Recombinant Bartonella quintana sn-glycerol-3-phosphate import ATP-binding protein UgpC (ugpC)

What is Bartonella quintana and why is it significant for UgpC research?

Bartonella quintana is a facultative, intracellular, gram-negative rod belonging to the α2 subgroup of proteobacteria. This pathogen has significant clinical importance as it causes trench fever and is responsible for various clinical conditions including endocarditis, bacteremia, and bacillary angiomatosis. B. quintana has a 1.6-Mb genome, which is a shortened derivative of the larger 1.9-Mb genome of B. henselae . The bacterium is primarily transmitted by the human body louse, Pediculus humanus corporis, with humans serving as the reservoir . Understanding its transport systems, including the UgpC protein, is crucial for elucidating bacterial survival mechanisms and identifying potential therapeutic targets.

What is the role of UgpC in bacterial glycerol-3-phosphate transport?

UgpC functions as the nucleotide binding subunit in the ATP binding cassette (ABC) transporter complex responsible for importing sn-glycerol-3-phosphate (G3P). Based on homologous systems studied in E. coli, UgpC likely forms a homodimer that provides the energy for substrate translocation through ATP hydrolysis . The complete transport system includes periplasmic binding proteins and transmembrane subunits that work together to enable G3P uptake, which serves as both a carbon and phosphate source under phosphate-limited conditions. The UgpC component is critical as it couples ATP hydrolysis to the conformational changes required for substrate transport across the membrane.

How does the UgpC protein structurally compare between B. quintana and other bacteria?

While detailed structural information specific to B. quintana UgpC is limited, insights can be drawn from homologous systems. In E. coli, the Ugp transporter consists of the periplasmic binding protein UgpB, the transmembrane subunits UgpA and UgpE, and a homodimer of the nucleotide binding subunit UgpC . The B. quintana UgpC likely shares conserved ATP-binding cassette domains common to this protein family, including Walker A and B motifs for nucleotide binding and signature sequences that distinguish ABC transporters. Based on comparative genomics, B. quintana's genome is a shortened version derived from larger bacterial genomes, suggesting possible conservation of essential transport mechanisms across related species .

What challenges exist in expressing and purifying recombinant B. quintana UgpC?

Expression and purification of recombinant B. quintana UgpC present several technical challenges:

• Codon usage bias: B. quintana's genome has a different codon usage pattern compared to common expression hosts like E. coli, potentially leading to poor expression levels. Codon optimization or use of specialized strains harboring rare tRNAs may be necessary.

• Protein solubility: As a component of a membrane-associated complex, UgpC may exhibit solubility issues when expressed alone. Co-expression with other Ugp components or use of solubility tags may be required.

• Functional integrity: Ensuring the recombinant protein maintains its native ATPase activity requires careful buffer optimization during purification. The functional characterization of the E. coli UgpAEC complex showed UgpB/G3P-stimulated ATPase activity in proteoliposomes , suggesting similar assays could be developed for B. quintana UgpC.

• Post-translational modifications: If B. quintana UgpC undergoes specific post-translational modifications, these may not be replicated in heterologous expression systems, potentially affecting protein function.

How can researchers design experiments to investigate substrate specificity of B. quintana UgpC?

To investigate substrate specificity of B. quintana UgpC, researchers should consider these methodological approaches:

• ATPase activity assays: Using purified recombinant UgpC to measure ATP hydrolysis rates in the presence of various potential substrates. Based on studies of the E. coli system, G3P and glycerophosphocholine are likely candidates, with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM respectively for the binding protein component .

• Isothermal titration calorimetry (ITC): To determine binding affinities of UgpC for different nucleotides and measure how these interactions are influenced by potential substrate molecules.

• Reconstitution studies: Reconstituting the complete UgpABCE transport system in proteoliposomes to assess transport of radiolabeled substrates, similar to functional studies performed with the E. coli transporter .

• Mutational analysis: Creating site-directed mutations in putative substrate-binding regions based on homology modeling with the E. coli system. The crucial role of specific residues (like Trp-169 in E. coli UgpB) highlights the importance of key amino acids in substrate recognition .

• Crystallography: Determining the crystal structure of UgpC in various nucleotide-bound states to elucidate the molecular basis of ATP binding and hydrolysis during the transport cycle.

What is the correlation between B. quintana UgpC function and bacterial virulence in clinical contexts?

The relationship between UgpC function and B. quintana virulence appears complex and multifaceted:

• Nutrient acquisition: UgpC likely plays a crucial role in phospholipid acquisition during infection, particularly in phosphate-limited environments within the human host. This ability to scavenge essential nutrients contributes to bacterial survival and persistence.

• Host adaptation: B. quintana has undergone genome reduction compared to related species , suggesting selective pressure to maintain essential transport systems like UgpC during host adaptation. This specialization may contribute to its effectiveness as a human pathogen.

• Clinical manifestations: B. quintana is associated with various clinical conditions including endocarditis, which was recently reported in a patient without traditional risk factors in Los Angeles . The ability to establish persistent infection likely depends partly on efficient nutrient acquisition systems like the UgpC-containing transporter.

• Geographic distribution: While historically associated with trench warfare and homelessness, B. quintana infections can occur in the general population , suggesting its transport systems are versatile enough to support bacterial survival in diverse host environments.

• Immune evasion: The intracellular lifestyle of B. quintana may be supported by efficient nutrient acquisition via ABC transporters, potentially contributing to immune evasion and chronic infection.

What expression systems are most effective for producing functional recombinant B. quintana UgpC?

Several expression systems can be considered for optimal production of functional B. quintana UgpC:

Based on studies with related ABC transporters, the E. coli C41/C43 strains may offer a good balance of yield and functionality for B. quintana UgpC, particularly when expressed at lower temperatures (18°C) with controlled induction using 0.1-0.5 mM IPTG.

What purification strategies work best for isolating B. quintana UgpC while maintaining its functional integrity?

A systematic purification approach is recommended to obtain functional B. quintana UgpC:

• Affinity tags selection: A C-terminal His6 or His8 tag is often preferred for ABC proteins as it minimally interferes with the N-terminal nucleotide-binding domain. For challenging cases, a dual-tag approach (His-MBP) can enhance solubility and purification efficiency.

• Cell lysis optimization: Gentle lysis using lysozyme treatment (1 mg/ml, 30 min, 4°C) followed by mild sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and 5 mM MgCl2.

• Affinity chromatography: Immobilized metal affinity chromatography using Ni-NTA resin with a gradient elution (20-500 mM imidazole) to separate UgpC from contaminants while minimizing protein denaturation.

• Size exclusion chromatography: Critical for separating monomeric, dimeric, and aggregated forms of UgpC, using a Superdex 200 column equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT.

• Stability considerations: Addition of 5% glycerol and 1 mM ATP throughout purification can significantly enhance protein stability, as demonstrated for related ABC transporters.

The similar purification approach used for the UgpAEC complex from E. coli, which displayed UgpB/G3P-stimulated ATPase activity when reconstituted in proteoliposomes , provides a valuable reference for developing B. quintana UgpC purification protocols.

What functional assays can definitively characterize the ATP hydrolysis activity of purified B. quintana UgpC?

Multiple complementary assays can be employed to comprehensively characterize UgpC ATP hydrolysis:

• Colorimetric phosphate release assays: The malachite green assay provides sensitive detection of inorganic phosphate released during ATP hydrolysis. Typically, reactions contain 0.1-1 μM purified UgpC, 1-5 mM ATP, and buffer containing essential magnesium (5 mM MgCl2). This quantitatively measures enzyme kinetics with parameters like Km and Vmax.

• Coupled enzyme assays: The pyruvate kinase/lactate dehydrogenase coupled assay provides continuous monitoring of ATPase activity by linking ATP regeneration to NADH oxidation, which can be measured spectrophotometrically at 340 nm.

• ADP-Glo™ assay: This luminescence-based assay offers high sensitivity for detecting ADP produced during ATP hydrolysis and is particularly useful for high-throughput screening of conditions affecting UgpC activity.

• Reconstitution assays: Incorporating purified UgpC along with other components of the transport system into proteoliposomes allows measurement of substrate-stimulated ATPase activity, as demonstrated with the E. coli UgpAEC complex .

• Binding assays: Fluorescence-based assays using fluorescent ATP analogs (TNP-ATP) or intrinsic tryptophan fluorescence quenching can characterize nucleotide binding without hydrolysis, providing insights into the first step of the catalytic cycle.

How should researchers interpret discrepancies between in vitro and in vivo UgpC activity data?

When confronted with discrepancies between in vitro and in vivo UgpC activity data, researchers should systematically analyze potential causes:

• Protein context differences: In vitro studies often use isolated UgpC, whereas in vivo the protein functions within a complete transporter complex. The isolated ATPase activity of UgpC may differ significantly from its activity when associated with UgpA and UgpE transmembrane domains and the UgpB binding protein.

• Regulatory influences: In vivo studies capture the influence of cellular regulatory systems absent in purified protein studies. For the E. coli system, it was observed that the UgpAEC complex activity was not inhibited by phosphate nor by regulatory proteins like PhoU , but other regulatory mechanisms may exist in B. quintana.

• Environmental parameters: Consider how parameters like pH, ionic strength, and macromolecular crowding differ between in vitro assays and the bacterial intracellular environment. Optimize in vitro conditions to better mimic physiological conditions.

• Substrate availability: In vivo, substrate concentrations and availability are dynamically regulated, whereas in vitro assays typically use fixed concentrations. Testing activity across a range of substrate concentrations can help bridge this gap.

• Statistical validation: Apply rigorous statistical analysis to determine if apparent discrepancies are statistically significant. Use appropriate statistical tests for comparing means (t-test, ANOVA) and consider the variability in both in vitro and in vivo measurements.

What strategies exist for overcoming expression challenges with B. quintana proteins in heterologous systems?

Researchers face several challenges when expressing B. quintana proteins in heterologous systems, which can be addressed through these strategies:

• Codon optimization: B. quintana's genome has distinct codon usage patterns. Custom synthesis of codon-optimized genes can significantly improve expression levels in E. coli or other hosts.

• Fusion tags approach: Expression as fusion proteins with solubility-enhancing partners such as MBP, SUMO, or Trx can dramatically improve yields of soluble protein. A systematic comparison of different fusion partners:

• Expression conditions optimization: Temperature (typically lowering to 16-20°C), inducer concentration (0.01-0.1 mM IPTG often preferable to standard 1 mM), and media composition (defined media vs. rich media) should be systematically optimized.

• Chaperone co-expression: Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor) can facilitate proper folding of challenging proteins.

• Alternative expression hosts: Beyond E. coli, consider Pseudomonas species which may provide a genomic background more compatible with Bartonella proteins, or eukaryotic systems for highly challenging targets.

How can comparative genomics inform functional characterization of B. quintana UgpC?

Comparative genomics offers valuable insights for functional characterization of B. quintana UgpC:

• Evolutionary context: B. quintana's genome is a shortened derivative of the B. henselae genome, which itself is a shortened version of Brucella melitensis chromosome I . This evolutionary relationship suggests that conserved genes like ugpC likely maintain essential functions despite genome reduction.

• Domain conservation analysis: Identifying highly conserved domains across UgpC homologs helps prioritize regions for functional studies. The nucleotide-binding domains of ABC transporters typically show higher conservation than other regions.

• Synteny examination: Analyzing the genomic context of ugpC across related species can reveal conserved operonic structures that suggest functional relationships with other genes.

• Host adaptation signatures: The B. quintana genome shows signs of accelerated degradation associated with host-restricted vectors . Examining which features of UgpC are preserved despite this degradation highlights functionally critical elements.

• Residue conservation mapping: Mapping conserved residues onto structural models can identify catalytic sites and protein-protein interaction interfaces crucial for UgpC function within the transport complex.

What novel therapeutic approaches might target B. quintana UgpC to treat persistent infections?

Emerging therapeutic strategies targeting UgpC could provide novel approaches for treating persistent B. quintana infections:

• ATP-competitive inhibitors: Developing small molecules that compete with ATP binding to UgpC could disrupt energy coupling for the transport process. Structure-based design targeting the nucleotide-binding pocket offers a promising approach.

• Allosteric modulators: Compounds binding to regulatory sites could lock UgpC in inactive conformations, preventing the conformational changes necessary for transport. These may offer greater selectivity than active site inhibitors.

• Interface disruptors: Peptides or small molecules designed to interfere with UgpC dimerization or its interaction with other transport components could disable the complete transport system while targeting protein interfaces that may be more species-specific than nucleotide binding sites.

• Adjunct therapy potential: UgpC inhibitors could enhance the efficacy of current treatments for B. quintana infections. For endocarditis cases, which often require valvular surgery and prolonged antibiotic treatment , transport inhibitors might reduce treatment duration or prevent recurrence.

• Delivery strategies: Targeted delivery systems could enhance the efficacy of UgpC inhibitors by increasing their concentration at infection sites. This is particularly relevant for treating B. quintana endocarditis, where bacterial vegetations on heart valves may be difficult to penetrate with conventional antibiotics .

How might environmental conditions affect UgpC expression and function in B. quintana during human infection?

Environmental factors likely modulate UgpC expression and function during infection:

• Nutrient limitation response: In phosphate-limited environments within the human host, UgpC expression is likely upregulated to enhance acquisition of glycerol-3-phosphate as an alternative phosphate source, similar to the response observed in E. coli .

• Temperature-dependent regulation: The shift from the cooler environment of the body louse vector (approximately 30°C) to human body temperature (37°C) may trigger changes in UgpC expression or activity as part of the bacterial adaptation to the mammalian host.

• pH adaptation: As B. quintana navigates different pH environments during infection, from the relatively neutral bloodstream to potentially acidic phagolysosomes within macrophages, UgpC activity may be modulated to maintain functional transport across these conditions.

• Oxygen tension effects: The microaerophilic nature of B. quintana suggests its metabolic systems, including transport proteins like UgpC, are optimized for low-oxygen environments. The oxygen gradient across tissues may influence UgpC expression patterns during infection.

• Host immunological pressure: The immune response against B. quintana may drive selective expression of transport systems less likely to be recognized by host pattern recognition receptors, potentially altering the relative importance of UgpC-mediated transport during different infection phases.