Recombinant Borrelia burgdorferi Uncharacterized HIT-like protein BB_0379 (BB_0379)

What is BB_0379 in Borrelia burgdorferi?

BB_0379 is an uncharacterized histidine triad (HIT)-like protein encoded in the genome of Borrelia burgdorferi, the causative agent of Lyme disease. Sequence analysis reveals that BB_0379 belongs to the conserved HIT protein family, characterized by histidine triads that typically form nucleotide-binding motifs. BLAST analysis shows significant sequence homology with HIT-like proteins from diverse species including Mycobacterium tuberculosis and Methanocaldococcus jannaschii, suggesting evolutionary conservation across bacterial domains .

What is the genomic context of BB_0379 in B. burgdorferi?

BB_0379 is located on the main chromosome of B. burgdorferi strain B31. While comprehensive transcriptomic analyses have revealed numerous novel transcripts in B. burgdorferi during various infection stages, BB_0379 has not been specifically highlighted in BbIVET (Borrelia burgdorferi In Vivo Expression Technology) screens that identify genes differentially expressed during mammalian infection . The gene encodes a protein of 139 amino acids in length, consistent with the typically compact size of HIT family proteins .

What structural characteristics define BB_0379?

Based on sequence alignment data, BB_0379 shares approximately 54% identity and 70% positive matches within a 48-amino acid region compared to other HIT-like proteins . The protein likely contains the characteristic nucleotide-binding pocket formed by conserved histidine residues typical of the HIT protein family. The conserved region appears to span amino acids 3-50 based on alignment data, suggesting this region may be critical for the protein's functional activity .

What expression systems are optimal for recombinant BB_0379 production?

For functional and structural studies of BB_0379, researchers should consider multiple expression strategies:

For initial testing, E. coli expression with an N-terminal His6-tag and a TEV protease cleavage site has proven successful for other HIT family proteins. Low-temperature induction (16-18°C) is recommended to enhance protein solubility.

What are the most effective purification strategies for BB_0379?

Purification of recombinant BB_0379 should follow a multi-step approach:

• Initial capture via immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Tag removal using TEV protease (if applicable)

• Ion exchange chromatography (typically anion exchange at pH 8.0)

• Size exclusion chromatography for final polishing

Buffer optimization is critical for HIT proteins. Testing should include various buffers (HEPES, Tris, phosphate) at pH 7.0-8.0 with stabilizing additives such as 5-10% glycerol, 1-5 mM DTT, and 100-250 mM NaCl. Quality control should include SDS-PAGE, mass spectrometry, and circular dichroism to confirm proper folding.

What functional assays can characterize BB_0379 activity?

Since BB_0379 belongs to the HIT protein family, the following functional assays are recommended:

• Nucleotide binding assays:

  • Isothermal titration calorimetry (ITC) with various nucleotides

  • Thermal shift assays to identify stabilizing ligands

  • Fluorescence-based assays using nucleotide analogs

• Enzymatic activity assessment:

  • Phosphoramidite hydrolase activity using colorimetric substrates

  • Nucleotide hydrolysis assays monitored by HPLC

  • Diadenosine tetraphosphate (Ap4A) hydrolysis assay

• Structural studies:

  • X-ray crystallography with and without potential substrates

  • NMR for solution structure and dynamics

  • Hydrogen-deuterium exchange mass spectrometry

How does BB_0379 compare to other HIT-like proteins in bacterial pathogens?

Sequence alignment reveals significant conservation between BB_0379 and HIT-like proteins from diverse bacterial species:

This high degree of conservation across phylogenetically distant bacterial species suggests BB_0379 may serve a fundamental cellular function rather than a Borrelia-specific role .

What is the potential role of BB_0379 in B. burgdorferi pathogenesis?

While BB_0379 has not been directly implicated in virulence, several hypotheses can be formulated based on functions of other HIT proteins:

• Metabolic regulation: HIT proteins often regulate nucleotide metabolism, which could be crucial during different phases of the B. burgdorferi life cycle.

• Stress response: HIT proteins in other bacteria participate in stress response pathways, which may be important for B. burgdorferi survival during host adaptation.

• Signal transduction: Some HIT proteins function in cellular signaling, potentially contributing to the complex gene regulation networks that B. burgdorferi employs during host transition.

Research approaches for testing these hypotheses should include targeted gene deletion studies similar to those employed for other B. burgdorferi genes identified through transcriptomic analyses .

How might BB_0379 function during tick-mammalian transmission?

B. burgdorferi undergoes dramatic gene expression changes during transmission between tick vector and mammalian hosts. While BB_0379 has not been specifically identified in BbIVET screens , its potential role can be investigated using methods developed for studying B. burgdorferi transcripts during the infectious cycle .

The peptidoglycan cell wall of B. burgdorferi contributes to structural integrity and serves as an antigen in Lyme disease patients . If BB_0379 influences cell envelope maintenance or remodeling, it could impact the pathogen's adaptation during host transition. Studies of other B. burgdorferi proteins have demonstrated the importance of cell envelope components for survival in different host environments .

What methods are available for creating BB_0379 mutants?

Several genetic approaches can be employed to study BB_0379 function:

• Allelic exchange mutagenesis: The standard approach for generating B. burgdorferi knockouts uses antibiotic resistance cassettes to replace the target gene.

• Conditional expression systems: For essential genes, tetracycline-inducible promoters can control expression levels.

• Complementation strategies: Trans-complementation using shuttle vectors or chromosomal integration at alternate loci can confirm phenotype specificity.

• Site-directed mutagenesis: To investigate specific domains or residues, particularly the conserved histidine triad region.

The BbIVET system methodology provides a framework for construction of genetic tools in B. burgdorferi, although modifications may be necessary based on the specific requirements of BB_0379 studies .

What phenotypic assays are most informative for a BB_0379 knockout?

A comprehensive phenotypic analysis should include:

• In vitro growth assays:

  • Growth kinetics under various conditions (temperature, pH, nutrient limitation)

  • Morphological assessment via electron microscopy

  • Susceptibility to antimicrobial compounds

• Tick-phase assessment:

  • Acquisition rates by larval ticks

  • Survival during molting

  • Transmission efficiency to naive mice

• Mammalian infection parameters:

  • Tissue burden quantification at various time points

  • Long-term persistence assessment

  • Immune response characterization

Similar approaches have been employed for other B. burgdorferi virulence factors, such as BpiP (BB_0167), which showed decreased mouse colonization and reduced acquisition/transmission by ticks .

How can gene expression regulation of BB_0379 be studied?

To elucidate BB_0379 regulation:

• Promoter mapping:

  • 5' RACE to identify transcriptional start sites

  • Reporter gene fusions to determine promoter activity

  • In vivo imaging to track temporal expression

• Regulatory network identification:

  • ChIP-seq to identify transcription factor binding

  • RNA-seq under various conditions to determine co-regulated genes

  • Proteomics approaches to identify post-translational regulation

• Host signal response:

  • Controlled exposure to host factors

  • Microfluidic systems for single-cell expression analysis

  • Tick feeding studies with reporter strains

Similar approaches have been used in BbIVET studies, which identified DNA sequences capable of promoting gene expression during infection .

What interacting partners might BB_0379 have in B. burgdorferi?

Identifying protein-protein interactions for BB_0379 requires multiple complementary approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Expression of tagged BB_0379 in B. burgdorferi

  • Crosslinking to capture transient interactions

  • Quantitative comparison with control pulldowns

• Bacterial two-hybrid screening:

  • Construction of a B. burgdorferi genomic library

  • Screening for interactions with BB_0379 bait

  • Validation by co-immunoprecipitation

• Computational prediction:

  • Structural modeling to identify interaction interfaces

  • Network analysis based on co-expression data

  • Comparative analysis with known HIT protein interactomes

Based on HIT protein functions in other organisms, potential interacting partners may include nucleotide metabolism enzymes, stress response proteins, or cell envelope components.

How might structural biology approaches inform BB_0379 function?

Structural characterization can provide critical insights:

Structural comparison with characterized HIT proteins could reveal conserved catalytic mechanisms or Borrelia-specific adaptations that inform functional hypotheses.

What is the relationship between BB_0379 and host immune recognition?

Understanding the immunological aspects of BB_0379:

• Antigenicity assessment:

  • Epitope mapping using synthetic peptides

  • Serum reactivity testing from Lyme disease patients

  • Animal immunization studies

• Immune evasion potential:

  • Expression analysis during different infection phases

  • Structural features that may mask immunogenic epitopes

  • Comparison with known immune evasion strategies in B. burgdorferi

• Diagnostic applications:

  • Evaluation as a biomarker for different stages of infection

  • Inclusion in multiplex serological assays

  • Differentiation between active infection and past exposure

Research on other B. burgdorferi proteins has shown that bacterial components like peptidoglycan can serve as persistent antigens in Lyme disease patients , suggesting the importance of studying potential immunological roles of BB_0379.

Current knowledge gaps and future research directions

Despite sequence conservation suggesting important cellular functions, BB_0379 remains largely uncharacterized. Critical knowledge gaps include:

• The enzymatic activity and substrate specificity of BB_0379

• Its expression pattern during the tick-mammal infectious cycle

• Its subcellular localization and potential interaction partners

• Its contribution to B. burgdorferi fitness and pathogenesis