Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni UvrABC system protein B (uvrB), partial

What is the UvrB protein in Leptospira interrogans?

UvrB is a critical component of the UvrABC nucleotide excision repair (NER) system in Leptospira interrogans. It functions as part of a DNA damage recognition and repair complex that helps protect the bacterial genome from various types of DNA damage. The UvrB protein typically works in conjunction with UvrA and UvrC to identify and excise damaged DNA segments, allowing for subsequent repair synthesis. Like other leptospiral proteins characterized through recombinant expression, UvrB likely plays an important role in bacterial survival under stress conditions, particularly those causing DNA damage.

How does UvrB contribute to bacterial survival?

UvrB contributes to bacterial survival by maintaining genomic integrity through the repair of DNA damage. Similar to how ClpB assists in stress response by repairing damaged proteins, UvrB helps repair damaged DNA, particularly damage caused by UV radiation, chemical mutagens, and oxidative stress. In pathogenic bacteria like Leptospira interrogans, this repair system enables survival in diverse and often hostile environments. As demonstrated with other leptospiral proteins, such systems may be essential for persistence both in the environment and during host infection, where bacteria face various stress conditions .

Why is studying UvrB in Leptospira interrogans important?

Studying UvrB in Leptospira interrogans is important because DNA repair mechanisms contribute significantly to bacterial pathogenesis and stress adaptation. Leptospirosis is an emerging zoonotic disease with significant public health impact, and understanding the molecular mechanisms of pathogen survival is crucial for developing interventions. Research on other leptospiral proteins has shown that stress response systems are often required for virulence, as demonstrated with the ClpB chaperone, which when inactivated resulted in attenuated virulence . Similar investigations of UvrB could reveal its potential role in pathogenesis and survival during infection.

What approaches can be used to clone and express recombinant UvrB?

To clone and express recombinant UvrB from Leptospira interrogans, researchers can employ similar approaches to those used for other leptospiral proteins. This typically involves:

• Gene amplification using PCR with primers designed based on the genome sequence of L. interrogans serovar copenhageni

• Cloning into an appropriate expression vector, such as pCR2.1 followed by subcloning into an expression vector

• Expression in E. coli systems with optimization of induction conditions

• Purification using affinity chromatography, typically with a His-tag system

For complementation studies, the cloning approach used for the ClpB gene could be adapted, where the gene is amplified with flanking restriction sites (e.g., AscI), cloned into a vector like pCR2.1, and verified by sequencing before introduction into mutant strains .

How can researchers verify the identity and purity of recombinant UvrB?

Verification of recombinant UvrB identity and purity can be accomplished through:

• SDS-PAGE analysis to confirm the expected molecular weight

• Western blotting using antibodies against the protein or attached tags

• Mass spectrometry for precise molecular weight determination and peptide mapping

• N-terminal sequencing to confirm the correct translation start site

• Enzymatic activity assays specific to UvrB function

Similar approaches were used for verification of other leptospiral recombinant proteins like LIC11051 and LIC11505, where SDS-PAGE and Western blotting confirmed protein expression and identity .

What expression systems optimize yield and functionality of recombinant UvrB?

For optimal expression of functional recombinant UvrB, consider:

Expression optimization should include testing various induction temperatures (15-37°C), inducer concentrations, and solubilization strategies if inclusion bodies form. For leptospiral proteins, expression at lower temperatures often improves solubility and proper folding, as has been observed with other DNA repair-related proteins.

What assays can effectively measure UvrB nucleotide excision repair activity?

UvrB nucleotide excision repair activity can be assessed through several complementary approaches:

• In vitro DNA binding assays: Electrophoretic mobility shift assays (EMSA) using damaged DNA substrates

• Helicase activity assays: Measuring ATP-dependent unwinding of DNA duplexes

• Reconstituted repair assays: Using purified UvrA, UvrB, UvrC, and damaged DNA substrates to measure incision products

• Fluorescence-based real-time assays: Monitoring repair of fluorescently labeled damaged DNA substrates

• Surface plasmon resonance: Measuring kinetics of UvrB binding to damaged DNA and other repair proteins

These approaches allow for quantitative assessment of both the DNA damage recognition and processing activities of UvrB in a controlled environment.

How can protein-protein interactions of UvrB be characterized?

Characterization of UvrB protein-protein interactions should employ multiple complementary techniques:

• Co-immunoprecipitation: Using antibodies against UvrB to pull down interacting partners from cell lysates

• Bacterial two-hybrid assays: For in vivo detection of protein interactions

• Microscale thermophoresis: For quantitative measurement of binding affinities

• Cross-linking coupled with mass spectrometry: To identify interaction interfaces

• Fluorescence resonance energy transfer (FRET): For monitoring interactions in real-time

Similar approaches have been used to study interactions of other leptospiral proteins, such as the leucine-rich repeat proteins LIC11051 and LIC11505, which demonstrated interactions with host components .

What methods determine subcellular localization of UvrB in Leptospira?

To determine the subcellular localization of UvrB in Leptospira, researchers can use:

• Subcellular fractionation: Separating membrane, cytoplasmic, and secreted fractions followed by Western blotting with anti-UvrB antibodies

• Immunofluorescence microscopy: Using fluorescently-labeled antibodies to visualize UvrB location

• Immuno-electron microscopy: For high-resolution localization studies

• Reporter gene fusions: Creating UvrB-GFP fusions to track localization in live cells

• Proteinase K accessibility assays: To determine surface exposure

These techniques were effectively employed for localizing LRR-proteins in Leptospira, showing that proteins like LIC11051 were primarily secreted while LIC11505 was found in both secreted and membrane fractions .

How does UvrB expression change under various stress conditions?

UvrB expression likely changes under stress conditions that cause DNA damage or threaten genomic integrity. To evaluate these changes:

• Quantitative RT-PCR: Measure transcript levels under various stresses (oxidative stress, UV exposure, antibiotic treatment)

• Western blotting: Quantify protein levels under stress conditions

• Promoter-reporter fusions: Monitor expression in real-time during stress

• RNA-seq analysis: Examine transcriptome-wide changes that include UvrB

Research on the ClpB chaperone in Leptospira showed altered expression under stress conditions, with the protein playing a crucial role in the general stress response . UvrB likely follows similar expression patterns when Leptospira encounters DNA-damaging stresses.

What role does UvrB play in Leptospira's response to oxidative stress?

UvrB likely plays a significant role in Leptospira's response to oxidative stress by:

• Repairing oxidative DNA damage (8-oxoguanine, thymine glycols, strand breaks)

• Coordinating with other DNA repair systems to maintain genomic integrity

• Potentially regulating expression of other stress response genes

The role of UvrB in oxidative stress response can be studied using similar approaches to those used for the ClpB chaperone, which showed increased susceptibility to oxidative stress in a clpB mutant . Generating a uvrB mutant and testing its survival under oxidative stress conditions would provide direct evidence of UvrB's role in this stress response.

Can UvrB mutants be generated and what phenotypes would they exhibit?

UvrB mutants can likely be generated using random transposon mutagenesis approaches similar to those used for the ClpB mutant in Leptospira . Expected phenotypes may include:

• Increased sensitivity to UV radiation

• Heightened susceptibility to DNA-damaging agents (H₂O₂, mitomycin C, ciprofloxacin)

• Reduced survival under oxidative stress conditions

• Potential growth defects at elevated temperatures

• Attenuated virulence in animal models

The ClpB mutant of L. interrogans exhibited growth defects at 30°C and 37°C, showed increased susceptibility to oxidative stress, and was attenuated in virulence . UvrB mutants would likely display similar stress-related defects, particularly in response to DNA-damaging conditions.

How does UvrB contribute to Leptospira pathogenesis?

UvrB likely contributes to Leptospira pathogenesis in several ways:

• Maintaining genomic integrity during host-induced stress (immune response, oxidative burst)

• Supporting bacterial persistence through DNA damage repair during infection

• Potentially influencing expression of virulence factors through regulatory networks

• Enabling adaptation to changing environments during the infection process

Studies of the ClpB chaperone demonstrated that stress response systems are essential for Leptospira virulence, with the clpB mutant showing attenuated virulence in animal models . A similar approach could determine UvrB's specific contribution to pathogenesis.

What animal models are appropriate for studying UvrB's role in virulence?

Appropriate animal models for studying UvrB's role in virulence include:

The hamster model was effectively used to demonstrate attenuated virulence of the clpB mutant in acute leptospirosis , making it a strong candidate for parallel studies with UvrB mutants.

How can complementation studies confirm UvrB's role in virulence?

Complementation studies to confirm UvrB's role in virulence should follow a methodology similar to that used for the ClpB protein:

• Create a full-length wild-type uvrB gene construct in an appropriate shuttle vector

• Introduce the construct into the uvrB mutant strain

• Confirm restoration of UvrB expression by Western blotting

• Test for restoration of stress survival phenotypes in vitro

• Evaluate recovery of virulence in animal models

The complementation approach used for the clpB mutant, which involved amplifying the gene, cloning it with flanking restriction sites, and introducing it back into the mutant strain, successfully restored both stress survival and virulence phenotypes . This provides a proven methodology for UvrB studies.

How can CRISPR-Cas9 technology be applied to study UvrB function?

CRISPR-Cas9 technology can be applied to study UvrB function through:

• Precise gene editing to create point mutations in functional domains

• Knock-in of tagged versions of UvrB for localization and interaction studies

• CRISPRi for controlled downregulation of UvrB expression

• Creation of UvrB variants with specific domain deletions

• Multiplex targeting of UvrB and interacting partners

While CRISPR-Cas9 systems are still being optimized for Leptospira, they offer significant advantages over random transposon mutagenesis by enabling precise genetic modifications without polar effects on neighboring genes.

What high-throughput approaches can identify UvrB interaction networks?

To identify UvrB interaction networks, researchers can employ:

• Chromatin immunoprecipitation sequencing (ChIP-seq): To identify DNA binding sites

• RNA-seq of UvrB mutants: To identify genes regulated by UvrB-dependent processes

• Proximity-dependent biotin identification (BioID): For identifying protein interaction partners

• Protein microarrays: To screen for interactions with host proteins

• Chemical crosslinking coupled with mass spectrometry: To capture transient interactions

These approaches could reveal UvrB's role in broader cellular networks beyond the immediate DNA repair functions, similar to how LRR proteins were found to interact with multiple host components .

How does UvrB function compare between pathogenic and saprophytic Leptospira species?

Comparative analysis of UvrB between pathogenic and saprophytic Leptospira species should examine:

• Sequence conservation and divergence in functional domains

• Expression patterns under various stress conditions

• Interaction capabilities with other repair proteins

• Subcellular localization differences

• Contribution to stress survival in different species

This comparative approach would be similar to studies of LRR proteins, which showed significant differences between pathogenic and saprophytic Leptospira strains, with pathogenic strains containing more LRR proteins than saprophytic ones . Such differences in DNA repair systems could contribute to the enhanced survival and virulence of pathogenic Leptospira.

What are the most promising research gaps regarding UvrB in Leptospira?

The most promising research gaps regarding UvrB in Leptospira include:

• Detailed structural analysis of Leptospira UvrB compared to other bacterial species

• Comprehensive mapping of UvrB's role in various stress responses beyond UV damage

• Investigation of potential regulatory functions beyond DNA repair

• Exploration of UvrB as a potential antimicrobial target

• Understanding the evolutionary adaptation of the UvrABC system in different Leptospira species

Addressing these gaps would significantly advance our understanding of how DNA repair systems contribute to Leptospira pathogenesis and environmental persistence.

How can UvrB research contribute to leptospirosis control strategies?

UvrB research can contribute to leptospirosis control strategies through:

• Identification of potential antimicrobial targets in the DNA repair pathway

• Development of attenuated vaccine strains through UvrB modification

• Understanding bacterial persistence mechanisms to prevent chronic infections

• Identifying environmental interventions that target DNA repair-dependent survival

• Diagnostic approaches based on DNA repair protein detection

Similar to findings with the ClpB chaperone, which was shown to be essential for virulence , targeting UvrB could potentially provide novel approaches for controlling this important zoonotic pathogen.

What technological advancements will enhance future UvrB research?

Key technological advancements that will enhance future UvrB research include:

• Cryo-electron microscopy: For high-resolution structural analysis of UvrB complexes

• Single-molecule techniques: To observe UvrB activity in real-time

• Advanced genetic manipulation tools: For more precise genome editing in Leptospira

• Microfluidic systems: For studying UvrB function under controlled stress conditions

• In vivo imaging technologies: To track DNA repair dynamics during infection