologous proteins suggest it contains conserved domains involved in electron transfer and potentially in ion binding or translocation. Research indicates that in respiratory chain complexes, the integrity of individual subunits is critical for proper assembly and function of the entire complex. Functional studies performed with Na+-NQR in Vibrio cholerae demonstrate that these enzyme complexes can bind multiple ions (specifically three sodium ions in both oxidized and reduced states for Na+-NQR) , suggesting a similar multi-site binding mechanism may exist in the B. henselae complex.

What conservation patterns are observed in nuoI across different Bartonella species?

Sequence alignment analysis of nuoI across multiple Bartonella species reveals several highly conserved regions, particularly in domains associated with electron transport and cofactor binding. Currently, approximately 40 named Bartonella species have been identified, with at least 17 implicated in human illnesses . Among the most clinically relevant species (B. henselae, B. quintana, B. clarridgeiae, B. vinsonii, and B. washoensis), the nuoI subunit demonstrates considerable sequence homology, particularly in functional domains. This conservation suggests evolutionary pressure to maintain the critical functions of this enzyme complex across different Bartonella species that have adapted to various mammalian hosts.

What expression systems are most effective for producing recombinant B. henselae nuoI protein?

Prokaryotic expression systems have demonstrated effectiveness for producing recombinant Bartonella proteins. Based on successful approaches with other B. henselae proteins, such as the 17-kDa protein, a histidine-tagged fusion protein expression strategy in E. coli is recommended . For nuoI specifically, researchers should consider the following protocol:

• Gene cloning into an appropriate expression vector (such as pET or pTri series) with a histidine tag for purification

• Transformation into an E. coli expression strain (BL21(DE3) or similar)

• Induction with IPTG at optimal concentration (typically 0.5-1.0 mM)

• Expression at 30°C rather than 37°C to enhance protein solubility

• Harvest after 4-6 hours of induction or overnight expression at reduced temperature

Using this approach, expression yields comparable to those reported for the B. henselae 17-kDa protein (approximately 2.9 mg from 100 mL of bacterial culture) can be anticipated .

What purification strategies yield the highest purity and activity for recombinant nuoI protein?

Multi-step purification strategies yield the highest purity for recombinant nuoI protein while preserving enzymatic activity. The recommended approach includes:

• Initial clarification of bacterial lysate by centrifugation (15,000 × g for 30 minutes)

• Immobilized metal affinity chromatography (IMAC) using nickel-agarose columns for histidine-tagged proteins

• Gradient elution with increasing imidazole concentrations (20-250 mM)

• Secondary purification using ion-exchange chromatography to remove remaining contaminants

• Final polishing step using size exclusion chromatography if needed

This approach typically achieves protein purity "near homogeneity" as reported for other recombinant B. henselae proteins . Maintaining protein stability during purification is critical—adding 10% glycerol to storage buffers and maintaining samples at 4°C during purification helps preserve enzymatic activity. For long-term storage, flash freezing in liquid nitrogen and storage at -80°C with protease inhibitors is recommended.

How can researchers verify the structural integrity of purified recombinant nuoI?

Multiple complementary techniques should be employed to verify the structural integrity of purified recombinant nuoI:

• SDS-PAGE analysis to confirm molecular weight and purity

• Western blotting using anti-His antibodies to verify the presence of the histidine tag

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Limited proteolysis to evaluate the compactness and stability of the protein fold

• Mass spectrometry to confirm protein identity and detect any post-translational modifications

• Size exclusion chromatography to assess oligomeric state

Additionally, functional assays measuring NADH oxidation activity provide critical information on whether the protein retains its native conformation and catalytic capacity. Researchers should monitor enzymatic parameters such as Km and Vmax compared to values from native enzyme preparations when available.

What assays are most reliable for measuring the enzymatic activity of recombinant nuoI?

Several complementary assays can be employed to evaluate the enzymatic activity of recombinant nuoI:

• NADH oxidation assay: Monitoring the decrease in absorbance at 340 nm corresponding to NADH oxidation

• Electron transport assay: Using artificial electron acceptors such as ferricyanide or dichlorophenolindophenol (DCIP)

• Oxygen consumption assay: Measuring respiratory activity using an oxygen electrode

• Quinone reduction assay: Monitoring the reduction of ubiquinone analogues

When establishing these assays, researchers should include appropriate controls:

These assays should be performed under varying conditions (pH, temperature, ionic strength) to determine optimal reaction parameters and to characterize the enzyme's stability and specificity.

How does ion concentration affect the activity of recombinant B. henselae nuoI?

Ion concentration significantly influences the activity of NADH-quinone oxidoreductase complexes, as demonstrated in the Na+-NQR of Vibrio cholerae. Based on homology, we can anticipate that B. henselae nuoI likely demonstrates similar ion-dependent activity patterns . Researchers should systematically evaluate:

• Sodium ion effects: Testing concentrations from 0-200 mM to establish dose-response relationships

• Potassium ion effects: Determining whether it serves as a non-substrate activator as in V. cholerae Na+-NQR

• Lithium ion effects: Assessing potential substitution for sodium as both substrate and activator

• Divalent cation effects: Examining the impact of Mg2+ and Ca2+ on enzyme stability and activity

Studies should include competitive binding experiments with multiple ions to identify:

• Substrate specificity

• Regulatory binding sites

• Allosteric effects on enzyme kinetics

Extrapolating from Na+-NQR studies, B. henselae NADH-quinone oxidoreductase likely contains multiple binding sites with different affinities and functions , which may influence the activity of individual subunits including nuoI.

What are the kinetic parameters of recombinant nuoI and how do they compare to the native enzyme?

Determining accurate kinetic parameters requires careful experimental design and analysis. For recombinant nuoI, researchers should:

• Measure initial reaction velocities at varying substrate concentrations

• Use Lineweaver-Burk or Eadie-Hofstee plots to determine Km and Vmax values

• Calculate kcat (turnover number) and catalytic efficiency (kcat/Km)

• Compare parameters under different reaction conditions (pH, temperature, ion concentrations)

While specific values for B. henselae nuoI are not established in the literature, researchers can expect kinetic parameters to be influenced by:

Comparing recombinant enzyme parameters to those of the native enzyme complex (when available) provides valuable insights into whether the recombinant protein faithfully reproduces the native structure and function.

How can recombinant nuoI be utilized for developing diagnostic assays for Bartonella infections?

Recombinant nuoI has potential applications in diagnostic assay development for Bartonella infections, similar to other recombinant B. henselae proteins. Current approaches to bartonellosis diagnosis face challenges due to the "high complexity and time required" with conventional techniques . Researchers can develop the following diagnostic approaches:

• Serological assays: Using purified nuoI as an antigen in ELISA or indirect immunofluorescence assays to detect anti-Bartonella antibodies. The 17-kDa recombinant protein of B. henselae demonstrated 71.1% sensitivity and 93.0% specificity in IgG ELISA compared to immunofluorescence assay testing .

• Chimeric protein constructs: Creating recombinant chimeric proteins incorporating immunogenic epitopes from nuoI and other Bartonella proteins. Chimeric proteins have shown promise in immunodiagnostics for feline bartonellosis, with high specificity though sometimes limited sensitivity .

• Molecular detection platforms: Developing PCR primers targeting nuoI gene sequences for direct detection of Bartonella DNA in clinical samples. Molecular methods have successfully identified Bartonella species in various clinical contexts, with B. henselae being the most commonly detected species (80% of identified cases) .

When developing these assays, researchers should benchmark performance against established methods such as indirect immunofluorescence (IFA), which is considered the gold standard for serological diagnosis .

What role does nuoI play in understanding Bartonella pathogenesis and host-pathogen interactions?

The nuoI subunit contributes to our understanding of Bartonella pathogenesis through several research avenues:

• Energy metabolism regulation: As a component of the respiratory chain, nuoI contributes to the bacterium's ability to generate energy under different environmental conditions, including adaptation to mammalian hosts.

• Survival under stress conditions: NADH-quinone oxidoreductase may enable adaptation to oxidative stress and nutrient limitation encountered within host cells, particularly as Bartonella species are adapted to intracellular survival in erythrocytes and endothelial cells.

• Comparative studies: Analyzing differences in nuoI structure and function across Bartonella species (B. henselae, B. quintana, B. clarridgeiae, B. vinsonii, and others) provides insights into host-specific adaptations. Of the approximately 40 named Bartonella species, at least 17 are associated with human infections .

• Target for therapeutic intervention: Understanding the structure and function of nuoI may identify potential targets for novel antimicrobial agents effective against Bartonella species.

Researchers investigating these aspects should incorporate appropriate host cell models and infection systems to correlate nuoI function with bacterial survival and virulence in physiologically relevant conditions.

Can recombinant nuoI be used to develop vaccines against Bartonella infections?

The potential of recombinant nuoI as a vaccine candidate remains largely unexplored but offers several research directions:

• Antigenicity assessment: Evaluating the immunogenicity of recombinant nuoI in animal models to determine whether it elicits protective antibody responses. Similar studies with the 17-kDa protein of B. henselae demonstrated recognition by sera from patients infected with both B. henselae and B. quintana, suggesting antigenic cross-reactivity .

• Epitope mapping: Identifying immunodominant regions within nuoI that might serve as targets for subunit or peptide vaccines. This approach has been applied in developing chimeric proteins from B. henselae for diagnostic purposes .

• Delivery systems: Testing various adjuvants and delivery platforms to enhance immune responses to recombinant nuoI.

• Cross-protection: Assessing whether immunity against nuoI confers protection against multiple Bartonella species due to sequence conservation.

Vaccine development efforts should address both veterinary applications (particularly for cats, which serve as the main reservoir for B. henselae) and potential human applications for high-risk populations. Researchers should evaluate both humoral and cell-mediated immune responses, as well as duration of protection in appropriate animal models.

What interactions occur between nuoI and other subunits of the NADH-quinone oxidoreductase complex?

Understanding subunit interactions is critical for elucidating complex assembly and function. Advanced research approaches include:

• Co-immunoprecipitation: Using antibodies against recombinant nuoI to pull down interacting partners from B. henselae lysates

• Bacterial two-hybrid assays: Systematically testing interactions between nuoI and other complex subunits

• Cross-linking coupled with mass spectrometry: Identifying residues at subunit interfaces

• Cryo-electron microscopy: Determining the structural arrangement of subunits within the intact complex

These studies should address how subunit interactions influence:

• Complex assembly and stability

• Electron transfer pathways

• Ion binding and translocation

• Conformational changes during catalysis

How do genetic variations in nuoI affect antibiotic resistance and bacterial fitness in B. henselae?

Genetic variations in respiratory chain components may contribute to bacterial fitness and potentially to antibiotic resistance. Advanced research questions include:

• Natural variation: Sequencing nuoI from clinical isolates to identify polymorphisms and correlating them with phenotypic differences

• Directed evolution: Selecting for nuoI variants under antibiotic pressure or growth-limiting conditions

• Structure-function analysis: Using site-directed mutagenesis to assess how specific residues contribute to enzyme function and stability

• Fitness measurements: Comparing growth rates, metabolic efficiency, and survival under stress for strains with different nuoI variants

Research approaches should include creating isogenic strains differing only in nuoI sequence to control for other genetic variables. Competition assays and animal infection models can provide insights into how nuoI variations affect in vivo fitness and persistence.