Recombinant Bartonella henselae ATP-dependent protease ATPase subunit HslU (hslU)

What is the biological function of HslU in Bartonella henselae?

HslU functions as the ATPase subunit of the HslVU protease complex in B. henselae, a bacterial homolog of the eukaryotic proteasome. This ATP-dependent protease system plays a critical role in protein quality control and stress response mechanisms. HslU provides energy through ATP hydrolysis that powers the proteolytic activity of the HslV component .

The primary functions of HslU include:

• ATP binding and hydrolysis to drive conformational changes essential for protease activation

• Recognition and unfolding of substrate proteins

• Translocation of unfolded substrates to HslV for degradation

• Allosteric activation of HslV's proteolytic sites through C-terminal interactions

In B. henselae specifically, HslU likely contributes to the bacterium's remarkable environmental persistence and pathogenicity. Studies have demonstrated that B. henselae can survive in various fluid matrices and even after desiccation, suggesting robust protein quality control systems like the HslVU complex are important for its survival in diverse environments .

How does the structure and composition of the HslVU complex enable its function?

The HslVU protease complex consists of two components that work together through a specific structural arrangement:

Structure of Individual Components

HslV (Protease Component):

• Forms a barrel-shaped dodecamer composed of two hexameric rings

• Similar to the β-subunits of eukaryotic proteasomes

• Contains threonine proteolytic active sites within its central chamber

HslU (ATPase Component):

• Forms hexameric rings with a central pore

• Contains three distinct domains: N-terminal nucleotide-binding domain, I-domain (insertion domain), and C-terminal domain

• The C-terminal tails are critical for HslV activation

Functional Assembly

The complex assembles with HslU hexameric rings capping one or both ends of the HslV barrel, creating "singly capped" or "doubly capped" HslVU particles. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease .

The interaction between components involves insertion of the C-termini of HslU into specific pockets in HslV, which is essential for activating the protease function. This insertion mechanism has been demonstrated through site-directed mutagenesis, peptide activation studies, and fluorescence experiments, showing that disruption of this interaction invariably leads to inactive enzyme complexes .

ATP binding to HslU drives conformational changes that promote proper complex formation and activation. Experimental evidence shows that ATP stimulates peptidase activity up to 150-fold, whereas other nucleotides have no effect .

What methodologies are most effective for expressing recombinant B. henselae HslU?

Based on studies with similar proteins, several expression strategies can be employed for producing recombinant B. henselae HslU:

Expression Systems

E. coli-Based Expression:

• BL21(DE3) or similar strains typically provide efficient expression

• pET vector systems with T7 promoter offer high-level inducible expression

• Codon-optimized constructs may improve expression of B. henselae proteins in E. coli

Fusion Tags:

• N-terminal His6 tag facilitates purification via immobilized metal affinity chromatography

• MBP (Maltose Binding Protein) or GST (Glutathione S-transferase) fusions can enhance solubility

• Tag removal using specific proteases (TEV, thrombin) should be considered for functional studies

Optimization Strategies

Induction Conditions:

• Lower temperature induction (16-20°C) often improves folding and solubility

• Reduced IPTG concentration (0.1-0.5 mM) may decrease inclusion body formation

• Extended expression times at lower temperatures (overnight at 18°C) can increase yields of soluble protein

Buffer Components:

• Include ATP or non-hydrolyzable ATP analogs (1-5 mM) to stabilize the protein

• Add magnesium ions (5-10 mM MgCl₂) as cofactors for ATP binding

• Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation

Co-expression Approaches:

• Co-expression with chaperones (GroEL/ES, DnaK/J) may improve folding

• Co-expression with HslV partner could stabilize HslU through complex formation

• Dual expression vectors or compatible plasmid systems can facilitate this approach

The target protein should be evaluated for proper folding and oligomeric state, as functional HslU typically forms hexamers. Size exclusion chromatography and ATPase activity assays are essential quality control steps before proceeding to interaction studies .

How can researchers accurately measure the ATPase activity of recombinant HslU?

Several complementary methods can be employed to accurately measure the ATPase activity of recombinant B. henselae HslU:

Colorimetric Phosphate Detection Methods

Malachite Green Assay:

• Detects free inorganic phosphate released during ATP hydrolysis

• Malachite green forms a complex with phosphomolybdate, producing a measurable color change

• Protocol outline:

  • Incubate purified HslU (0.1-1 μM) with ATP (1-5 mM) in reaction buffer

  • At defined time points, stop reaction with acid

  • Add malachite green reagent and measure absorbance at 620-650 nm

  • Calculate reaction rate using a phosphate standard curve

Coupled Enzyme Assays

Pyruvate Kinase/Lactate Dehydrogenase System:

• ADP produced by HslU is converted back to ATP by pyruvate kinase

• This reaction converts phosphoenolpyruvate to pyruvate

• Lactate dehydrogenase then reduces pyruvate to lactate, oxidizing NADH to NAD⁺

• The decrease in NADH is monitored at 340 nm

• Advantages: Continuous real-time measurement, high sensitivity

Critical Experimental Controls

• No-enzyme control: Accounts for spontaneous ATP hydrolysis

• Heat-inactivated enzyme control: Confirms enzymatic nature of activity

• Nucleotide specificity controls: Test other nucleotides (GTP, CTP) to verify ATP specificity

• HslV addition: Determine whether HslV presence affects ATPase activity

Kinetic Analysis

For comprehensive characterization, determine:

• Km for ATP (typically in the μM to low mM range)

• Vmax and kcat values

• Effects of temperature, pH, and salt concentration

• Influence of HslV on ATPase activity

• Impact of potential substrates on ATP hydrolysis rates

These assays reveal essential information about the enzymatic properties of HslU and can be used to compare wild-type protein with mutant variants or to evaluate the effects of potential inhibitors .

What is known about the interaction between HslU and HslV in the protease complex?

The interaction between HslU and HslV is critical for forming a functional protease complex with several key features:

Key Interaction Mechanism

The most significant interaction involves insertion of the C-terminal tails of HslU into specific binding pockets in HslV. This interaction has been demonstrated through multiple experimental approaches:

• Site-directed mutagenesis: Mutations that disrupt the interaction between HslU C-termini and HslV invariably lead to inactive enzyme complexes

• Peptide activation studies: Synthetic peptides derived from the C-terminus of HslU can bind to HslV with approximately 10⁻⁵ M affinity

• Functional replacement: These peptides can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of folded substrates

Activation Mechanism

The binding of HslU causes two primary effects on HslV:

• Allosteric activation: Conformational changes in HslV that activate its catalytic sites

• Potential channel opening: While allosteric activation is essential, channel opening may also occur to facilitate substrate entry

ATP Dependence

ATP plays a crucial role in the interaction:

• ATP binding to HslU promotes conformational changes that facilitate HslV binding

• ATP hydrolysis is essential for substrate processing, though not for initial complex formation

• Experiments show that ATP stimulates peptidase activity up to 150-fold, while other nucleotides have no effect

Structural Evidence

Electron microscopy has revealed that the HslVU complex forms ring-shaped particles similar to the eukaryotic proteasome. The arrangement involves HslU hexamers capping one or both ends of the HslV dodecamer, creating a sealed chamber for controlled proteolysis .

This interaction mechanism differs from the activation mechanisms of related proteases like ClpP and the eukaryotic proteasome, highlighting the unique features of the HslVU system that could potentially be exploited for therapeutic development .

How does B. henselae HslU compare to homologs in other bacterial species?

The HslU protein from B. henselae shares significant structural and functional similarities with homologs in other bacterial species, while also potentially possessing unique adaptations related to B. henselae's lifestyle:

Conserved Features

Primary Structure:

Functional Conservation:

• ATP-dependent activation of HslV appears mechanistically similar across species

• The proteolytic specificity pattern shows conservation, with B. henselae HslVU likely hydrolyzing peptides with hydrophobic residues at the P1 position, similar to E. coli HslVU

Potential Unique Adaptations

B. henselae HslU may feature adaptations related to the organism's unique lifestyle:

• Environmental Stability: B. henselae shows remarkable environmental stability, surviving in various fluids and after desiccation. Its HslVU system might be adapted to maintain protein quality control under these diverse conditions .

• Host Adaptation: As an intracellular pathogen that can persist in mammalian cells, B. henselae HslU might have evolved specific substrate preferences related to its intracellular lifestyle .

• Vector Compatibility: Given that B. henselae can survive in flea gut and feces, its stress response systems including HslU may have special adaptations for this environment .

Evolutionary Context

B. henselae belongs to the alpha-2 proteobacteria subclass, and its HslU likely shows highest similarity to homologs within this group. Recent sequence typing studies of B. henselae have identified multiple sequence types (STs) circulating in various host species, which may potentially have subtle variations in their HslU proteins that could affect protein function or regulation .

Understanding these similarities and differences is valuable for both fundamental research and potential therapeutic development targeting B. henselae-specific features of the protein .

What role might HslU play in B. henselae pathogenicity and environmental persistence?

HslU likely contributes significantly to both B. henselae pathogenicity and its remarkable environmental persistence:

Contribution to Environmental Persistence

Recent research has demonstrated extraordinary environmental stability of B. henselae, including:

• Survival in feline whole blood, serum, and urine for up to 7 days

• Viability in bovine milk and physiologic saline for extended periods

• Remarkable ability to survive desiccation and subsequent reconstitution in various biological fluids

The HslVU protease system likely plays a crucial role in this environmental resilience by:

• Eliminating damaged proteins that accumulate during environmental stress

• Maintaining protein homeostasis during transitions between environments

• Supporting adaptation to nutrient fluctuations through controlled protein turnover

Potential Roles in Pathogenicity

Several aspects of B. henselae pathogenicity may involve HslU:

• Intracellular Survival: As a facultative intracellular pathogen, B. henselae must adapt to the intracellular environment, where HslU-mediated protein quality control could be essential .

• Vasoproliferative Activity: B. henselae causes vasoproliferative lesions (bacillary angiomatosis) through factors like Bartonella angiogenic factor A (BafA) and Bartonella adhesin A (BadA). Proper folding and regulation of these virulence factors might depend on functional proteolytic systems .

• Strain-Specific Variation: Different B. henselae strains show variation in pathogenicity. Recent research has identified multiple sequence types (STs) with varying virulence potential. The HslVU system might contribute to these strain-specific differences in pathogenicity .

• Biofilm Formation: B. henselae can form biofilms associated with culture-negative endocarditis. Protein quality control systems are often essential for biofilm development and maintenance .

• Stress Response During Infection: During infection, B. henselae faces numerous stressors including host immune responses, temperature changes, and oxidative stress. HslU helps degrade damaged proteins resulting from these stressors, supporting bacterial persistence .

Understanding HslU's role in these processes could provide insights into B. henselae's ability to cause both acute infections like CSD and more serious chronic infections affecting the cardiovascular, neurocognitive, and rheumatologic systems .

What assays are most suitable for evaluating the proteolytic activity of the HslVU complex?

Several complementary assays can effectively evaluate the proteolytic activity of the recombinant B. henselae HslVU complex:

Fluorogenic Peptide Substrate Assays

Z-Gly-Gly-Leu-AMC Assay:

• This fluorogenic peptide is rapidly hydrolyzed by the HslVU complex in the presence of ATP

• Release of AMC (7-amino-4-methylcoumarin) produces measurable fluorescence (Ex: 380 nm, Em: 460 nm)

• Protocol outline:

  • Combine purified HslV and HslU (typically in 1:2 molar ratio) in reaction buffer

  • Add ATP (1-5 mM) to activate the complex

  • Add Z-Gly-Gly-Leu-AMC substrate (50-100 μM)

  • Monitor fluorescence increase over time

  • Calculate reaction rates under various conditions

This assay is particularly useful because research on E. coli HslVU has shown that Z-Gly-Gly-Leu-AMC is rapidly hydrolyzed by the complex, making it an ideal substrate for kinetic studies .

Protein Substrate Degradation Assays

SDS-PAGE Based Analysis:

• Incubate the HslVU complex with model protein substrates (e.g., casein, denatured proteins)

• Sample at various time points and analyze by SDS-PAGE

• Visualize protein degradation by Coomassie staining or Western blotting

• Advantages: Demonstrates activity against full protein substrates, closer to physiological function

Critical Controls and Variations

For comprehensive characterization, include:

• Component Controls:

  • HslV alone to demonstrate ATP and HslU dependence

  • HslU alone to confirm no inherent proteolytic activity

  • Heat-inactivated complex as negative control

• ATP Requirement Assessment:

  • Reactions without ATP to confirm ATP dependence

  • ATP analogs (AMP-PNP, ATP-γS) to distinguish between ATP binding and hydrolysis requirements

  • ATP regeneration system for extended assays

• Inhibition Studies:

  • Test proteasome inhibitors that affect threonine proteases

  • Use anti-HslV antibodies to confirm specificity

  • The research shows that unlike eukaryotic proteasomes, HslVU lacks tryptic-like and peptidyl-glutamyl-peptidase activities, providing a basis for specificity testing

These assays collectively provide a comprehensive assessment of the proteolytic function of the HslVU complex, its substrate specificity, and the requirements for its activation, offering insights into its role in B. henselae protein quality control.

How can site-directed mutagenesis be applied to study functional domains of B. henselae HslU?

Site-directed mutagenesis offers a powerful approach to dissect the structure-function relationships in B. henselae HslU by targeting specific residues in key domains:

Key Functional Domains for Mutagenesis

1. Nucleotide-Binding Domain:

• Walker A motif mutations (e.g., conserved lysine to alanine) should eliminate ATP binding

• Walker B motif mutations (conserved aspartate to alanine) should permit binding but prevent hydrolysis

• These mutations would help distinguish between ATP binding and hydrolysis requirements for various functions

2. I-Domain (Insertion Domain):

• Target residues potentially involved in substrate recognition

• Mutate exposed hydrophobic or charged patches that might interact with unfolded proteins

• These studies would provide insights into substrate specificity

3. C-Terminal Region:

• The C-terminus is critical for HslV interaction and activation

• Create C-terminal truncations or point mutations in terminal residues

• Research has shown that synthetic peptides derived from HslU C-terminus can activate HslV, confirming the importance of this region

Experimental Design for Mutant Analysis

1. Functional Assays:

2. Interaction Studies:

• Size exclusion chromatography to assess complex formation

• Surface plasmon resonance to measure binding kinetics with HslV

• Co-immunoprecipitation to detect complex formation in solution

• These approaches would reveal how specific mutations affect HslU-HslV interactions

3. Structural Analysis:

Specific Mutation Strategies

1. Alanine Scanning:

• Systematically replace individual residues with alanine

• Focus on conserved residues identified through alignment with well-characterized homologs

• This approach has proven valuable in studying the HslVU complex from other bacteria

2. Charge Reversal:

• Change positively charged residues to negative and vice versa

• Particularly useful for studying electrostatic interactions in HslU-HslV binding

• Research shows that mutations disrupting the interaction between HslU C-termini and HslV lead to inactive enzyme complexes

3. Conservative Substitutions:

• Replace residues with chemically similar ones to subtly alter function

• Compare effects with more dramatic substitutions to distinguish between structural and functional roles

This systematic mutagenesis approach would provide detailed insights into how B. henselae HslU functions and potentially reveal species-specific adaptations that could be targeted for therapeutic development.

What are the potential applications of recombinant B. henselae HslU in diagnostic or therapeutic development?

Recombinant B. henselae HslU offers several promising applications in both diagnostics and therapeutics:

Diagnostic Applications

1. Serological Diagnostics:

• Recombinant HslU could serve as an antigen for detecting anti-B. henselae antibodies

• Potential advantages over current serological tests:

  • Higher specificity if using B. henselae-specific epitopes

  • Reduced cross-reactivity compared to whole-cell antigens

• Current diagnostic challenges include cross-reactivity between B. henselae and other pathogens like Brucella melitensis, Coxiella burnetii, and Rickettsia typhi

2. Epitope Mapping:

• Identifying immunodominant epitopes in HslU could lead to more specific diagnostic peptides

• Comparing to other immunogenic B. henselae proteins like SucB (dihydrolipoamide succinyltransferase) could improve test panels

3. Recombinant Antigen Panels:

• Combining HslU with other recombinant B. henselae antigens like Pap31

• Research on recombinant Pap31 has shown 72% sensitivity and 61% specificity for human bartonellosis

• A multi-antigen approach could improve diagnostic accuracy

Therapeutic Applications

1. Inhibitor Development:

• ATP-dependent proteases represent potential antibiotic targets

• The structural differences between bacterial HslU and human proteasome components could allow selective targeting

• Targeting strategies might include:

  • ATP-competitive inhibitors that prevent the energy-dependent functions of HslU

  • Compounds that disrupt the HslU-HslV interaction, particularly targeting the critical C-terminal region

  • Allosteric inhibitors that lock HslU in an inactive conformation

2. Vaccine Development:

• Recombinant HslU could potentially serve as a component in a subunit vaccine

• Protein quality control systems are often essential for bacterial survival under stress conditions

• Immune responses targeting HslU might impair the bacterium's ability to withstand host-induced stresses

3. Structure-Based Drug Design:

• Detailed structural information about B. henselae HslU could guide rational drug design

• Features unique to B. henselae HslU compared to human AAA+ ATPases would be primary targets

• The ATP-binding pocket offers a well-defined binding site for small molecule inhibitors

Given the expanding spectrum of clinical manifestations associated with B. henselae infection beyond classical CSD - including serious cardiovascular, neurocognitive, and rheumatologic conditions - developing improved diagnostics and novel therapeutics targeting this pathogen is becoming increasingly important .

How can researchers optimize the expression and purification of functionally active recombinant HslU?

Optimizing the expression and purification of functionally active recombinant B. henselae HslU requires careful attention to several critical factors:

Expression System Optimization

1. Vector Selection:

• Use pET vectors with T7 promoter for high-level expression

• Consider codon optimization for B. henselae genes expressed in E. coli

• Include appropriate fusion tags (His6, MBP, GST) to facilitate purification and enhance solubility

• Include a precision protease cleavage site for tag removal if needed for functional studies

2. Expression Host:

• E. coli BL21(DE3) or derivatives are typically effective

• Consider specialized strains for problematic expression:

  • Rosetta strains for rare codon usage

  • Arctic Express for cold-temperature expression

  • SHuffle strains if disulfide bonds are present

3. Induction Conditions:

• Test range of temperatures (16-37°C) with lower temperatures often improving solubility

• Optimize IPTG concentration (0.1-1 mM)

• Extended induction times at lower temperatures can increase yields

• Auto-induction media can provide gentle expression for improved folding

Purification Strategy

1. Initial Capture:

• Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

• Optimize imidazole concentration in binding and washing buffers to reduce non-specific binding

• Include ATP (1-5 mM) in lysis and purification buffers to stabilize the protein structure

2. Secondary Purification:

• Ion exchange chromatography to remove contaminants and nucleic acids

• Size exclusion chromatography to isolate properly folded hexameric HslU

• Consider hydroxyapatite chromatography if contaminating proteins persist

3. Buffer Optimization:

• Include stabilizing components:

  • ATP or non-hydrolyzable ATP analogs (1-5 mM)

  • Magnesium ions (5-10 mM MgCl₂)

  • Glycerol (10-20%) to prevent aggregation

  • Reducing agents (1-5 mM DTT or β-mercaptoethanol)

Functional Validation

1. ATPase Activity:

• Measure ATP hydrolysis rates using malachite green or coupled enzyme assays

• Compare with published values for HslU from other bacteria

2. Oligomeric State Analysis:

• Size exclusion chromatography coupled with multi-angle light scattering

• Native PAGE to visualize hexamer formation

• Analytical ultracentrifugation for definitive oligomeric state determination

3. HslV Activation:

• Test ability to activate recombinant HslV using fluorogenic peptide substrates

• Verify ATP dependence of activation

• Compare activity to well-characterized systems like E. coli HslVU

This optimization framework provides a systematic approach to producing functionally active B. henselae HslU suitable for structural, biochemical, and drug development studies. The methods can be further refined based on specific experimental outcomes and requirements.

Heat Shock and Stress Response Regulation

As a component of the heat shock locus (hsl), HslU expression is likely regulated as part of the bacterial stress response network:

• Heat Shock Regulation: In most bacteria, heat shock proteins including HslVU are regulated by dedicated heat shock sigma factors that recognize specific promoter elements upstream of these genes.

• Stress-Responsive Expression: Research on related proteases in E. coli has shown upregulation during various stresses:

  • Heat shock (temperature elevation)

  • Oxidative stress

  • Nutrient limitation

  • Exposure to antibiotics or host defense factors

Relevance to B. henselae Lifestyle

B. henselae has a complex lifecycle involving transitions between different environments:

• Mammalian Host Adaptation: During infection, B. henselae faces several stressors:

  • Temperature shifts (environmental to body temperature)

  • Immune response pressures

  • Nutrient limitations

  • Intracellular adaptation requirements

• Environmental Persistence: Studies have demonstrated remarkable environmental stability of B. henselae:

  • Survival in feline blood, serum, and urine for extended periods

  • Viability after desiccation and reconstitution

  • These conditions likely trigger stress responses involving HslU

• Vector Transition: B. henselae is linked epidemiologically to cats and the cat flea vector Ctenocephalides felis, suggesting adaptation to different host environments with potential regulation of stress response systems during transitions

Potential Regulatory Mechanisms

• Transcriptional Regulation: Heat shock sigma factors likely control expression level in response to environmental cues.

• Post-translational Regulation: Activity might be modulated through:

  • ATP availability affecting complex formation

  • Interactions with other cellular components

  • Substrate availability and competition

• Spatial Regulation: Localization within the bacterial cell may change during different growth phases or stress conditions.

Investigating the specific regulation of HslU in B. henselae during infection cycles would provide valuable insights into how this pathogen adapts to diverse environments and maintains protein homeostasis during transitions between hosts. This understanding could potentially reveal intervention points for therapeutic development .