Bartonella 26kDa

What is the Bartonella 26kDa protein and what is its significance in research?

The Bartonella 26kDa (P26) protein is an immunodominant antigen expressed during Bartonella infection, particularly identified in Bartonella henselae. It is expressed as a preprotein that undergoes cleavage at a putative peptide cleavage site to form the mature protein. The protein's significance lies in its strong immunoreactivity, making it valuable for diagnostic applications. It has been identified as one of the most important antigens used for the diagnosis of Cat Scratch Disease and other forms of Bartonellosis .

When produced recombinantly in E. coli, it forms a single, non-glycosylated polypeptide chain with a molecular mass of approximately 25kDa. While the precise biological function of P26 remains unknown, its phylogenetic conservation and immunogenicity make it an important target for Bartonella research .

What are the structural characteristics of the Bartonella 26kDa protein?

The Bartonella 26kDa protein is characterized by:

• Molecular weight: ~25kDa (calculated molecular weight of recombinant protein is 25kDa)

• Isoelectric point: pH 7.27

• Structure: Single, non-glycosylated polypeptide chain

• Post-translational processing: Expressed as a preprotein that is cleaved to form the mature protein

• Homology: Has nucleotide identity with orthologs in Bartonella spp, Brucella spp, and several plant-associated bacteria

When analyzing the protein by SDS-PAGE, it typically appears as a distinct band around 25-26kDa, with purity levels >80% achievable through appropriate purification methods .

How does Bartonella 26kDa protein compare to similar proteins in other bacterial species?

Bartonella henselae outer membrane protein p26 shares significant nucleotide identity with orthologs in other Bartonella species, Brucella species, and several plant-associated bacteria . Notably, similar proteins in the Brucellae genus, designated as BP26, have also been described as immunodominant antigens with serodiagnostic potential in infected cattle, sheep, goats, and humans .

This conservation suggests evolutionary importance, though the functional roles may differ between species. The immunological cross-reactivity between these homologous proteins can be both beneficial for broad-spectrum diagnostics and challenging when trying to achieve species-specific detection .

What are the optimal methods for recombinant expression of Bartonella 26kDa protein?

For optimal recombinant expression of Bartonella 26kDa protein:

• Expression system: Escherichia coli is the preferred heterologous expression system, demonstrating good yield and proper folding of the target protein .

• Fusion tags: Incorporating a polyhistidine tag (either N-terminal or C-terminal) facilitates purification and detection. Most commonly, a deca-histidine (10xHis) tag is fused to the N-terminus of the protein .

• Expression construct: Full-length cDNA coding for Bartonella henselae 26kDa protein should be used to ensure complete antigenic properties .

• Induction conditions: While not explicitly stated in the search results, standard IPTG induction protocols for E. coli expression systems are typically employed.

• Expected yield: With optimized conditions, significant yields can be achieved (as evidenced by final concentrations ranging from 4.9 mg/mL to 45.3 mg/mL for similar Bartonella proteins) .

What purification strategies yield the highest purity Bartonella 26kDa protein for research applications?

A two-step purification strategy is recommended for obtaining high-purity Bartonella 26kDa protein:

• Affinity chromatography:

  • Use HisTrap FF columns or similar Ni-NTA based matrices

  • Equilibration buffer: 25 mM HEPES, pH 7.0, 300 mM NaCl, 5% Glycerol, 30 mM Imidazole, 1 mM DTT

  • Elution buffer: Same as equilibration buffer with 500 mM imidazole

• Size exclusion chromatography (polishing step):

  • Buffer: 20 mM HEPES, pH 7.0, 300 mM NaCl, 5% glycerol and 1 mM TCEP

  • This step removes aggregates and impurities that co-purify in the affinity step

Using this approach, purity levels >90% can be achieved as assessed by Coomassie-stained SDS-polyacrylamide gels, with final concentrations of 20-45 mg/mL depending on optimization .

What are the optimal storage conditions to maintain Bartonella 26kDa protein stability and activity?

For optimal stability and preservation of immunological activity, Bartonella 26kDa protein should be stored under the following conditions:

• Short-term storage (2-4 weeks): 4°C in an appropriate buffer

• Long-term storage: -70°C or below, with -20°C acceptable for intermediate timeframes

• Buffer composition:

  • Neutral to slightly alkaline pH (approximately 7.0-8.0)

  • 20% glycerol as a cryoprotective agent

  • Commonly used: 20mM HEPES buffer pH-8.0, 200mM NaCl and 20% Glycerol

• Additional recommendations:

  • Aliquot in 100-200 μL volumes to avoid repeated freeze-thaw cycles

  • Flash freezing in liquid nitrogen prior to -80°C storage enhances stability

  • Multiple freeze-thaw cycles should be strictly avoided as they can compromise protein integrity and activity

How effective is Bartonella 26kDa protein as a diagnostic antigen for Bartonella infections?

The Bartonella 26kDa protein (P26) has demonstrated significant efficacy as a diagnostic antigen, particularly for feline Bartonella infections:

• Sensitivity and specificity:

  • In a validation study, rP26-based capture ELISA correctly identified 33 of 35 (94.3%) bacteremic cats as seropositive

  • 100% of uninfected cats (24/24) were correctly identified as seronegative

• Species specificity:

  • Highly effective for detecting B. henselae and B. clarridgeiae infections

  • Limited utility for B. koehlerae infections, as antisera from B. koehlerae-infected cats showed consistently low reactivity

• Immunoreactivity:

  • P26 binds both IgG- and IgM-type antibodies, allowing for detection of both acute and chronic infections

  • Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae show strong reactivity with rP26

These characteristics make P26 a valuable antigen for serodiagnostic applications, particularly when species-specific diagnosis is not critical or when focusing specifically on B. henselae and B. clarridgeiae infections.

What immunoassay formats have been optimized for Bartonella 26kDa-based diagnostics?

Several immunoassay formats have been developed and optimized for Bartonella 26kDa-based diagnostics:

• Capture ELISA:

  • Effectively detects IgG antibodies against rP26

  • Shows kinetic profiles similar to indirect fluorescence assay (IFA) using whole Bartonella cells

  • Particularly useful for monitoring antibody responses over time in experimental settings

• Immunoblot/Western blot:

  • Provides qualitative confirmation of antibody specificity

  • Useful for characterizing cross-reactivity with other Bartonella species

  • Good correlation with bacteremia status

• Immunodot analyses:

  • Rapid screening method for positive/negative determination

  • Used for functionality testing of recombinant P26

• Indirect fluorescence assay (IFA):

  • While not directly using P26, IFA serves as a reference method for comparison

  • rP26 capture ELISA shows comparable kinetic profiles to IFA for B. henselae and B. clarridgeiae antisera

Each format has advantages in different research and diagnostic contexts, with capture ELISA being particularly valuable for quantitative analysis of antibody responses.

How does the cross-reactivity profile of Bartonella 26kDa protein affect its diagnostic utility?

The cross-reactivity profile of Bartonella 26kDa protein significantly impacts its diagnostic applications:

• Cross-reactivity within Bartonella species:

  • Strong cross-reactivity between B. henselae and B. clarridgeiae antisera

  • Minimal cross-reactivity with B. koehlerae antisera, limiting its utility for detecting this species

• Orthologous proteins in other genera:

  • P26 shares nucleotide identity with orthologs in Brucella spp. and some plant-associated bacteria

  • This may potentially lead to false positives in regions where Brucella infections are endemic

• Diagnostic implications:

  • Excellent for general Bartonella screening, especially for B. henselae and B. clarridgeiae

  • Less suitable for species-specific diagnosis without additional confirmatory tests

  • May require complementary antigens for comprehensive Bartonella diagnostics that include B. koehlerae

• Research applications:

  • The differential reactivity pattern makes P26 valuable for studying species-specific immune responses

  • Useful for investigating the evolution of antigen recognition across Bartonella species

Understanding these cross-reactivity patterns is essential when designing diagnostic strategies and interpreting serological results in both research and clinical settings.

How can Bartonella 26kDa protein be utilized in vaccine development research?

While the search results don't directly address vaccine applications, the immunodominant nature of Bartonella 26kDa protein suggests several research strategies:

• Subunit vaccine candidate:

  • As an immunodominant antigen that elicits strong antibody responses, P26 represents a potential subunit vaccine component

  • Its recombinant production in E. coli is well-established, facilitating vaccine manufacturing

• Adjuvant optimization studies:

  • Research could focus on identifying optimal adjuvant combinations to enhance P26 immunogenicity

  • Different delivery systems (e.g., liposomes, nanoparticles) could be evaluated for improving P26 presentation to the immune system

• Cross-protection assessment:

  • Given its conservation across some Bartonella species, researchers should investigate whether P26-based vaccines confer cross-protection

  • The lack of reactivity with B. koehlerae suggests potential limitations in broad-spectrum protection

• Immune response characterization:

  • Studies should characterize both humoral and cell-mediated responses to P26

  • Identification of protective epitopes within P26 would be valuable for rational vaccine design

• Challenge models:

  • Establishing appropriate animal models to evaluate P26-based vaccine efficacy against Bartonella challenge

  • Monitoring both serological responses and protection against bacteremia would be essential metrics

These approaches would provide a comprehensive evaluation of P26's potential as a vaccine antigen while addressing the current knowledge gaps in this area.

What structural modifications of Bartonella 26kDa protein have been investigated to enhance its immunological properties?

• Epitope mapping and enhancement:

  • Identifying immunodominant epitopes within P26 through techniques like peptide arrays or phage display

  • Engineering variants with optimized epitope presentation or multiple copies of key epitopes

• Fusion protein constructs:

  • Creating chimeric proteins combining P26 with other Bartonella antigens to broaden immunological coverage

  • Fusion with immunological adjuvants (like flagellin or heat-shock proteins) to enhance immunogenicity

• Post-translational modification engineering:

  • While native P26 is non-glycosylated, exploring the effects of targeted glycosylation on stability and immunogenicity

  • Investigating phosphorylation or other modifications that might affect antigen processing and presentation

• Structure-guided modifications:

  • Using structural biology approaches to identify regions amenable to modification without disrupting key epitopes

  • Applying insights from the study of FIC domains in Bartonella effector proteins to potentially enhance P26 functionality

• Stability engineering:

  • Introducing disulfide bonds or other stabilizing elements to enhance shelf-life and thermostability

  • Optimizing formulation conditions based on the established buffer preferences (neutral to slightly alkaline pH with glycerol)

These research avenues would contribute to our understanding of P26 structure-function relationships while potentially yielding variants with enhanced diagnostic or vaccine properties.

How does the Bartonella 26kDa protein interact with the host immune system at the molecular level?

• Antibody recognition:

  • P26 binds both IgG and IgM antibodies in infected hosts

  • Species-specific differences in recognition (strong with B. henselae and B. clarridgeiae antisera, weak with B. koehlerae antisera) suggest structural determinants of antibody binding

• Antigen processing and presentation:

  • As P26 is expressed as a preprotein that undergoes cleavage, research could investigate how this processing affects presentation to the immune system

  • Studies on the cellular uptake and processing pathways would illuminate how P26 epitopes are presented via MHC molecules

• Pattern recognition receptor interactions:

  • Investigation of P26 interactions with innate immune receptors like Toll-like receptors

  • Evaluation of downstream signaling and cytokine production in response to P26 exposure

• Comparative immunology:

  • The differential reactivity across host species (feline vs. human) offers an opportunity to study host-specific immune recognition

  • Research on the evolutionary aspects of P26 recognition could provide insights into host-pathogen co-evolution

• Methodological approaches:

  • Surface plasmon resonance to characterize binding kinetics with antibodies

  • Cryo-EM or X-ray crystallography to determine structural details of P26-antibody complexes

  • Flow cytometry to assess cellular responses to P26 stimulation

  • Cytokine profiling to characterize the immune signature elicited by P26

These investigations would significantly advance our understanding of how P26 contributes to Bartonella pathogenesis and host immune responses.

How does Bartonella 26kDa protein compare to similar proteins in outer membrane vesicles (OMVs)?

Comparative analysis of Bartonella 26kDa protein with proteins in outer membrane vesicles (OMVs) reveals several interesting relationships:

• Protein profile similarities:

  • OMVs from B. melitensis exhibit major protein bands at approximately 20 and 23 kDa, which are in a similar molecular weight range as the 26kDa protein

  • These proteins may share structural or functional characteristics with Bartonella 26kDa protein

• Core OMV proteome components:

  • The core proteome of Brucella OMVs contains several key proteins including Omp31, Omp25, GroL, and Omp16

  • While not directly stated, the Bartonella 26kDa protein likely belongs to a similar class of outer membrane proteins that are commonly incorporated into OMVs

• Detergent sensitivity differences:

  • OMVs from smooth strains (with complete LPS) show greater resistance to detergent and enzyme treatments compared to rough strains

  • This suggests that the native environment of membrane proteins like P26 significantly affects their stability and accessibility

• Immunomodulatory potential:

  • OMVs containing outer membrane proteins can induce cytokine production (TNFα and IL-6) and cytoskeletal rearrangements

  • The presence of proteins like P26 in OMVs may contribute to these immunomodulatory effects during infection

Understanding these relationships provides insights into how P26 might function in its native membrane environment and how it might contribute to Bartonella pathogenesis through OMV-mediated processes.

What functional differences exist between Bartonella 26kDa protein and its orthologs in Brucella species?

The functional differences between Bartonella 26kDa protein and its Brucella orthologs (BP26) represent an important area of comparative research:

• Immunological profiles:

  • Both proteins are immunodominant in their respective host-pathogen systems

  • Brucella BP26 has demonstrated serodiagnostic potential in cattle, sheep, goats, and humans

  • Bartonella P26 shows strong reactivity with feline antisera, particularly from B. henselae and B. clarridgeiae infections

• Evolutionary relationship:

  • The proteins share significant nucleotide identity, suggesting a common ancestral origin

  • Divergent evolution has likely optimized each protein for its specific pathogen's lifestyle and host range

• Structural differences:

  • While detailed structural comparisons are not provided in the search results, differences in epitope presentation likely account for the species-specific serological responses

  • These structural variations could be targeted for species-specific diagnostic development

• Research approaches to investigate differences:

  • Comparative genomics to identify sequence divergence patterns

  • Recombinant expression of both proteins followed by head-to-head immunological characterization

  • Cross-absorption studies to identify unique and shared epitopes

  • Crystal structure determination to map structural differences

• Diagnostic implications:

  • Understanding these differences is crucial for developing tests that can differentiate between Bartonella and Brucella infections

  • The shared antigenic determinants may be exploited for broader diagnostic coverage in regions where both pathogens are endemic

This comparative analysis helps researchers better understand pathogen evolution and develop more precise diagnostic tools.

How do the expression patterns of Bartonella 26kDa protein vary across different Bartonella species and strains?

The expression patterns of 26kDa protein across different Bartonella species and strains show notable variations:

• Species-specific expression:

  • The protein is well-characterized in B. henselae, where it serves as an immunodominant antigen

  • B. clarridgeiae appears to express a similar immunoreactive protein, as evidenced by cross-reactivity in serological tests

  • B. koehlerae likely expresses a more divergent form, given the limited cross-reactivity observed with antisera from B. koehlerae-infected cats

• Expression conditions:

  • P26 is expressed during natural infection, making it valuable for serodiagnosis

  • The protein is likely expressed under conditions relevant to host colonization, though specific regulatory mechanisms are not detailed in the search results

• Genomic basis for variation:

  • Comparative genomic studies of Bartonella species have revealed both a core genome (>900 genes common to all species) and species-specific genomic elements

  • These genomic differences likely contribute to variations in P26 expression patterns and antigenic properties

• Research methodologies to study expression:

  • Quantitative PCR to measure gene expression levels across species/strains

  • Western blotting with species-specific antisera to detect protein expression

  • Mass spectrometry to identify and quantify P26 variants

  • Reporter gene fusions to study regulatory elements controlling expression

• Diagnostic implications:

  • Understanding these expression patterns is crucial for designing comprehensive diagnostic tests

  • Multiplexed assays incorporating antigens from different species may be necessary for broad-spectrum detection

This variability in expression patterns provides insights into the evolutionary adaptations of different Bartonella species and informs the development of more comprehensive diagnostic approaches.

What are the most significant knowledge gaps in our understanding of Bartonella 26kDa protein?

Despite considerable research on Bartonella 26kDa protein, several significant knowledge gaps remain:

• Biological function:

  • The natural function of P26 in Bartonella biology remains unknown

  • Its role in bacterial physiology, host cell interaction, or virulence has not been clearly established

• Structural details:

  • High-resolution crystal or cryo-EM structures are not available in the literature

  • Detailed epitope mapping and structural analysis would enhance our understanding of immunogenicity

• Host-pathogen interactions:

  • The specific interactions between P26 and host cellular components remain poorly characterized

  • Whether P26 contributes to immune evasion or modulation is unclear

• Regulation of expression:

  • The regulatory mechanisms controlling P26 expression during different stages of infection are not well defined

  • Environmental signals that modulate its expression have not been systematically investigated

• Evolutionary history:

  • While orthologs exist in related bacteria, the evolutionary trajectory of P26 across the Bartonella genus and related alpha-proteobacteria requires further exploration

Addressing these knowledge gaps would significantly advance our understanding of Bartonella pathogenesis and potentially open new avenues for diagnostic and therapeutic interventions.

What emerging technologies could accelerate research on Bartonella 26kDa protein?

Several emerging technologies show promise for advancing Bartonella 26kDa protein research:

• AlphaFold and other AI-based structure prediction:

  • Machine learning approaches can predict protein structures with increasing accuracy

  • These could provide structural insights into P26 and guide experimental studies

• CRISPR-Cas9 genome editing:

  • Precise genetic manipulation of Bartonella to create P26 knockouts or variants

  • Introduction of tagged versions for in vivo tracking during infection

• Single-cell proteomics:

  • Characterizing P26 expression at the single-cell level during infection

  • Identifying heterogeneity in expression across bacterial populations

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize P26 localization in bacterial cells

  • Correlative light and electron microscopy to study P26 in the context of host-pathogen interactions

• High-throughput epitope mapping:

  • Peptide arrays and phage display libraries to comprehensively map immunogenic regions

  • Systems serology to characterize multidimensional antibody responses to P26

• Recombinant antibody technologies:

  • Development of high-affinity single-domain antibodies for detection and functional studies

  • Antibody engineering to create improved diagnostic reagents

These technologies could overcome current technical limitations and accelerate progress in understanding P26's role in Bartonella biology and pathogenesis.

How might research on Bartonella 26kDa protein contribute to broader understanding of bacterial pathogenesis?

Research on Bartonella 26kDa protein has significant implications for our broader understanding of bacterial pathogenesis:

• Evolutionary insights:

  • The conservation of P26 across Bartonella species and its homology with proteins in Brucella provides a window into the evolution of alpha-proteobacterial pathogens

  • Understanding how these proteins diverged while maintaining immunodominance could reveal fundamental principles of pathogen evolution

• Host adaptation mechanisms:

  • The differential reactivity of P26 with antisera from different host species offers insights into host-specific adaptation

  • This could illuminate how pathogens evolve to occupy different ecological niches

• Diagnostic paradigms:

  • The successful use of P26 in serodiagnosis provides a model for identifying and validating immunodominant antigens in other bacterial pathogens

  • The cross-reactivity patterns inform broader strategies for developing species-specific diagnostics

• Immune evasion strategies:

  • Understanding how immunodominant antigens like P26 persist despite strong antibody responses could reveal immune evasion mechanisms

  • This may have parallels in other stealth pathogens that establish persistent infections

• Vaccine development principles:

  • Lessons from P26 research could inform rational antigen selection for vaccines against other intracellular pathogens

  • The balance between conservation and variation in P26 illustrates key challenges in bacterial vaccine development