Recombinant Coxiella burnetii Uncharacterized protein CBU_0041 (CBU_0041), partial

What is Coxiella burnetii and what role does CBU_0041 play in its pathogenesis?

Coxiella burnetii is an intracellular gram-negative bacterium that causes Q fever in humans. This pathogen has a unique intracellular lifestyle involving acid activation of metabolism within a phagolysosome-like compartment called the Coxiella-containing vacuole (CCV). A critical virulence determinant is its Dot/Icm type 4B secretion system (T4BSS), which translocates effector proteins directly into the host cell cytoplasm to manipulate host cell functions and promote bacterial growth. CBU_0041 (CirA) is one of these T4BSS effectors with defined host cell functions, though its specific mechanisms are still being characterized .

What are the basic structural characteristics of CBU_0041?

CBU_0041 (CirA) is a T4BSS substrate classified as a hypothetical protein with a length of 710 amino acids. While its complete three-dimensional structure remains to be determined experimentally, it has been identified as a substrate with defined host cell effector functions. According to translocation efficiency studies, CBU_0041 demonstrates approximately 70% translocation efficiency when tested in Legionella systems, suggesting it is efficiently delivered to host cells during infection .

How is CBU_0041 identified in experimental settings?

In experimental settings, CBU_0041 can be identified and studied using fusion protein approaches. A common technique involves fusing CBU_0041 at its N-terminus with CyaA, a reporter tag that catalyzes the production of cyclic AMP (cAMP) when delivered to the host cell cytoplasm. This allows researchers to quantitatively measure protein translocation into host cells. Using this approach, CBU_0041 has been confirmed as a substrate secreted by C. burnetii through its T4BSS .

What approaches can be used to predict the structure and function of uncharacterized proteins like CBU_0041?

For uncharacterized proteins such as CBU_0041, structure prediction is critical for functional annotation. Contemporary approaches include:

• Multiple sequence alignment to identify conserved domains and reduce alignment gaps

• Bioinformatic tools for 3D structure prediction

• Experimental validation of computational predictions using X-ray crystallography and NMR spectroscopy

These in silico/computational methods have improved significantly with newer statistical bioinformatics tools that better handle the challenges of protein structure prediction . For a large protein like CBU_0041 (710 amino acids), domain-based analysis focusing on functional regions may be more practical than whole-protein structural analysis.

How can researchers study CBU_0041's role in host-pathogen interactions?

Understanding CBU_0041's role in host-pathogen interactions requires multiple complementary approaches:

• Protein-protein interaction studies using co-immunoprecipitation or yeast two-hybrid systems

• Subcellular localization studies using fluorescently-tagged CBU_0041

• Functional assays in infection models using wild-type and mutant variants

• Comparative studies with other characterized T4SS effectors

• Knockout/complementation studies to assess phenotypic effects

These approaches can help determine whether CBU_0041 modulates host cell processes like vesicular trafficking, immune response, or cell death pathways, which are common targets of bacterial effectors .

What is known about CBU_0041's immunogenic potential?

While specific immunogenicity data for CBU_0041 is limited, research indicates that T4SS effectors represent an important class of C. burnetii antigens that can induce CD8+ T-cell responses. In studies of T4SS-related proteins, researchers have identified CD8+ T-cell epitopes by scanning proteins for 9-mer peptides predicted to have high-affinity binding capacity for MHC class I molecules. For CBU_0041 specifically, Table 1 in the research indicates that 6 predicted peptides were synthesized and tested, though their specific immunogenic properties are not detailed in the provided results .

What expression systems are optimal for producing recombinant CBU_0041?

For recombinant expression of C. burnetii proteins like CBU_0041, several expression systems have proven effective:

• E. coli expression systems with appropriate tags (His, GST, MBP) for purification

• Attenuated Listeria monocytogenes as a vaccine vector for immunological studies

• Cell-free expression systems for potentially toxic proteins

When selecting an expression system for CBU_0041, researchers should consider:

• Codon optimization for the host organism

• Appropriate tags for purification and detection

• Protein solubility and proper folding

• Potential toxicity to the expression host

• Required post-translational modifications

For immunological studies specifically, attenuated L. monocytogenes has been successfully used as an antigen-delivery platform to induce robust CD8+ T-cell responses against C. burnetii antigens .

How can researchers identify and validate CD8+ T-cell epitopes in CBU_0041?

The identification and validation of CD8+ T-cell epitopes follows a systematic approach:

• Epitope Prediction: Scan the CBU_0041 sequence for 9-mer peptides predicted to have high-affinity binding capacity for MHC class I molecules (H2 Db and Kb) using consensus approaches on the Immune Epitope Database and Analysis Resource.

• Peptide Synthesis: Synthesize the predicted peptides with high purity (>90%).

• ELISPOT Assay Screening:

  • Challenge mice with C. burnetii or vaccinate with whole-cell vaccine

  • Isolate CD8+ T cells from spleens using microbeads

  • Incubate purified CD8+ T cells with APCs and individual peptides

  • Measure antigen-specific IFN-γ recall responses

  • Calculate stimulation index (SI) by dividing the number of spot-forming cells in peptide-stimulated cells by those in medium-stimulated cells (SI > 2 is considered positive)

• Validation by Intracellular Cytokine Staining:

  • Incubate splenocytes with positive peptides

  • Add Golgistop to prevent cytokine secretion

  • Stain cells with antibodies against CD3e, CD8, and IFN-γ

  • Analyze by flow cytometry to quantify IFN-γ-producing CD8+ T cells

This methodical approach has successfully identified immunodominant epitopes from C. burnetii T4SS effector proteins .

What methods can be used to study the translocation of CBU_0041 into host cells?

Several methodologies are available for studying the translocation of T4SS effectors like CBU_0041:

• CyaA Reporter System:

  • Fusion of CBU_0041 with Bordetella pertussis adenylate cyclase (CyaA)

  • Measurement of cAMP production in host cells as indicator of translocation

  • Quantifiable and sensitive detection of protein delivery to the cytosol

• Fluorescence-Based Approaches:

  • GFP fusion proteins for visualization of localization

  • Split-GFP complementation to detect translocation events

• Biochemical Fractionation:

  • Infection of cells followed by subcellular fractionation

  • Western blot analysis to detect CBU_0041 in cytosolic fractions

• Comparative Analysis in Wild-Type versus ΔicmS Mutant:

  • Assessment of translocation efficiency in the presence or absence of the IcmS chaperone

  • Provides insights into the regulatory mechanisms affecting CBU_0041 secretion

For C. burnetii specifically, researchers have effectively used the CyaA reporter system in THP-1 macrophages infected for 48 hours to demonstrate effector translocation .

What genetic manipulation approaches can be used to study CBU_0041 function?

Several genetic approaches can be employed to elucidate CBU_0041 function:

• Gene Knockout/Deletion:

  • Creation of CBU_0041 deletion mutants in C. burnetii

  • Assessment of mutant phenotypes during infection

  • Complementation studies to confirm phenotype is due to CBU_0041 loss

• Domain Mapping:

  • Creation of truncated variants to identify functional domains

  • Site-directed mutagenesis of conserved residues

  • Analysis of effects on translocation and function

• Heterologous Expression:

  • Expression in model systems like yeast or mammalian cells

  • Assessment of effects on cellular processes

  • Identification of interacting host factors

• Reporter Fusions:

  • Fusion with reporter proteins to track localization and dynamics

  • Split reporter systems to detect protein-protein interactions

These approaches, combined with functional assays measuring bacterial replication, vacuole formation, or host cell responses, can provide comprehensive insights into CBU_0041's role in C. burnetii pathogenesis.

What bioinformatic approaches can predict functional domains in CBU_0041?

To predict functional domains in uncharacterized proteins like CBU_0041, researchers can employ:

• Sequence Homology Analysis:

  • BLAST searches against characterized proteins

  • Multiple sequence alignment with homologs

  • Identification of conserved motifs or domains

• Structural Prediction:

  • Ab initio modeling approaches

  • Homology modeling using related proteins as templates

  • Secondary structure prediction

• Functional Site Prediction:

  • Analysis of potentially functional residues

  • Prediction of protein-protein interaction sites

  • Identification of potential catalytic residues

• Machine Learning Approaches:

  • Neural network-based predictions

  • Support vector machine algorithms for function prediction

The integration of multiple prediction methods typically provides more reliable functional insights than any single approach .

How should researchers design experiments to determine CBU_0041's role in C. burnetii pathogenesis?

A comprehensive experimental design to elucidate CBU_0041's role should include:

• Comparative Phenotypic Analysis:

  • Infection studies using wild-type C. burnetii vs. CBU_0041 mutant

  • Assessment of bacterial replication, CCV formation, and host cell responses

  • Time-course experiments to identify temporal effects

• Host Response Analysis:

  • Transcriptomic profiling of infected vs. uninfected cells

  • Proteomic analysis to identify altered host pathways

  • Cytokine/chemokine profiling to assess immune modulation

• Cellular Localization Studies:

  • Immunofluorescence microscopy to track CBU_0041 within infected cells

  • Co-localization studies with cellular organelle markers

  • Live-cell imaging to monitor dynamics during infection

• Animal Model Studies:

  • Comparison of wild-type and CBU_0041 mutant in mouse models

  • Assessment of bacterial burden, pathology, and immune responses

  • Evaluation of protection in vaccine studies

This multi-faceted approach allows for comprehensive characterization of CBU_0041's role in pathogenesis from molecular to organismal levels.

How can CBU_0041 be incorporated into vaccine development strategies?

CBU_0041, as a T4SS effector, represents a potential target for vaccine development based on several considerations:

• T-Cell Epitope Identification:

  • Identification of CD8+ T-cell epitopes within CBU_0041

  • Screening of epitopes for immunogenicity using ELISPOT and flow cytometry

  • Selection of epitopes with strong IFN-γ responses

• Vaccine Vector Selection:

  • Attenuated Listeria monocytogenes as an antigen-delivery platform

  • Expression of CBU_0041 epitopes in the L. monocytogenes vector

  • Assessment of CD8+ T-cell responses in immunized mice

• Protection Studies:

  • Challenge immunized mice with C. burnetii

  • Measurement of bacterial burden and disease parameters

  • Correlation of protection with specific immune responses

Research has demonstrated that T4SS effectors represent an important class of C. burnetii antigens that can induce CD8+ T-cell responses. Immunization with recombinant L. monocytogenes vaccines expressing C. burnetii T4SS effector epitopes has induced robust CD8+ T-cell responses and conferred measurable protection against C. burnetii infection in mice .

What quality control measures are essential when working with recombinant CBU_0041?

When working with recombinant CBU_0041, researchers should implement several quality control measures:

• Protein Purity Assessment:

  • SDS-PAGE analysis to confirm size and purity

  • Mass spectrometry to verify protein identity

  • Endotoxin testing for preparations used in immunological studies

• Structural Integrity Verification:

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate stability

  • Limited proteolysis to identify structural domains

• Functional Validation:

  • Activity assays based on predicted function

  • Binding studies with potential interaction partners

  • Cellular assays to confirm biological activity

• Batch Consistency:

  • Lot-to-lot comparison of physicochemical properties

  • Standardized production and purification protocols

  • Storage stability testing under various conditions

These measures ensure that experimental outcomes reflect the true properties of CBU_0041 rather than artifacts of protein preparation or handling.

What are the technical challenges in studying CBU_0041 and how can they be addressed?

Researchers face several challenges when studying CBU_0041:

• Protein Size and Solubility:

  • At 710 amino acids, full-length expression may be challenging

  • Solution: Domain-based expression approaches or solubility-enhancing tags

• Biosafety Considerations:

  • C. burnetii is a BSL-3 pathogen requiring specialized facilities

  • Solution: Recombinant approaches in model systems or heterologous expression

• Functional Prediction:

  • Limited homology to characterized proteins complicates functional prediction

  • Solution: Integrative bioinformatic approaches and systematic experimental screening

• Structural Determination:

  • Large size may complicate crystallization or NMR studies

  • Solution: Divide-and-conquer approach focusing on predicted domains

• Host Cell Effects:

  • Potential redundancy with other effectors may mask phenotypes

  • Solution: Combinatorial knockout approaches or overexpression strategies

Addressing these challenges requires integrated approaches combining computational prediction, biochemical characterization, and cellular studies .

What future research directions could advance understanding of CBU_0041?

Future research on CBU_0041 should focus on:

• Comprehensive Structure-Function Analysis:

  • High-resolution structural determination of CBU_0041 domains

  • Structure-guided mutagenesis to identify key functional residues

  • Correlation of structural features with biological activities

• Host Target Identification:

  • Unbiased proteomics approaches to identify interacting partners

  • CRISPR screens to identify host factors required for CBU_0041 function

  • Validation of interactions using biochemical and cellular approaches

• In Vivo Significance:

  • Development of animal models to assess CBU_0041's role in pathogenesis

  • Tissue-specific effects of CBU_0041 during infection

  • Contribution to chronic infection and persistence

• Therapeutic Applications:

  • Exploration of CBU_0041 as a vaccine component or diagnostic marker

  • Development of inhibitors targeting CBU_0041 function

  • Structure-based drug design approaches