Borrelia DbpA

What is the molecular structure and function of Borrelia DbpA?

DbpA is a non-glycosylated polypeptide with a calculated molecular mass of 19,916 Dalton, commonly expressed with a -6x His tag at the N-terminus in recombinant forms . Structurally, DbpA contains several key features including a substrate binding pocket for decorin interaction, an exposed flexible loop between α-helix 1 and α-helix 2 (residues 64-81), and a lysine-rich C-terminal tail involved in attachment to glycosaminoglycans . These structural elements are essential for DbpA's primary function: binding to decorin, a proteoglycan found in mammalian extracellular matrix, particularly in joint tissues. This adhesion capability is crucial for B. burgdorferi's colonization of specific host tissues during infection.

How does DbpA contribute to Lyme disease pathogenesis?

DbpA plays a critical role in Lyme disease progression by facilitating tissue colonization and contributing to inflammatory pathology. Studies using mouse models demonstrate that B. burgdorferi strains expressing both DbpA and DbpB cause early and prominent joint swelling, while strains expressing only one adhesin or neither caused negligible joint manifestations . This indicates DbpA's particular importance in arthritis development. The protein's ability to bind decorin allows B. burgdorferi to establish infection in decorin-rich tissues like joints, potentially explaining the arthritis-promoting effects. Additionally, research suggests DbpA may contribute to bacterial persistence after antibiotic treatment, as DbpA-expressing bacteria leave DNA residues in joint tissues even after antibiotic clearance .

How is DbpA expressed during different phases of B. burgdorferi's life cycle?

DbpA expression follows a distinct pattern throughout B. burgdorferi's enzootic cycle. Unlike OspA, which is primarily expressed in the tick vector and downregulated during mammalian infection, DbpA is predominantly expressed during the mammalian phase of infection . This differential expression is evident from studies showing DbpA is not detectable on B. burgdorferi in ticks but becomes upregulated during mammalian infection . Within the mammalian host, DbpA expression shows tissue-specific and temporal variation. Using bioluminescent reporter strains, researchers have observed that DbpA expression varies across tissues, with each tissue displaying a distinct expression pattern . In the heart, for example, bosR (which may influence DbpA regulation) shows peak expression during the first week of infection, while dbpBA expression remains detectable at various timepoints across different tissues .

What techniques are most effective for studying DbpA expression in vivo?

The most effective approach for studying DbpA expression in vivo involves combining multiple complementary techniques. Bioluminescent reporter strains have proven particularly valuable, as they allow real-time in vivo imaging to track spatiotemporal DbpA expression during infection . This non-invasive method can be supplemented with quantitative PCR (qPCR) to measure dbpA transcript levels in harvested tissues. Importantly, fluctuating borrelial load must be monitored and used for normalization when evaluating expression levels, typically by quantifying a constitutively expressed gene like flaB . Immunohistochemistry using DbpA-specific antibodies provides information about protein localization within tissues, while Western blotting allows semi-quantitative protein detection. For detailed analysis of regulatory mechanisms, researchers can employ techniques described in search result , including gene knockouts and complementation studies to investigate transcriptional regulators like RpoS and RpoN.

How should researchers design experiments to evaluate DbpA's role in arthritis development?

When designing experiments to evaluate DbpA's role in arthritis development, researchers should implement a multi-faceted approach. Based on published studies, the following methodology is recommended:

• Use multiple B. burgdorferi strains with varying DbpA expression profiles:

  • Wild-type strains expressing both DbpA and DbpB

  • Strains expressing DbpA alone

  • Strains expressing DbpB alone

  • Strains deficient in both DbpA and DbpB

• Infection and monitoring protocol:

  • Infect mice via needle inoculation with standardized doses

  • Monitor joint swelling regularly (typically using digital calipers)

  • Extend observations up to 15 weeks post-infection to capture both early and late arthritis manifestations

• Analysis parameters:

  • Histopathological assessment of joint tissues

  • Quantification of bacterial load in joints using culture and PCR

  • Immunohistochemical detection of DbpA in joint tissues

  • Analysis of immune responses, including cytokine profiles and cellular infiltration

This experimental design allows for direct comparison of arthritis development between DbpA-expressing and non-expressing strains, helping establish causality rather than mere correlation.

What are the key considerations when producing recombinant DbpA for functional studies?

Production of functional recombinant DbpA requires careful attention to several critical factors. Recombinant DbpA is typically produced in E. coli expression systems with an N-terminal 6x His tag to facilitate purification . The purification process employs proprietary chromatographic techniques, resulting in a sterile filtered clear solution . For optimal stability, the protein should be formulated in an appropriate buffer system - typically 20mM HEPES buffer at pH 8.0 with 20% glycerol . Storage conditions significantly impact protein integrity; for short-term use (2-4 weeks), the protein can be stored at 4°C, while longer storage requires freezing at -20°C . Multiple freeze-thaw cycles should be avoided to prevent degradation . Quality control should include verification of decorin-binding activity, as improper folding may yield protein that lacks functional activity despite appearing pure by standard biochemical criteria. Researchers should also consider the strain-specific sequence variations in DbpA when designing recombinant constructs, as these variations may affect binding properties and immunological responses.

How is DbpA expression regulated at the transcriptional level?

The transcriptional regulation of DbpA involves a complex regulatory network with multiple components. Research indicates that DbpA expression is controlled by a regulatory cascade involving the alternative sigma factors RpoS and RpoN . Experimental evidence suggests that RpoN (σ54) regulates the expression of RpoS, which in turn controls dbpA transcription . This hierarchical regulation is part of B. burgdorferi's adaptive response as it transitions between tick vector and mammalian host environments. The regulatory pathway can be visualized as:

Environmental signals (temperature, pH, nutrients) → RpoN activation → RpoS expression → dbpA transcription

How does DbpA expression vary across different tissues during infection?

DbpA expression demonstrates significant tissue-specific variation during infection, as revealed by in vivo imaging studies using bioluminescent reporter strains . Each tissue exhibits a distinct dbpBA expression pattern, suggesting that B. burgdorferi adapts its gene expression in response to unique tissue microenvironments . In the heart, for example, bosR (which may influence DbpA regulation) shows highest expression during the first week of infection, potentially correlating with the initial colonization phase . While specific expression patterns for each tissue are not fully detailed in the search results, research indicates that dbpBA is "readily detectable at all time points with each tissue displaying a distinct expression pattern" . This spatiotemporal regulation likely reflects the bacterium's adaptation to different immune pressures, nutrient availability, and structural components in various tissues. Understanding these tissue-specific expression patterns is crucial for developing targeted therapeutic approaches and for comprehending the differential pathology observed in various organs during Lyme disease.

Which DbpA epitopes are most immunogenic in humans with Lyme disease?

Serological analysis has identified specific regions of DbpA that elicit strong antibody responses in humans with Lyme disease. Using enzyme-linked immunosorbent assays (ELISA) and multiplex immunoassays (MIA), researchers identified 12 DbpA-derived peptides with significantly elevated antibody reactivity in B. burgdorferi-seropositive sera compared to healthy controls . The two most immunogenic regions were:

• Residues 64-81: This peptide, corresponding to an exposed flexible loop between DbpA's α-helix 1 and α-helix 2, showed the strongest reactivity with >80-fold IgG and 10-fold IgM elevation compared to controls . This sequence is identical between B. burgdorferi strains B31 and 297, and structurally overhangs DbpA's substrate binding pocket.

• C-terminal region: The lysine-rich tail of DbpA also elicited strong antibody responses, with >80-fold IgG and 3-5-fold IgM elevation . This region is implicated in attachment to glycosaminoglycans.

These findings suggest that these highly immunogenic epitopes could be targeted by the immune system to limit B. burgdorferi's ability to attach to and colonize distal tissues during early infection stages .

What are the challenges in developing DbpA-based vaccines for Lyme disease?

Despite DbpA's strong immunogenicity and its role in mammalian infection, several challenges complicate its development as a Lyme disease vaccine. The most significant obstacle is the discrepancy between needle inoculation and tick challenge models. While DbpA immunization protects mice from infection when challenged via needle inoculation with in vitro-cultivated spirochetes, it fails to protect mice infested with B. burgdorferi-infected ticks . This lack of protection correlates with the finding that DbpA is not detectable on B. burgdorferi in ticks, suggesting it's unavailable as an antibody target during tick feeding . This temporal expression pattern creates a fundamental challenge: by the time DbpA is expressed during mammalian infection, the initial transmission has already occurred.

Additional challenges include:

• Strain variation in DbpA sequences, potentially limiting cross-protection against diverse B. burgdorferi strains

• The need to generate functionally blocking antibodies rather than merely binding antibodies

• Potential antigenic competition when combining DbpA with other antigens in multivalent vaccines

These findings suggest that while DbpA alone "may not be suitable as a Lyme disease vaccine," it could potentially contribute to a multivalent approach combining antigens expressed at different stages of the B. burgdorferi life cycle .

How do anti-DbpA antibodies potentially protect against B. burgdorferi dissemination?

Antibodies targeting DbpA may protect against B. burgdorferi dissemination through several mechanisms related to the protein's key functions in bacterial adhesion and tissue colonization. Analysis of serological data suggests that antibodies against specific DbpA epitopes - particularly residues 64-81 (the flexible loop region) and the C-terminal lysine-rich tail - could "limit B. burgdorferi's ability to attach to and colonize distal tissues during the early stages of infection" . This protective effect likely operates through multiple mechanisms:

• Blocking decorin binding: Antibodies targeting the substrate binding pocket region may directly interfere with DbpA's ability to bind decorin, preventing bacterial adherence to tissues rich in this proteoglycan.

• Inhibiting glycosaminoglycan interactions: Antibodies against the C-terminal lysine-rich tail may disrupt interactions with glycosaminoglycans, further limiting tissue colonization.

• Complement-mediated killing: Bound antibodies may facilitate complement fixation and subsequent bacterial lysis.

• Opsonization: Anti-DbpA antibodies could enhance phagocytosis of B. burgdorferi by neutrophils and macrophages.

How does DbpA contribute to post-antibiotic persistence of B. burgdorferi DNA?

Research indicates that DbpA plays a significant role in the persistence of B. burgdorferi DNA following antibiotic treatment. In mouse studies, B. burgdorferi DNA was "detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria" after ceftriaxone treatment, while this was not observed in mice infected with DbpA and B deficient strains . This suggests that the expression of these decorin binding proteins is crucial for the persistence phenomenon. Several potential mechanisms might explain DbpA's contribution:

• Enhanced tissue adhesion: DbpA's binding to decorin may allow B. burgdorferi to establish persistent reservoirs in decorin-rich tissues like joints, potentially protecting bacterial components from complete clearance.

• Protection of bacterial remnants: The strong adhesive properties of DbpA may incorporate bacterial DNA into extracellular matrix structures, shielding it from degradation even after the bacteria are killed.

• Altered immune responses: DbpA-mediated tissue localization might influence local immune responses, potentially affecting the clearance of bacterial components after antibiotic treatment.

Importantly, immunosuppression studies using anti-TNF-alpha in antibiotic-treated mice did not result in bacterial regrowth, suggesting "that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria" .

What methods can distinguish between viable B. burgdorferi and DbpA-containing remnants?

Distinguishing viable B. burgdorferi from DbpA-containing remnants requires a combination of complementary techniques with different specificities:

• Culture methods: Successfully culturing B. burgdorferi from tissues provides definitive evidence of viable bacteria, but has limited sensitivity, especially after antibiotic treatment .

• Molecular detection approaches:

  • Standard PCR detects bacterial DNA but cannot distinguish between viable bacteria and DNA remnants

  • RNA detection, particularly of short-lived mRNAs, can indicate transcriptional activity suggesting viability

  • PCR following treatment with membrane-impermeable DNA intercalating dyes can selectively detect DNA from intact cells

• Functional testing:

  • Immunosuppression studies (e.g., with anti-TNF-alpha) can determine if bacterial regrowth occurs when immune pressure is reduced, indicating viable but non-cultivable forms

  • Transplantation of tissues into naïve animals to test for transmissibility

• Imaging approaches:

  • Immunofluorescence using antibodies against DbpA combined with viability stains

  • FISH (fluorescent in situ hybridization) with DbpA-specific probes and viability indicators

What are the implications of DbpA-mediated persistence for clinical Lyme disease management?

The discovery that DbpA contributes to the persistence of B. burgdorferi DNA in joints after antibiotic treatment has several important implications for clinical Lyme disease management:

• Understanding treatment failures: The persistence of bacterial DNA, particularly in joint tissues, may help explain why some patients continue to experience symptoms after standard antibiotic therapy, especially arthritis-related manifestations.

• Therapeutic targets: DbpA-mediated adhesion represents a potential therapeutic target that could be addressed alongside antibiotics. Inhibitors of DbpA-decorin interactions might enhance bacterial clearance and reduce DNA persistence.

• Biomarker development: Detection of DbpA or DbpA-containing bacterial remnants could serve as biomarkers to monitor treatment efficacy and distinguish between active infection and post-treatment inflammatory responses.

• Immunomodulatory approaches: If persistent bacterial components drive ongoing inflammation through DbpA-specific immune responses, targeted immunomodulatory therapies might benefit patients with persistent symptoms.

• Treatment duration considerations: The persistence phenomenon suggests that treatment strategies focused solely on killing bacteria might be insufficient; longer treatment courses or combination approaches targeting both bacteria and their remnants might be necessary in some cases.

While the mouse study indicates that persistent DNA represents non-viable remnants rather than live bacteria , these remnants may still have immunological and clinical significance that warrants further investigation for optimizing treatment approaches.