Borrelia p66

What is the basic structural organization of Borrelia P66?

P66 is a bifunctional integral outer membrane protein of Borrelia burgdorferi with a complex β-barrel structure. According to PRED-TMBB prediction models, P66 contains 22-24 transmembrane domains forming 11-12 extracellular loops. The protein self-oligomerizes into higher-order complexes in the bacterial outer membrane, forming heptamers or octamers rather than the traditional trimers seen in other bacterial porins. This unusual oligomeric structure creates a pore with an estimated diameter of 1.9 nm and a central constriction of 0.8 nm . Surface-exposed regions have been experimentally confirmed through proteinase K digestion studies, which demonstrate degradation of wild-type P66 while periplasmic proteins remain protected .

How are the functional domains of P66 organized?

P66 contains several critical domains with distinct functions:

• Highly conserved domains at positions near E33 and D303 are essential for proper oligomerization and porin function

• The integrin binding region includes the domain near T187

• Surface-exposed loops contain both conserved and non-conserved domains

• The C-terminal region contains a surface-exposed loop that is recognized by sera from Lyme disease patients
  Experimental studies using c-Myc epitope tag insertions have confirmed surface exposure of domains C-terminal to E33, T187, and E334 . Mutations in conserved domains, particularly near E33 and D303, disrupt both oligomerization and porin function, suggesting these regions are structurally critical .

What techniques are available for studying P66 surface exposure?

Researchers have employed several complementary approaches to characterize P66 surface exposure:

• Proteinase K accessibility assays: This method involves treating intact bacterial cells with increasing concentrations of proteinase K (typically 0, 1, 10, 50 μg/ml), followed by immunoblot analysis. Surface-exposed proteins are degraded, while periplasmic proteins remain protected. Studies show that at 10 μg/ml proteinase K, approximately 97% of P66 is cleaved in B. burgdorferi B31 A3 wild-type, producing a characteristic ~50 kDa degradation product . This approach requires appropriate controls, including monitoring integrity of periplasmic proteins like flagellin to confirm outer membrane integrity.

• Epitope tagging: Insertion of c-Myc epitope tags into specific domains allows assessment of their surface exposure through immunofluorescence microscopy and flow cytometry. This technique has been used to demonstrate surface exposure of regions C-terminal to amino acids E33, T187, and E334 .

• Monoclonal antibody binding: Surface-exposed loops can be identified through binding of monoclonal antibodies to intact bacteria, with epitope mapping to determine precise binding locations .

What genetic approaches facilitate functional studies of P66?

Advanced genetic manipulation techniques for Borrelia include:

• Targeted gene disruption: The p66 gene can be disrupted by inserting antibiotic resistance cassettes (e.g., kanamycin resistance gene) through homologous recombination, creating the Δp66 mutant (p66::kanR) .

• Complementation studies: Reintroducing wild-type p66 on a shuttle vector (e.g., pBSV2G) into Δp66 mutants restores function and confirms phenotype specificity .

• Site-directed mutagenesis: Creating specific amino acid substitutions or insertions allows mapping of functional domains. For example, c-Myc epitope tag insertions at specific sites have identified regions critical for oligomerization and porin function .

• Chromosomal modification: Direct modification of the p66 gene in its native location maintains natural expression levels and regulation, avoiding artifacts from plasmid-based complementation .

How does P66 contribute to Borrelia infection establishment?

P66 plays a critical role in establishing Borrelia infection through multiple mechanisms:

What role does P66 play in Borrelia dissemination?

P66 facilitates bacterial dissemination through the following mechanisms:

• Extravasation: The integrin-binding function of P66 promotes extravasation (movement from blood vessels into tissues), enabling bacterial spread throughout the host .

• Tissue invasion: By binding to integrins expressed on various cell types, P66 may facilitate adhesion to and invasion of different tissues, allowing Borrelia to establish persistent infection in multiple organ systems .

• Maintaining viability during dissemination: The domains critical for porin function (particularly near E33 and D303) are also important for dissemination, suggesting that metabolite exchange through P66 pores may support bacterial survival during dissemination .
  Experimental evidence from murine infection models demonstrates that P66 mutants with disrupted oligomerization and porin function (E33 and D303 mutants) exhibit decreased infectivity and dissemination capabilities compared to wild-type bacteria .

How conserved is P66 across Borrelia species?

Comparative analysis of P66 across Borrelia species reveals:

Which regions of P66 show the greatest interspecies variation?

Analysis of P66 sequences across Borrelia species reveals distinct patterns of conservation and variation:

• Variable surface-exposed loops: The surface-exposed loops show the highest degree of variation between species, both in amino acid sequence and size. These loops vary between 35 and 45 amino acids in length across different Borrelia species .

• Conserved structural elements: Two conserved hydrophobic regions flank the variable surface-exposed loops in all species examined. These likely represent transmembrane domains essential for maintaining the β-barrel structure .

• Functional implications: The high variability in surface-exposed regions suggests these domains are subject to selective pressure, possibly from host immune responses. This variation may contribute to species-specific differences in host range, tissue tropism, or immune evasion strategies .

What experimental approaches can determine the critical in vivo function of P66?

Despite extensive research, the precise function of P66 that makes it essential for infection remains unclear. Several experimental approaches can address this question:

• Domain-specific mutants: Creating targeted mutations in specific functional domains (integrin-binding, porin function) can help dissect which activities are essential for infection. Research has shown that integrin-binding deficient mutants can still establish infection, suggesting other functions are critical .

• Complementation with chimeric proteins: Constructing chimeric proteins combining domains from P66 of different Borrelia species with varying pathogenicity could identify regions responsible for host specificity and virulence .

• Conditional expression systems: Developing regulated expression systems for P66 would allow researchers to determine when during infection P66 is most critical, providing temporal insights into its function .

• In vivo tracking studies: Using fluorescently labeled bacteria with various P66 mutations could reveal specific defects in dissemination, tissue localization, or survival at different infection stages .

• Metabolite supplementation: If porin function is the critical activity, supplementing infection studies with potential metabolites that might normally pass through P66 pores could potentially rescue infectivity of porless mutants .

How might the unusual oligomeric structure of P66 contribute to its function?

P66 forms heptamers or octamers rather than the typical trimers seen in most bacterial porins, raising intriguing questions about structure-function relationships:

• Pore characteristics: The unusual oligomeric structure creates a pore with distinct dimensions (1.9 nm diameter with a 0.8 nm central constriction). Research could explore how these dimensions affect the specificity of molecules that can pass through the pore .

• Structural stability: The oligomeric structure may provide stability in the particularly fluid outer membrane of Borrelia, which contains fewer integral membrane proteins than typical gram-negative bacteria .

• Multifunctional capacity: The larger oligomeric complex might facilitate multiple simultaneous functions (integrin binding and metabolite transport) that wouldn't be possible with a simpler structure .

• Experimental approaches: Techniques such as native PAGE, cross-linking studies, and atomic force microscopy could further characterize the oligomeric structure and stability. Cryo-electron microscopy might eventually reveal the detailed 3D structure of the complex .

How is P66 utilized in Lyme disease diagnostics?

P66 has important applications in Lyme disease diagnostics:

• Serological testing: P66 is one of the diagnostic antigens used for Lyme disease serology. Patient antibodies against P66 develop during infection, making it a valuable marker for diagnostic testing .

• Western blot confirmation: P66 antibodies are among those detected in confirmatory Western blot assays for Lyme disease diagnosis .

• Epitope considerations: The surface-exposed C-terminal region of P66 contains an antigen commonly recognized by sera from Lyme disease patients. This region's variability between Borrelia species must be considered when developing species-specific diagnostic tests .

• Temporal dynamics: P66 is produced during mammalian infection, and antibody responses to it develop as infection progresses. Understanding the timing of this response is important for interpreting diagnostic test results .

What potential exists for P66-targeted therapeutics?

As an essential virulence factor with surface accessibility, P66 presents several potential therapeutic targets: