Recombinant Borrelia burgdorferi Uncharacterized protein BB_0381 (BB_0381)

What is the Borrelia burgdorferi BB_0381 protein?

BB_0381 is an uncharacterized protein found in Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680), the causative agent of Lyme disease. The protein consists of 474 amino acids with the UniProt accession number P94250. While classified as "uncharacterized," structural analysis suggests potential roles in cellular processes that may contribute to pathogenesis, though specific functions remain to be fully elucidated through experimental validation .

What is the amino acid sequence of BB_0381?

The full amino acid sequence of BB_0381 is:
MNKKMFPKIYYYDQDFIDIYNKSLSWIQDKVILQKVADRGKKDKNYYSENCDYIDQMQACMSSFFLVYSNGEYSSTSAIDKFYQLQEESGAIRARYDNNNAIIDLDENEENIGFPIFAWAEYNLYHKTGNKKRISEVLPILDKYYKWIESKFLKENGLYSIDVNKIFYKNSPRVDAYYPIPDFNSLQVHNAYCISKLADILNDKNLSLEYKKRFFSLKVKINSLMWSEKDGFYYDLDVNENILEIIKTIVGFFPMLSEIPSEDRIERMIFYLKSTNHFGTPNPFPTLSVSEPGFSEDGNGYYGSYVTYMNFFVIKGLEYCGRANIAREFTIRHLYYILDTLMPFNKIKGHIWEAYRPMQEGPAYFDSNKKTYTEKGLICYLALFSISLMIENIIGLTISLPDKTVYWNIPTLEIMGIESLSLKKNQTTIICNKGKRGWEIKMESEKLYYFTINIL-NKKEKTLPIPSGRCSMLLDKL

How is recombinant BB_0381 protein produced for laboratory studies?

Recombinant BB_0381 is typically produced using standard bacterial expression systems, most commonly Escherichia coli, similar to the production of other recombinant Borrelia proteins such as BB0238 . The process involves:

• Gene synthesis or PCR amplification of the bb_0381 gene sequence

• Cloning into an appropriate expression vector with a fusion tag (determined during production process)

• Transformation into competent E. coli cells

• Induction of protein expression using IPTG or similar inducers

• Cell lysis and protein purification via affinity chromatography based on the fusion tag

• Buffer exchange to Tris-based buffer with 50% glycerol for stability

This expression system allows for production of sufficient quantities of the protein for structural and functional analyses.

What are the optimal storage conditions for recombinant BB_0381?

For optimal stability and activity retention, recombinant BB_0381 should be stored at -20°C. For extended storage periods, conservation at -80°C is recommended. Working aliquots should be maintained at 4°C for up to one week. It's important to note that repeated freezing and thawing cycles should be avoided as they can lead to protein degradation and loss of structural integrity . Researchers should consider preparing small working aliquots to minimize freeze-thaw cycles.

What structural domains are predicted in BB_0381?

While the search results don't provide specific information about the domain structure of BB_0381, structural prediction approaches similar to those used for other Borrelia proteins (such as X-ray crystallography and AlphaFold analysis used for BB0238 ) would be applicable. Based on similar proteins studied in Borrelia, BB_0381 may contain:

• Potential protein-protein interaction domains

• Possible regulatory regions

• Structural motifs that might indicate function

Computational analysis using tools like AlphaFold, SWISS-MODEL, or Phyre2 would provide insights into the potential domain organization and structural features of this uncharacterized protein.

What experimental approaches are recommended for functional characterization of BB_0381?

Since BB_0381 is currently uncharacterized, a multi-faceted experimental approach is recommended:

• Structural Analysis:

  • X-ray crystallography to determine 3D structure

  • AlphaFold or similar prediction tools for structural modeling

  • Circular dichroism for secondary structure analysis

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with potential partners

  • Bacterial two-hybrid system

  • Pull-down assays with B. burgdorferi lysates

• Functional Assays:

  • Gene knockout studies using similar approaches to those used for other B. burgdorferi genes

  • Complementation studies to verify phenotypes

  • In vivo expression technology (IVET) to assess expression during infection

  • Mouse infection models to evaluate virulence contributions

• Expression Analysis:

  • qRT-PCR to measure transcription under various conditions

  • Western blotting to assess protein levels

  • Comparative expression analysis in vitro versus in vivo

How should factorial design be implemented for optimizing BB_0381 expression?

When optimizing recombinant BB_0381 expression, a systematic factorial design approach would yield the most reliable results. Based on bioengineering principles:

• Full Factorial Design (for initial screening):

  • Variables: Temperature (20°C, 25°C, 37°C), Inducer concentration (0.1mM, 0.5mM, 1mM IPTG), Media type (LB, TB, 2YT)

  • This yields 27 experimental conditions to identify significant factors

• Box-Behnken Design (for optimization):

  • Once significant factors are identified, apply Box-Behnken design with 3-4 factors and 3 levels each

  • This reduces experimental runs while maintaining statistical power

• Central Composite Design (for response surface generation):

  • Final optimization to determine precise optimal conditions

  • Enables modeling of quadratic effects and interactions between factors

This systematic approach enables efficient optimization while minimizing resource expenditure.

How might BB_0381 contribute to Borrelia burgdorferi pathogenesis?

While the specific function of BB_0381 remains uncharacterized, research patterns in other Borrelia proteins suggest several potential pathogenic mechanisms:

• Immune Evasion: Similar to BB0238, BB_0381 might facilitate evasion of host cellular immunity, potentially contributing to bacterial persistence in the host . This could involve interference with complement activation or inhibition of phagocytosis.

• Protein-Protein Interactions: BB_0381 might interact with other essential borrelial proteins, forming complexes that contribute to virulence, similar to how BB0238 interacts with BB0323 and BB0108 .

• Regulatory Functions: The protein could play a role in gene regulation, potentially under the control of global regulators like RpoS that influence expression of genes during mammalian infection .

• Host Adaptation: BB_0381 might be differentially expressed in tick versus mammalian hosts, potentially helping the bacterium adapt to different environments during its complex life cycle .

Experimental approaches such as gene knockout studies followed by mouse infectivity assays would be necessary to confirm any pathogenic role.

How can phylogenomic approaches be used to identify regulatory elements associated with BB_0381?

Phylogenomic approaches offer powerful insights into regulatory elements controlling BB_0381 expression. Based on methodologies applied to other Borrelia genes:

• Identification of Conserved Intergenic Regions:

  • Search for perfectly conserved intergenic blocks (PCIBs) within 125 nucleotides upstream or downstream of the bb_0381 gene across multiple Borrelia genomes

  • Compare these sequences against randomly permuted intergenic space alignments to identify statistically significant conservation

• Promoter Element Analysis:

  • Analyze sequences that map to regions just upstream of and in the same orientation to the bb_0381 gene

  • Consider the possibility of promoter elements within the gene itself, as found in other bacterial pathogens

• Comparative Analysis:

  • Examine regulatory patterns across the Lyme disease species group (Borrelia burgdorferi sensu lato) using multiple genomes (23 or more for statistical significance)

  • Identify shared regulatory elements that indicate evolutionary conservation of expression control

• Experimental Validation:

  • Confirm predicted regulatory elements through reporter gene assays

  • Perform electrophoretic mobility shift assays to identify protein-DNA interactions

  • Use site-directed mutagenesis to validate functional importance of conserved sequences

This combinatorial approach can reveal previously unrecognized regulatory mechanisms controlling BB_0381 expression during different phases of the Borrelia life cycle.

What are the challenges in determining the structure-function relationship of BB_0381?

Determining structure-function relationships for uncharacterized proteins like BB_0381 presents several significant challenges:

• Structural Determination Barriers:

  • Potential difficulties in protein crystallization

  • Problems with protein solubility and stability

  • Challenges in obtaining sufficient quantities of purified protein

  • Potential flexibility in regions that may resist crystallization

• Functional Annotation Limitations:

  • Lack of identifiable conserved domains with known functions

  • Limited homology to characterized proteins

  • Absence of obvious enzymatic motifs or active sites

  • Potential for novel or Borrelia-specific functions

• Experimental Approach Complications:

  • Challenges in generating viable knockout mutants if the protein is essential

  • Difficulties in establishing appropriate functional assays without functional hypotheses

  • Challenges in recreating in vivo conditions in laboratory settings

  • Limited availability of validated interaction partners to guide functional studies

• Bioinformatic Prediction Constraints:

  • Limitations in prediction accuracy for proteins with no close homologs

  • Challenges in distinguishing between structural similarity and functional similarity

  • Potential for incorrect functional assignment based on partial structural homology

Addressing these challenges requires integrated approaches combining structural biology, molecular genetics, biochemistry, and in vivo infection models.

What methodologies are recommended for studying BB_0381 expression in different host environments?

To effectively study BB_0381 expression across different environments, particularly comparing in vivo vs. in vitro conditions:

• Quantitative RT-PCR Method:

  • Isolate RNA from B. burgdorferi under various conditions (in vitro culture, infected tick tissues, infected mouse tissues)

  • Perform reverse transcription followed by quantitative PCR

  • Use appropriate reference genes (e.g., flaB, recA) for normalization

  • Calculate fold-change in expression compared to a standard condition

• In Vivo Expression Technology (IVET):

  • Apply IVET screening to identify promoters active during mammalian infection

  • Validate BB_0381 expression using reporter systems

  • Compare expression levels between in vivo and in vitro conditions

• RNA-Seq Analysis:

  • Perform transcriptome analysis of B. burgdorferi under different conditions

  • Compare BB_0381 expression patterns relative to other genes

  • Identify co-regulated genes that may provide functional insights

• Western Blot Protocol:

  • Generate specific antibodies against recombinant BB_0381

  • Extract proteins from bacteria grown in different conditions

  • Perform quantitative Western blotting with appropriate loading controls

  • Analyze relative protein abundance across conditions

How can protein-protein interaction partners of BB_0381 be identified and validated?

A comprehensive approach to identifying and validating protein-protein interactions for BB_0381 includes:

• Initial Screening Methods:

  • Bacterial two-hybrid system adapted for Borrelia proteins

  • Yeast two-hybrid screening using BB_0381 as bait

  • Co-immunoprecipitation with anti-BB_0381 antibodies

  • Proximity-dependent biotin identification (BioID)

  • Pull-down assays using purified recombinant BB_0381

• Confirmation Techniques:

  • Biolayer interferometry to measure binding kinetics

  • Surface plasmon resonance to confirm direct interactions

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction analysis

  • Co-localization studies using fluorescent fusion proteins

• Functional Validation:

  • Co-expression and co-purification of interaction partners

  • Mutational analysis of binding interfaces

  • Competition assays with peptides derived from binding regions

  • Evaluation of functional consequences when interactions are disrupted

• Structural Studies of Complexes:

  • X-ray crystallography of co-crystallized proteins

  • Cryo-electron microscopy for larger complexes

  • NMR studies of labeled proteins in complex

  • Cross-linking mass spectrometry to map interaction surfaces

This systematic approach, similar to that used for identifying BB0238 interactions with BB0323 and BB0108 , provides multiple layers of evidence to validate true interaction partners.

How should researchers analyze and interpret contradictory functional data for BB_0381?

When confronted with contradictory data regarding BB_0381 function, researchers should implement a systematic approach to resolution:

• Critical Evaluation of Methodology:

  • Examine differences in experimental conditions between studies

  • Assess genetic backgrounds of bacterial strains used

  • Evaluate completeness of plasmid profiles in mutant strains

  • Consider differences in host systems or models used

• Statistical Reanalysis:

  • Perform power analysis to determine if sample sizes were adequate

  • Re-evaluate statistical methods applied to the data

  • Consider potential confounding variables

  • Implement meta-analysis techniques when multiple datasets exist

• Genetic Verification:

  • Confirm genotypes of all bacterial clones used in studies

  • Validate the presence/absence of all plasmids, as plasmid loss can dramatically affect phenotypes

  • Perform complementation studies to verify that phenotypes are specifically due to BB_0381

• Reconciliation Framework:

  • Consider that contradictory results may reflect different aspects of a multifunctional protein

  • Develop integrated models that accommodate seemingly contradictory observations

  • Design decisive experiments specifically targeting the contradictions

The importance of genetic verification is highlighted by previous research errors, such as the case with ΔbbK46 mutants where phenotypes were initially attributed to gene deletion but later found to be due to plasmid loss .

What statistical approaches are most appropriate for analyzing BB_0381 structural data?

Structural data analysis for BB_0381 requires specialized statistical approaches:

• Crystallographic Data Analysis:

  • R-factor and Rfree evaluation to assess model quality

  • Ramachandran plot analysis to verify protein geometry

  • B-factor analysis to identify flexible regions

  • Validation through MolProbity or similar structure validation tools

• Computational Structure Prediction Assessment:

  • Confidence scores from AlphaFold predictions (pLDDT scores)

  • Template modeling (TM) scores when comparing to known structures

  • Root-mean-square deviation (RMSD) analysis for structural alignments

  • Statistical significance of structural similarities using methods like DALI Z-scores

• Functional Site Prediction:

  • Conservation analysis using Jensen-Shannon divergence

  • Computational prediction of binding sites with statistical significance testing

  • Electrostatic potential analysis with comparison to random models

  • Cavity detection algorithms with statistical assessment of significance

• Molecular Dynamics Analysis:

  • Principal component analysis of simulation trajectories

  • Clustering algorithms to identify predominant conformations

  • Statistical assessment of hydrogen bond networks and salt bridges

  • Correlation analysis to identify allosteric networks

These approaches provide rigorous statistical frameworks for interpreting structural data, similar to the methods that would have been applied in the structural characterization of BB0238 .

What are the key unexplored questions regarding BB_0381 that warrant further investigation?

Several critical areas remain unexplored regarding BB_0381 and merit focused research attention:

• Temporal Expression Patterns:

  • How does BB_0381 expression change throughout the infectious cycle?

  • Is expression regulated by environmental factors such as temperature, pH, or nutrient availability?

  • Does RpoS or other global regulators control BB_0381 expression similar to other virulence factors?

• Structural Biology Questions:

  • What is the three-dimensional structure of BB_0381?

  • Do post-translational modifications affect its function?

  • Are there functionally important conformational changes in different environments?

• Host-Pathogen Interaction:

  • Does BB_0381 interact directly with host molecules?

  • Is it involved in immune evasion mechanisms like other Borrelia proteins?

  • Does it contribute to tissue tropism or dissemination within the host?

• Therapeutic and Diagnostic Potential:

  • Could BB_0381 serve as a target for new antimicrobial strategies?

  • Does it have potential as a diagnostic marker for Lyme disease?

  • Could antibodies against BB_0381 be protective in animal models?

Addressing these questions through methodologies such as in vivo expression technology, structural biology approaches, and host-pathogen interaction studies would significantly advance our understanding of this uncharacterized protein.

How can academic research laboratories best collaborate on BB_0381 characterization?

Effective collaboration on BB_0381 characterization can be structured through the following framework:

• Research Consortium Development:

  • Establish a multi-institutional working group with complementary expertise

  • Implement standardized protocols for protein production and characterization

  • Create a shared repository for bacterial strains, plasmids, and reagents

  • Develop a centralized database for experimental results

• Resource Sharing Mechanisms:

  • Distribute specialized tasks based on institutional strengths

  • Share specialized equipment access through collaborative agreements

  • Implement material transfer agreements optimized for academic collaboration

  • Consider applying for shared research credits through programs like those at UCSD

• Integrated Research Design:

  • Coordinate research objectives to minimize redundancy

  • Design experiments with built-in independent validation

  • Implement regular review of ongoing research through virtual meetings

  • Develop a unified publication strategy with appropriate authorship agreements

• Student and Early-Career Researcher Involvement:

  • Create opportunities for undergraduate research participation

  • Establish exchange programs for graduate students between collaborating labs

  • Develop mentorship structures across institutional boundaries

  • Provide academic credit opportunities through programs similar to BISP 199

This collaborative approach maximizes resource utilization while creating valuable training opportunities for the next generation of researchers.