Recombinant Treponema denticola Enolase (eno)

What is Treponema denticola Enolase (TdEno) and why is it significant for research?

Treponema denticola Enolase (TdEno) is a highly conserved metabolic enzyme produced by the oral spirochete Treponema denticola, a major periodontal pathogen associated with chronic periodontitis. The significance of TdEno stems from its remarkable homology with human alpha-enolase (ENO1) - it has the highest score among enolases from human-associated bacteria in similarity searches . This molecular mimicry creates an interesting immunological phenomenon where antibodies produced against TdEno can cross-react with the host's own ENO1 protein .

TdEno is approximately 52 kDa in size when expressed with a His tag and shares significant structural similarity with enolases from other species, with 40-90% identity between enolases from different organisms . This conservation across species makes it a valuable target for studying bacterial-host interactions and potential autoimmune mechanisms in periodontal disease.

How is recombinant TdEno typically produced for research purposes?

Recombinant TdEno can be produced through established molecular biology techniques. Based on the methodology described in the literature, the process typically follows these steps:

• Gene amplification: A 1.3 kb gene fragment encoding TdEno is amplified from the genomic DNA of T. denticola (typically strain ATCC 35405) using PCR with specific primers (5ʹ-GGATCCTCTGATATTATTTATATTG-3ʹ and 5ʹ-TTATTTTTTTATCAGGTTTGC-3ʹ) .

• Cloning: The PCR product is first cloned into a TA vector, then subcloned into an expression vector such as pQE-30 using appropriate restriction sites (BamHI and KpnI) .

• Protein expression: The cloned gene is expressed in E. coli (strain SG 13009 has been successfully used) by induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for approximately 4 hours .

• Protein purification: The His-tagged recombinant protein is purified using Ni-NTA affinity chromatography from clear cell lysates, followed by refolding through dialysis against an appropriate refolding buffer .

• Endotoxin removal: Critical for immunological studies, endotoxin is removed from the recombinant protein using Triton X-114 extraction to prevent the capture of LPS-binding antibodies during subsequent assays. The final preparation should contain <0.1 EU per 1 μg of protein as determined by LAL test .

This methodological approach yields functional recombinant TdEno suitable for immunological and biochemical studies.

What is the relationship between TdEno and human alpha-enolase (ENO1)?

The relationship between TdEno and human alpha-enolase (ENO1) is characterized by significant molecular mimicry, which has important immunological implications. Key aspects of this relationship include:

• Structural homology: TdEno has the highest sequence homology with human ENO1 among all enolases from human-associated bacteria . This molecular similarity is the basis for potential cross-reactive immune responses.

• Cross-reactivity: Research has demonstrated strong positive correlations between anti-TdEno and anti-ENO1 antibody levels in both human subjects and animal models. Specifically, affinity-purified anti-TdEno antibodies recognize ENO1 in a dose-dependent manner in dot blot analyses, confirming cross-reactivity between these proteins .

• Autoantibody production: The immune response against TdEno can lead to the production of autoantibodies against ENO1 through molecular mimicry. This is evidenced by observations in mice immunized with TdEno, which developed not only anti-TdEno antibodies but also antibodies against mouse Eno1 (mEno1) .

• Temporal pattern: In animal studies, levels of both anti-TdEno and anti-mEno1 antibodies began to decline 4 weeks after the final immunization boost, suggesting that anti-mEno1 antibodies are byproducts of the anti-TdEno antibody response rather than representing independent autoimmune reactions .

This relationship between bacterial and human enolases may have implications for understanding the links between infection and autoimmunity in various conditions, including periodontitis and rheumatoid arthritis.

How does molecular mimicry between TdEno and ENO1 impact autoimmune responses?

The molecular mimicry between Treponema denticola Enolase (TdEno) and human alpha-enolase (ENO1) creates a complex immunological situation with several noteworthy impacts on autoimmune responses:

• Cross-reactive antibody production: When the immune system mounts a response against TdEno during T. denticola infection, the antibodies produced can cross-react with host ENO1 due to the structural similarities between these proteins. Research has confirmed this through multiple lines of evidence:

  • Strong positive correlation between anti-TdEno and anti-ENO1 antibody levels in human subjects

  • Recognition of ENO1 by affinity-purified anti-TdEno antibodies in dot blot assays

  • Production of both anti-TdEno and anti-mEno1 (mouse ENO1) antibodies in mice immunized with TdEno

• Differential antibody response: Interestingly, immunization of mice with TdEno induced more than ten times higher levels of anti-mEno1 antibodies compared to anti-ENO1 antibodies (p < 0.0001), despite having the same degree of homology . This suggests that factors beyond simple sequence homology influence the cross-reactivity and production of autoantibodies.

• Inflammatory cytokine induction: TdEno immunization increased the expression of TNFα in gingival tissues, indicating that the immune response against TdEno (and potentially the resulting autoantibodies) can promote local inflammation . This may contribute to tissue damage in periodontal disease.

• Clonal selection considerations: The research suggests that during germinal center reactions, binding to antigens and signaling through B cell receptors is essential for clone selection and antibody production, even for autoantibodies . What determines the selection or deselection of B cell clones cross-reactive to autoantigens during antibody responses to bacterial antigens remains to be fully elucidated.

• Limited direct pathogenicity: Despite inducing autoantibodies, TdEno immunization alone did not significantly enhance alveolar bone destruction in mouse models, suggesting that the cross-reactive anti-ENO1 antibodies play a minimal role in the progression of periodontitis in the absence of other factors, such as rheumatoid arthritis .

This molecular mimicry mechanism between TdEno and ENO1 represents a potentially important link between bacterial infection and autoimmunity, though its clinical significance may depend on the presence of other disease-modifying factors.

What experimental models are most effective for studying TdEno's role in periodontitis?

Based on the current research, several experimental models have proven effective for studying TdEno's role in periodontitis, each with specific advantages for addressing different research questions:

• Human cross-sectional studies:

  • These studies compare antibody levels (anti-TdEno and anti-ENO1) between subjects with healthy periodontium and those with chronic periodontitis

  • Advantages: Direct relevance to human disease; can establish correlations between antibody levels and disease severity

  • Limitations: Cannot establish causality; influenced by individual variation and comorbidities

• Mouse immunization models:

  • A well-documented model involves subcutaneous immunization with purified recombinant TdEno

  • Protocol typically includes initial immunization followed by multiple boosters

  • Allows measurement of anti-TdEno and anti-mEno1 (mouse ENO1) antibody production

  • Can be combined with oral gavage of periodontal pathogens (e.g., P. gingivalis) to model periodontal disease

• Combined infection-immunization model:

  • This approach has been effectively used to study the interaction between TdEno immune responses and periodontal disease progression

  • Experimental groups typically include:
    a) Sham control
    b) P. gingivalis oral gavage alone (Pg)
    c) TdEno immunization alone (TdEno)
    d) Combined P. gingivalis oral gavage and TdEno immunization (Pg+TdEno)

  • This model allows assessment of alveolar bone loss, gingival tissue inflammation, and cytokine expression

• In vitro analysis of antibody cross-reactivity:

  • Techniques such as dot blot and ELISA using affinity-purified antibodies have been effective for studying the cross-reactivity between TdEno and ENO1/mEno1

  • These methods allow quantitative assessment of antibody binding and specificity

• Gingival tissue cytokine analysis:

  • Real-time PCR analysis of inflammatory cytokines (TNFα, IL-1β) in gingival tissues from experimental animals

  • Provides insights into local inflammatory responses associated with TdEno immunization

The combination of these models provides a comprehensive approach to studying TdEno's role in periodontitis, from molecular interactions to tissue-level effects and systemic immune responses. The mouse immunization model combined with P. gingivalis oral gavage appears particularly useful for investigating the specific contribution of TdEno to periodontal disease progression.

How does TdEno contribute to inflammatory cytokine production in periodontal tissues?

The contribution of Treponema denticola Enolase (TdEno) to inflammatory cytokine production in periodontal tissues appears to be multifaceted and involves both direct and indirect mechanisms:

What are the methodological challenges in studying the immunological effects of TdEno?

Researchers investigating the immunological effects of TdEno face several significant methodological challenges that can impact experimental outcomes and interpretations:

• Endotoxin contamination issues:

  • Recombinant proteins expressed in E. coli often contain lipopolysaccharide (LPS) contamination, which can independently trigger inflammatory responses

  • Proper endotoxin removal is critical using techniques such as Triton X-114 extraction

  • Verification of endotoxin levels (<0.1 EU per 1 μg protein) using LAL testing is essential to ensure valid immunological data

• Cross-reactivity validation challenges:

  • Demonstrating true cross-reactivity between anti-TdEno antibodies and ENO1 requires rigorous controls

  • Affinity purification of antibodies and dose-dependent binding assays are necessary to confirm specificity

  • Distinguishing between specific cross-reactivity and non-specific binding can be technically demanding

• Temporal dynamics of antibody responses:

  • Anti-TdEno and anti-ENO1/mEno1 antibody levels may fluctuate over time

  • In animal studies, antibody levels began to fall 4 weeks after the last boost

  • Experimental timelines must account for these dynamics to capture relevant immunological events

• Species-specific differences in immune responses:

  • Significant differences exist between human and mouse immune responses to TdEno

  • Immunization of mice with TdEno induced over ten times higher levels of anti-mEno1 antibodies compared to anti-ENO1 antibodies, despite similar homology

  • Translating findings from animal models to human disease requires careful consideration of these species-specific differences

• Multifactorial nature of periodontal disease:

  • Isolating the specific contribution of TdEno-induced immune responses in the context of complex periodontal microbiota is challenging

  • The combined effects of multiple bacterial antigens and host factors complicate interpretation

  • Study designs must account for this complexity through appropriate controls and combination approaches

• Sample size and statistical power considerations:

  • Human studies with relatively small sample sizes may fail to detect significant associations between anti-ENO1 antibody titers and periodontitis severity

  • Larger cohorts may be necessary to achieve statistical significance in cross-sectional human studies

• Causality vs. correlation determination:

  • Cross-sectional studies cannot determine if anti-ENO1 antibodies are the result of bacterial infection or a contributing factor to disease progression

  • Longitudinal studies and interventional approaches in animal models are necessary to establish causality

Addressing these methodological challenges requires careful experimental design, appropriate controls, and integration of multiple experimental approaches to provide a comprehensive understanding of TdEno's immunological effects.

What does current evidence suggest about TdEno's role in the progression of periodontitis?

Current evidence presents a nuanced picture of TdEno's role in periodontitis progression, with several key findings from both human and animal studies:

The current scientific consensus suggests that TdEno contributes to the immunopathology of periodontitis primarily through inflammatory cytokine induction rather than through direct tissue destruction mediated by autoantibodies. Its role appears to be more significant in the context of pre-existing autoimmune conditions, highlighting the complex interplay between periodontal infection and systemic autoimmunity.

How do TdEno-induced autoantibodies compare in different experimental systems?

The production and characteristics of TdEno-induced autoantibodies vary significantly across different experimental systems, providing important insights into the immune mechanisms involved:

These comparative observations across different experimental systems highlight the complex and context-dependent nature of TdEno-induced autoantibody responses. The significant quantitative differences between species and the dependence on systemic immunological context suggest that multiple factors modulate the production and pathogenic potential of these cross-reactive antibodies.

What is the relationship between TdEno research and investigations into rheumatoid arthritis?

The relationship between TdEno research and rheumatoid arthritis (RA) investigations represents an important intersection of periodontal and systemic autoimmune disease research:

This relationship highlights the complex interplay between oral microbial infections and systemic autoimmune conditions, suggesting that TdEno might serve as a molecular link between periodontitis and RA in susceptible individuals. Further research is needed to fully elucidate the mechanisms and clinical significance of this connection.

What are the optimal methods for analyzing TdEno protein-protein interactions with host molecules?

Analyzing protein-protein interactions between TdEno and host molecules requires sophisticated technical approaches to ensure accurate and physiologically relevant results. Based on current research methodologies, several optimal approaches can be recommended:

• Recombinant protein preparation and quality control:

  • Expression of TdEno in suitable systems (typically E. coli) with affinity tags for purification

  • Rigorous quality control including SDS-PAGE verification, endotoxin removal (Triton X-114 extraction), and confirmation of protein folding through functional assays

  • Endotoxin levels should be <0.1 EU per 1 μg protein as determined by LAL test to prevent interference in immunological assays

• Direct binding assays:

  • Dot blot analysis: Simple but effective method for preliminary assessment of protein-protein interactions

  • ELISA-based binding assays: Offers quantitative measurement of binding affinities between TdEno and potential host target proteins

  • Surface Plasmon Resonance (SPR): Provides real-time, label-free analysis of binding kinetics and affinity constants

  • Microscale Thermophoresis (MST): Allows detection of interactions in solution with minimal sample consumption

• Cross-reactivity assessment:

  • Affinity purification of anti-TdEno antibodies using immobilized recombinant TdEno

  • Testing purified antibodies against human ENO1 and other potential cross-reactive targets

  • Dose-response analysis to confirm specificity of cross-reactivity

  • Competition assays to verify binding to shared epitopes

• Structural analysis:

  • X-ray crystallography of TdEno alone and in complex with host target proteins

  • Molecular modeling and docking simulations to predict interaction interfaces

  • Epitope mapping using peptide arrays or hydrogen-deuterium exchange mass spectrometry

  • Site-directed mutagenesis to confirm key interacting residues

• Cell-based interaction studies:

  • Immunofluorescence microscopy to visualize co-localization of TdEno with host proteins

  • Co-immunoprecipitation from cell lysates exposed to TdEno

  • FRET/BRET assays to detect protein-protein interactions in living cells

  • Cell-based reporter assays to assess functional consequences of interactions

• Systems biology approaches:

  • Proteomics analysis of pull-down assays to identify novel interacting partners

  • Transcriptomics to assess host cell responses to TdEno exposure

  • Network analysis to place TdEno-host interactions in broader biological context

• In vivo validation:

  • Animal models with wild-type and mutant TdEno to confirm the physiological relevance of identified interactions

  • Tissue-specific analysis of protein complex formation

  • Correlation of interaction strength with disease parameters

These methodological approaches should be applied in a complementary manner, with initial screening followed by rigorous validation using multiple techniques. The integration of structural, biochemical, and cellular approaches provides the most comprehensive understanding of TdEno interactions with host molecules and their functional consequences.

How can researchers address data inconsistencies in TdEno autoimmunity studies?

Addressing data inconsistencies in TdEno autoimmunity studies requires systematic approaches to identify sources of variation and implement standardized methodologies. Based on current research challenges, the following strategies are recommended:

• Standardization of recombinant protein preparation:

  • Implement consistent expression systems and purification protocols for TdEno

  • Establish quality control benchmarks including purity (>95% by SDS-PAGE), endotoxin levels (<0.1 EU/μg), and functional activity

  • Consider the impact of tags (His, GST, etc.) on protein structure and activity

  • Document and report all production parameters to enable cross-study comparisons

• Experimental design considerations:

  • Use appropriate sample sizes based on power calculations to detect expected effect sizes

  • Include all necessary controls: positive controls, negative controls, and isotype controls for antibody studies

  • Blind experimenters to sample groups when measuring outcomes

  • Pre-register experimental protocols and analysis plans to reduce reporting bias

• Addressing biological variability:

  • Account for genetic variability in human studies through larger cohorts or stratification

  • In mouse studies, use standardized strains and housing conditions

  • Consider sex as a biological variable in both human and animal studies

  • Account for comorbidities and medications in human studies

• Methodological harmonization:

  • Establish standardized ELISA protocols for detecting anti-TdEno and anti-ENO1 antibodies

  • Use calibrated reference sera to enable cross-laboratory comparisons

  • Define consistent cutoff values for antibody positivity

  • Implement reporting standards for methodological details

• Data analysis approaches:

  • Use appropriate statistical tests based on data distribution

  • Account for multiple comparisons in complex datasets

  • Consider both the statistical and biological significance of findings

  • Explore correlations between multiple parameters rather than focusing on single comparisons

• Addressing contradictory findings:

  • When studies report inconsistent results, perform systematic meta-analyses

  • Investigate potential sources of heterogeneity in study populations and methods

  • Consider disease context (e.g., presence or absence of RA) when interpreting data

  • Distinguish between association and causation in human studies

• Reproducibility initiatives:

  • Conduct multi-center validation studies with standardized protocols

  • Share raw data, reagents, and detailed protocols to facilitate independent verification

  • Report both positive and negative findings to address publication bias

  • Consider direct replication studies for critical findings

• Integration of diverse experimental systems:

  • Triangulate evidence from in vitro, animal, and human studies

  • Develop mathematical models to reconcile apparently contradictory data

  • Consider temporal dynamics and disease progression when integrating data

  • Examine dose-response relationships rather than single-dose studies

By implementing these strategies, researchers can address inconsistencies in TdEno autoimmunity studies, leading to more robust and reproducible findings. This approach recognizes that apparent contradictions often reflect biological complexity rather than methodological failures, and seeks to integrate rather than dismiss conflicting data.

What future research directions could advance our understanding of TdEno's role in host-bacterial interactions?

Several promising research directions could significantly advance our understanding of TdEno's role in host-bacterial interactions and potentially lead to translational applications:

• Structural biology approaches:

  • Determine the high-resolution crystal structure of TdEno and compare it with human ENO1

  • Map the specific epitopes involved in cross-reactivity between TdEno and ENO1

  • Identify structural determinants that could be targeted to block pathological cross-reactivity while preserving beneficial immune responses

• Advanced immunological characterization:

  • Analyze the B cell receptor repertoire in response to TdEno immunization using single-cell sequencing

  • Investigate germinal center dynamics during anti-TdEno antibody production

  • Determine what factors influence the selection or deselection of B cell clones cross-reactive to autoantigens during the antibody response to bacterial antigens

  • Characterize T cell responses to TdEno and their role in orchestrating B cell responses

• Microbiome-host interaction studies:

  • Investigate how the oral microbiome composition influences the immune response to TdEno

  • Examine whether other periodontal pathogens modulate the cross-reactivity between TdEno and ENO1

  • Study the impact of microbiome-derived metabolites on TdEno expression and immunogenicity

• Systems biology integration:

  • Employ multi-omics approaches (transcriptomics, proteomics, metabolomics) to comprehensively characterize host responses to TdEno

  • Develop computational models to predict how TdEno-induced responses interact with other immunological pathways

  • Identify biomarker signatures associated with pathological responses to TdEno

• Clinical translational research:

  • Conduct longitudinal studies in humans to determine if anti-TdEno/anti-ENO1 antibodies predict disease progression

  • Stratify patients based on antibody profiles to identify subgroups that might benefit from targeted interventions

  • Develop point-of-care diagnostics for detecting anti-TdEno/anti-ENO1 antibodies in clinical settings

• Novel therapeutic approaches:

  • Design peptide decoys that neutralize cross-reactive antibodies without affecting protective immunity

  • Develop vaccination strategies targeting TdEno-specific epitopes that don't cross-react with human ENO1

  • Explore whether modulating the immune response to TdEno could benefit patients with both periodontitis and autoimmune conditions

• Mechanistic studies at tissue interfaces:

  • Investigate local immune responses to TdEno at the gingival tissue level

  • Examine how TdEno interacts with the vascular endothelium and potentially facilitates systemic spread

  • Study the role of TdEno in biofilm formation and bacterial community dynamics

• Comparative biology perspectives:

  • Compare immune responses to enolases from different oral pathogens

  • Investigate whether similar cross-reactivity mechanisms occur in other host-microbe interactions

  • Examine evolutionary aspects of enolase conservation and how this relates to host immune recognition

These future directions would address critical knowledge gaps while potentially leading to clinical applications. Particularly promising are approaches that integrate structural biology with immunological characterization to delineate the precise molecular mechanisms underlying TdEno-induced cross-reactivity with host ENO1.

What are the key takeaways for researchers planning to work with recombinant TdEno?

For researchers planning to work with recombinant Treponema denticola Enolase (TdEno), several key considerations should guide experimental design and interpretation:

• Protein production and quality control:

  • Use established protocols for cloning the 1.3 kb TdEno gene from T. denticola ATCC 35405 using specific primers

  • Express in E. coli systems (strain SG 13009 with pQE-30 vector has been validated) with IPTG induction

  • Purify using Ni-NTA affinity chromatography and proper refolding protocols

  • Critically, remove endotoxin contamination using Triton X-114 extraction and verify levels are <0.1 EU/μg protein via LAL test

  • Be aware that the His-tagged recombinant protein is approximately 52 kD in SDS-PAGE

• Cross-reactivity considerations:

  • Anticipate and control for cross-reactivity between TdEno and human/mouse enolases in immunological assays

  • Consider that antibodies raised against TdEno will recognize host enolases due to molecular mimicry

  • Include appropriate controls to distinguish specific from non-specific binding

• Temporal dynamics:

  • In animal studies, plan collection timepoints with awareness that antibody levels may begin to decline approximately 4 weeks after immunization

  • Consider longitudinal sampling to capture the full dynamics of immune responses

• Experimental model selection:

  • For studying periodontal disease mechanisms, the combined P. gingivalis oral gavage with TdEno immunization model provides valuable insights

  • For pure immunological studies of cross-reactivity, direct immunization protocols are sufficient

  • Consider that mouse models show stronger anti-self enolase responses than anti-human ENO1 responses

• Outcome measurement recommendations:

  • Assess both antibody responses (anti-TdEno, anti-ENO1/mEno1) and functional outcomes (alveolar bone loss, inflammatory markers)

  • Include TNFα and IL-1β expression analysis in gingival tissues via real-time PCR

  • Consider both local (tissue) and systemic (serum) inflammatory markers

• Interpretation caveats:

  • Recognize that TdEno-induced autoantibodies may play different roles depending on context (e.g., presence of RA)

  • Be cautious about concluding direct causality between antibody levels and disease outcomes

  • Consider that TdEno antibodies may have both protective (against bacterial enolases) and potentially harmful (cross-reactivity) effects

• Technical troubleshooting tips:

  • If experiencing inconsistent antibody detection, optimize ELISA conditions carefully

  • For recalcitrant protein expression, consider codon optimization for E. coli

  • When studying inflammatory effects, ensure endotoxin contamination is not confounding results

By considering these key points, researchers can design more robust experiments, anticipate technical challenges, and appropriately interpret their findings within the broader context of host-bacterial interactions in periodontal disease.

How can TdEno research inform broader studies of molecular mimicry in bacterial pathogenesis?

TdEno research provides a valuable model for understanding molecular mimicry in bacterial pathogenesis that can inform broader studies in this field:

• Methodological framework for establishing molecular mimicry:

  • The TdEno-ENO1 system demonstrates a step-wise approach to confirming molecular mimicry:
    a) Identify sequence/structural homology between bacterial and host proteins
    b) Demonstrate cross-reactivity of antibodies at the protein level
    c) Establish in vivo production of autoantibodies following bacterial antigen exposure
    d) Assess the functional consequences of cross-reactive antibodies

  • This methodological framework can be applied to investigate other potential molecular mimicry systems

• Context-dependent pathogenicity insights:

  • TdEno research reveals that molecular mimicry alone may not be sufficient to cause pathology

  • The finding that anti-ENO1 antibodies are associated with periodontitis severity specifically in RA patients but not in the general population suggests that:
    a) Pre-existing autoimmunity may sensitize to molecular mimicry effects
    b) Multiple "hits" may be required for molecular mimicry to translate into clinical pathology
    c) Genetic and environmental factors likely modify outcomes of molecular mimicry

• Bacterial-host protein homology considerations:

  • Despite TdEno having the highest homology with ENO1 among human-associated bacteria , this alone doesn't predict pathogenicity

  • This suggests researchers should look beyond simple sequence homology when investigating molecular mimicry

  • Conformational epitopes, post-translational modifications, and tissue accessibility of antigens may be equally important

• Immune response complexity:

  • The observation that mice produce more anti-mEno1 than anti-ENO1 antibodies despite similar homology highlights that:
    a) B cell selection in germinal centers involves complex regulation beyond antigen similarity
    b) Species-specific differences in immune tolerance may influence outcomes
    c) B cell receptor signaling strength may determine which cross-reactive clones expand

• Translational implications:

  • The minimal role of TdEno-induced autoantibodies in periodontitis progression in non-RA contexts suggests that:
    a) Therapeutic targeting of molecular mimicry should be context-specific
    b) Biomarkers of molecular mimicry (e.g., autoantibodies) must be interpreted cautiously
    c) Intervention strategies may need to address multiple factors beyond cross-reactive antibodies

• Research design considerations:

  • TdEno studies demonstrate the value of integrating:
    a) Human observational studies to establish clinical relevance
    b) Mechanistic animal models to establish causality
    c) In vitro studies to delineate molecular interactions

  • This multi-level approach should be adopted in other molecular mimicry investigations

• Evolutionary perspectives:

  • The high conservation of enolases across species (40-90% identity) raises questions about: a) Why bacteria maintain proteins with high homology to host counterparts b) How hosts balance immune protection against bacteria with self-tolerance c) Whether molecular mimicry represents an evolutionary adaptation or coincidence