Recombinant Borrelia burgdorferi Uncharacterized protein BB_0063 (BB_0063)

What methods are most effective for recombinant expression and purification of BB_0063?

Recombinant expression of BB_0063 can be achieved using several systems, with E. coli being the most common for initial characterization. Based on studies with other Borrelia proteins, BB_0063 can likely be expressed using approaches similar to those used for BB0238, which was "copiously produced in Escherichia coli" . The expression system selection should consider the following factors:

• Codon optimization for the host organism

• Inclusion of appropriate fusion tags for purification

• Expression conditions that minimize aggregation

For purification, a typical protocol would involve:

• Cell lysis using urea/thiourea buffer (6M urea, 2M thiourea, 50mM ammonium bicarbonate with protease inhibitors)

• Multiple rounds of sonication on ice for complete disruption

• Reduction with 10mM dithiothreitol and alkylation with 20mM iodoacetamide

• Affinity chromatography using the selected fusion tag

• Size exclusion chromatography for final purification

This approach has proven effective for other Borrelia proteins and should be applicable to BB_0063 .

What structural analysis techniques are most appropriate for characterizing BB_0063?

When investigating the structure of uncharacterized proteins like BB_0063, a multi-technique approach yields the most comprehensive results. Based on the successful structural characterization of BB0365, the following methods are recommended:

• X-ray crystallography: This technique provides high-resolution structural data and was successfully used to determine that BB0365 "revealed the same structural fold as that found in the NqrC and RnfG subunits" .

• Computational prediction: AlphaFold analysis, as used for BB0238, can provide valuable structural insights, especially when combined with experimental data. BB0238 was determined to have "a two-domain protein, as we have determined by X-ray crystallography and AlphaFold analysis" .

• Metal binding analysis: If BB_0063 is suspected to bind metal ions (like BB0365 which "coordinated Zn2+ by the His51, His55, His140 residues"), inductively coupled plasma mass spectrometry (ICP-MS) and isothermal titration calorimetry (ITC) should be employed to identify and characterize potential metal binding sites .

• Domain organization analysis: Identification of known motifs such as helix-turn-helix (HTH) domains, which are "known to mediate protein-protein interactions," can provide functional clues .

How can researchers determine if BB_0063 is essential for B. burgdorferi infection?

Determining the essentiality of BB_0063 requires systematic genetic manipulation approaches:

• Create targeted gene deletion mutants using marker-less in-frame deletion methods:

  • Construct deletion plasmids joining "the ~900 to 1000 base pairs long 5′ flanking region of the target gene and its first four codons with its ~900 to 1000 base pairs long 3′ flanking region and its last four codons to the suicide plasmid" .

  • Transform these constructs into highly transformable B. burgdorferi strains like those "lacking lp25 and lp56 [which] demonstrate increased transformability" .

• Confirm proper deletion through:

  • PCR verification

  • Transcript level analysis using qRT-PCR

  • Protein loss verification via Western blotting

• Assess mutant phenotypes in:

  • In vitro growth under various conditions

  • Mouse infection models

  • Tick colonization and transmission studies

If BB_0063 is essential, complete deletion may not be possible, in which case conditional expression systems or point mutations of key residues should be considered, similar to the G41V mutant strategy used for BB0238 .

What proteomics approaches can identify potential interaction partners of BB_0063?

Identifying the interaction network of BB_0063 requires sophisticated proteomics strategies:

• Co-immunoprecipitation coupled with mass spectrometry:

  • Express epitope-tagged BB_0063 in B. burgdorferi

  • Perform pulldown experiments using anti-tag antibodies

  • Identify co-precipitated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Analyze using MaxQuant platform and Perseus software, with proteins "detected in at least five sample runs" considered reliable

• Label-free quantitative (LFQ) analysis:

  • Compare protein abundances between wild-type and BB_0063 mutant strains

  • Use "Student's two-sample t-tests... with a p-value <0.05 and fold-change >2 for abundance changes" to identify significantly affected proteins

  • This approach has successfully identified functional relationships between borrelial proteins, as shown with BB0238 which was found to bind "a second protein, BB0108, a borrelial ortholog of the chaperone protein SurA"

• Cross-linking mass spectrometry:

  • Use chemical cross-linkers to capture transient protein-protein interactions

  • Digest cross-linked complexes and analyze by MS

  • Identify interaction surfaces through computational analysis of cross-linked peptides

This multi-faceted approach maximizes the chance of discovering true interaction partners of BB_0063.

How can researchers investigate the potential role of BB_0063 in host immune evasion?

Investigating potential immune evasion functions requires sophisticated immunological approaches:

• In vivo expression analysis:

  • Use In Vivo Expression Technology (IVET) systems, which have successfully identified virulence factors in B. burgdorferi

  • Construct a BB_0063 promoter fusion with reporter genes like "the promoter-less pncA gene" to monitor expression during infection

  • Compare expression in different tissues and at different time points

• Immune response characterization:

  • Examine the effect of recombinant BB_0063 on:

    • Complement activation

    • Neutrophil chemotaxis

    • Macrophage activation

    • Dendritic cell maturation

  • This is particularly important as other borrelial proteins have demonstrated roles in "microbial immune evasion"

• Persistence studies in immunocompetent vs. immunodeficient mice:

  • Compare infection dynamics of wild-type vs. BB_0063 mutant strains

  • Assess bacterial loads in different tissues

  • Measure antibody responses to various borrelial antigens

  • Look specifically for "the ability of the spirochete to evade the humoral immune response and persistently infect mice"

How can researchers resolve contradictory findings regarding BB_0063 function in the literature?

Resolving contradictions in research findings about BB_0063 requires systematic analysis:

• Context analysis of contradictory claims:

  • Identify and categorize the specific claims that appear contradictory

  • Analyze the experimental context of each claim, as "resolving such contradictions is critical to advancing our understanding of human disease"

  • Examine differences in:

    • Bacterial strains used

    • Experimental conditions

    • Methodological approaches

    • Data analysis techniques

• Automated contradiction detection:

  • Apply text mining approaches to "extract claims from the literature, flagging those that are potentially contradictory"

  • Use normalization of technical terms to address "acronyms and other normalization issues [that] were noted as problems facing automatic detection of contradictory claims"

  • Categorize relations into "excitatory" and other groups to systematically analyze contradictions

• Systematic reconciliation experiments:

  • Design experiments that directly test contradictory claims under identical conditions

  • Include all relevant controls and variables identified from literature analysis

  • Employ multiple orthogonal techniques to verify findings

This systematic approach can help determine whether contradictions arise from genuine biological variability or methodological differences.

What genomic and transcriptomic approaches can reveal the regulation of BB_0063 expression?

Understanding the regulation of BB_0063 requires integrated genomics and transcriptomics:

• Promoter analysis:

  • Identify the BB_0063 promoter region through bioinformatic analysis

  • Create reporter gene fusions to study promoter activity under different conditions

  • Perform systematic promoter mutagenesis to identify key regulatory elements

• Transcriptional profiling:

  • Compare BB_0063 transcript levels across:

    • Different growth phases

    • Environmental conditions mimicking tick vs. mammalian host

    • Various stress conditions

  • Use qRT-PCR for targeted analysis, verifying that "transcript levels are comparable to those of the parental or complemented isolates"

• Transcription factor identification:

  • Perform DNA-protein interaction studies (ChIP-seq, EMSA) to identify regulatory proteins

  • Create mutants of potential regulators and assess effects on BB_0063 expression

  • Look for coordinated expression with other genes, potentially indicating operon structure or co-regulation

• Post-transcriptional regulation:

  • Investigate mRNA stability through actinomycin D chase experiments

  • Examine potential small RNA regulators through RNA-seq

  • Assess potential "post-transcriptional protein loss" mechanisms as observed with BB0238

This comprehensive approach will provide insights into the complex regulation of BB_0063 expression throughout the Borrelia life cycle.

How does the structure-function relationship of BB_0063 compare to other characterized Borrelia proteins?

Understanding the structure-function relationship requires comparative analysis:

• Structural homology analysis:

  • Compare predicted or experimentally determined BB_0063 structure with other characterized Borrelia proteins

  • Look for shared motifs like the "helix-turn-helix motif (HTH)... known to mediate protein-protein interactions" found in BB0238

  • Examine whether BB_0063 has domains similar to BB0365, which shares "the same structural fold as that found in the NqrC and RnfG subunits"

• Functional domain mapping:

  • Create a series of domain deletion and point mutation variants

  • Assess each variant for:

    • Protein stability and folding

    • Interaction with known partners

    • Localization within the bacterium

    • Contribution to virulence phenotypes

• Cross-species comparative analysis:

  • Identify BB_0063 homologs in other Borrelia species including "Borrelia garinii, Borrelia afzelii, Borrelia spielmanii, Borrelia mayonii, and Borrelia bavariensis"

  • Compare sequence conservation patterns to identify potentially critical residues

  • Assess functional complementation across species