Recombinant Coxiella burnetii UPF0234 protein CBU_0114 (CBU_0114)

What are the optimal expression systems for producing recombinant CBU_0114 protein?

Based on extensive experimental data with C. burnetii proteins, several expression systems have been validated for CBU_0114 production. E. coli BL21(DE3) remains the most commonly used host for initial expression trials due to its high yield and simplicity . For proteins requiring post-translational modifications, eukaryotic systems such as P. pastoris or insect cells may be preferable .

Recommended expression systems for C. burnetii proteins:

For CBU_0114 specifically, expression using pET systems with IPTG induction at 1mM concentration for 4 hours at 37°C has shown consistent results, similar to other successfully expressed C. burnetii proteins .

What purification strategies yield the highest purity of recombinant CBU_0114?

A single-step Ni-NTA affinity chromatography approach can achieve relatively high purity for His-tagged CBU_0114, similar to other C. burnetii recombinant proteins . For higher purity required for structural or interaction studies, a multi-step purification strategy is recommended.

Recommended purification protocol:

• Express CBU_0114 with a C-terminal His₆-tag in E. coli BL21(DE3)

• Harvest cells 4 hours post-induction with 1mM IPTG

• Lyse cells using mechanical disruption in buffer containing 50mM Tris-HCl pH 8.0, 300mM NaCl, 10mM imidazole

• Purify using Ni-NTA chromatography with imidazole gradient elution

• For higher purity, perform size exclusion chromatography using Superdex 75 or 200 columns

This approach has demonstrated >90% purity for similar C. burnetii proteins based on SDS-PAGE analysis .

How can I validate the secretion of CBU_0114 via the Dot/Icm type IV secretion system?

Validation of CBU_0114 as a Dot/Icm T4SS substrate requires multiple complementary approaches. The gold standard involves creating fusion constructs with reporter proteins and demonstrating secretion in both heterologous and native systems.

Methodological approach:

• β-lactamase (BlaM) fusion assay:

  • Clone CBU_0114 into a BlaM fusion vector (e.g., pJB-CAT-BlaM)

  • Transform into L. pneumophila as a heterologous model or directly into C. burnetii

  • Infect host cells (THP-1 or J774A.1 cells) and detect translocation using CCF4-AM substrate

  • Translocation is indicated by substrate cleavage and fluorescence shift from green to blue

• CyaA fusion assay as a complementary method:

  • Clone CBU_0114 into a CyaA fusion vector (e.g., pJB-CAT-CyaA)

  • Measure cAMP production in infected cells as indicator of effector translocation

  • Use icmX-deficient strains as negative controls to confirm T4SS dependency

• Confirmation in C. burnetii:

  • Due to the challenges of genetic manipulation in C. burnetii, validate in both L. pneumophila and C. burnetii systems

  • Use axenic medium (ACCM-2 or ACCM-D) for C. burnetii cultivation

  • Compare translocation in wild-type and icmX-deficient strains

Recent studies have demonstrated that the C-terminal region of C. burnetii effectors contains a secretion signal similar to L. pneumophila effectors, which can be used to predict T4SS substrates .

What structural characteristics of CBU_0114 contribute to its potential function?

While specific structural data for CBU_0114 is limited, computational analyses and comparative studies with other UPF0234 family proteins provide insights into its structural features.

Key structural characteristics:

• Sequence analysis:

  • UPF0234 family proteins typically contain conserved domains that may participate in protein-protein interactions

  • Analysis for potential eukaryotic-like motifs, which are common in C. burnetii effectors

  • Examination of C-terminal region for T4SS translocation signals

• Structural prediction:

  • Secondary structure prediction suggests α-helical regions that may be involved in host protein binding

  • Tertiary structure modeling using homology-based approaches can predict potential binding pockets

  • Molecular dynamics simulations to assess structural stability in different pH environments (relevant for CCV)

• Experimental validation:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Limited proteolysis combined with mass spectrometry to identify stable domains

  • X-ray crystallography or NMR spectroscopy for high-resolution structural determination

Understanding these structural features is essential for predicting potential host targets and designing functional studies.

How can I assess CBU_0114's role in intracellular replication of C. burnetii?

Determining the role of CBU_0114 in C. burnetii pathogenesis requires a systematic approach combining genetic manipulation and cellular assays:

Experimental workflow:

• Generation of CBU_0114 mutant strains:

  • Create knockout mutant using Himar1 transposon mutagenesis or targeted gene deletion

  • Develop complemented strains to confirm phenotype specificity

  • Create overexpression strains to assess gain-of-function effects

• Intracellular replication assessment:

  • Infect multiple cell types (HeLa, THP-1, J774A.1, and iBMDM cells)

  • Monitor bacterial growth kinetics using qPCR targeting IS1111

  • Compare growth curves at days 1, 4, and 7 post-infection

  • Use fluorescence microscopy to assess CCV formation and size

• Coinfection experiments:

  • Perform competitive index assays with wild-type and mutant strains

  • Use fluorescent protein markers to distinguish strains

  • Quantify relative abundance over time using flow cytometry or microscopy

Expected results interpretation:
If CBU_0114 is essential for intracellular replication, the mutant strain will show:

• Reduced bacterial numbers in qPCR quantification

• Smaller CCV formation or altered CCV morphology

• Decreased competitive fitness in coinfection experiments

Recent work with other C. burnetii effectors (e.g., CBU2016) has shown that even non-essential effectors may contribute to specific aspects of infection, such as CCV expansion .

What approaches can determine the host cell targets of CBU_0114?

Identifying host targets of bacterial effectors is crucial for understanding their function in pathogenesis. For CBU_0114, a multi-faceted approach is recommended:

Comprehensive strategy:

• Protein-protein interaction studies:

  • Affinity purification-mass spectrometry (AP-MS) using tagged CBU_0114 expressed in host cells

  • Yeast two-hybrid screening against human cDNA libraries

  • Proximity-dependent biotin identification (BioID) to capture transient interactions

• Subcellular localization:

  • Express EGFP-CBU_0114 fusion proteins in host cells

  • Co-staining with organelle markers (mitochondria, ER, Golgi, lysosomes)

  • Live-cell imaging to track temporal dynamics of localization

• Functional screening:

  • Yeast growth inhibition assays to identify toxic phenotypes

  • siRNA screening of candidate interactors to identify genetic suppressors

  • Phosphoproteomic analysis to detect changes in host signaling pathways

• Validation of interactions:

  • Co-immunoprecipitation with candidate interacting proteins

  • FRET or BRET assays for direct interaction confirmation

  • Functional rescue experiments with interacting protein knockdowns/knockouts

Analytical framework for interpreting results:

What are the most effective methods for characterizing post-translational modifications of recombinant CBU_0114?

Comprehensive characterization of post-translational modifications (PTMs) is essential for understanding protein function, especially for bacterial effectors that may mimic or interfere with host cell processes:

Multi-level PTM characterization strategy:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics for site-specific PTM mapping

  • Top-down proteomics for intact protein analysis

  • Targeted MS/MS for quantification of specific modifications

  • Enrichment strategies for phosphopeptides, glycopeptides, and ubiquitinated peptides

• Biochemical detection methods:

  • ProQ Diamond staining for phosphorylation

  • Periodic acid-Schiff staining for glycosylation

  • Western blotting with PTM-specific antibodies

  • Enzymatic treatments (phosphatases, glycosidases) coupled with mobility shift assays

• Functional relevance assessment:

  • Site-directed mutagenesis of modified residues

  • Comparative analysis of PTMs in prokaryotic vs. eukaryotic expression systems

  • Temporal analysis of PTMs during infection cycle

  • Effects of PTM inhibitors on protein function

Example workflow for phosphorylation analysis of CBU_0114:

• Express and purify recombinant CBU_0114 from both prokaryotic and eukaryotic systems

• Perform in-gel digestion with multiple proteases to maximize sequence coverage

• Enrich phosphopeptides using TiO₂ or IMAC

• Analyze by LC-MS/MS with HCD and ETD fragmentation

• Validate sites by site-directed mutagenesis (S/T/Y to A or D/E)

• Assess functional consequences of mutations on protein-protein interactions or localization

Recent studies have shown that some C. burnetii effectors undergo host-mediated phosphorylation that regulates their activity or stability .

How does CBU_0114 potentially contribute to the formation or maintenance of the Coxiella-containing vacuole (CCV)?

Understanding the role of effector proteins in CCV biogenesis is critical for C. burnetii pathogenesis research. While specific data on CBU_0114 is limited, insights can be drawn from studies of other effectors:

Potential mechanisms for CBU_0114 in CCV biology:

• Membrane trafficking manipulation:

  • Possible interaction with host Rab GTPases or SNARE proteins

  • Modulation of endosome-lysosome fusion kinetics

  • Interference with autophagy machinery recruitment

  • Similar to Cig2/CvpB effector, which manipulates phosphoinositide metabolism

• Lysosomal function modulation:

  • Potential role in adjusting pH regulation within the CCV

  • Modulation of lysosomal enzyme activity

  • Protection against antimicrobial peptides or reactive oxygen species

  • Creation of optimal nutrient environment for bacterial replication

• Structural integrity maintenance:

  • Possible role in cytoskeletal rearrangements around the CCV

  • Contribution to membrane expansion during bacterial replication

  • Similar to CBU2016, which supports CCV expansion without affecting replication

Experimental approaches to test these hypotheses:

• Complementation experiments in CBU_0114 knockout strains with measurement of CCV size

• Fluorescence microscopy with markers for different endolysosomal compartments

• Live-cell imaging of CCV development in cells expressing CBU_0114-GFP

• Biochemical analysis of CCV composition in presence/absence of CBU_0114

What is the evidence for CBU_0114 involvement in immune evasion strategies of C. burnetii?

C. burnetii employs various strategies to modulate host immune responses. The potential role of CBU_0114 in these processes can be investigated through specific experimental approaches:

Potential immune evasion mechanisms:

• Modulation of inflammatory signaling:

  • Potential interference with NF-κB pathway activation

  • Regulation of cytokine production

  • Similar to other effectors that downregulate IL-17 signaling pathway

• Manipulation of cell death pathways:

  • Possible anti-apoptotic activity, similar to AnkG effector

  • Modulation of inflammasome activation

  • Interference with pyroptosis or necroptosis pathways

• Interference with host transcriptional responses:

  • Potential targeting of transcription factors

  • Modification of chromatin structure

  • Similar to NopA effector, which perturbs nuclear import of transcription factors

Experimental framework for immune modulation assessment:

For C. burnetii effectors like AnkG, specific interactions with host pathways (e.g., 7SK snRNP complex) have been documented, suggesting sophisticated mechanisms of immune modulation .

How does CBU_0114 compare to homologous proteins in other bacterial pathogens?

Comparative analysis provides evolutionary context and potential functional insights for CBU_0114:

Comparative genomic approach:

• Homology identification:

  • BLAST and HMM-based searches across bacterial genomes

  • Phylogenetic analysis to determine evolutionary relationships

  • Identification of conserved domains or motifs

  • Comparative study with other intracellular pathogens like Legionella

• Structural comparison:

  • Protein threading against known structures

  • Conservation mapping onto predicted structure

  • Identification of selective pressure on specific regions

  • Analysis of UPF0234 family proteins across species

• Functional inference:

  • Correlation of presence/absence with infection strategies

  • Identification of pathotype-specific variations

  • Integration with pan-genome analysis results

  • Comparison with C. burnetii virulence factors from different isolates

Table of potential homologs in related bacteria:

Pangenomic analysis has revealed that some C. burnetii genes are uniquely preserved across all isolates, suggesting essential roles in pathogenesis, while others show strain-specific variations .

What is the evidence for horizontal gene transfer or convergent evolution in the acquisition of CBU_0114?

Understanding the evolutionary origins of virulence factors provides insights into pathogen adaptation:

Evolutionary analysis framework:

• Genomic context analysis:

  • Examination of flanking regions for mobile genetic elements

  • GC content and codon usage deviation from genome average

  • Presence in genomic islands or regions of genomic plasticity

  • Comparison across C. burnetii isolates from different sources

• Phylogenetic incongruence:

  • Comparison of gene tree vs. species tree

  • Analysis of nucleotide substitution patterns

  • Assessment of selection pressure using dN/dS ratios

  • Dating of acquisition events through molecular clock analysis

• Functional convergence:

  • Identification of structurally similar proteins with low sequence identity

  • Analysis of independently evolved effectors targeting same host pathways

  • Comparison with effectors from distantly related intracellular pathogens

  • Examination of similar motifs acquired through different evolutionary paths

C. burnetii shares phylogenetic relationships with Legionella, Francisella, and Pseudomonas within the γ-Proteobacteria subdivision, which may explain similarities in secretion systems and some effector proteins .

What is the potential of CBU_0114 as a diagnostic biomarker for Q fever?

The utility of C. burnetii proteins as diagnostic markers depends on their immunogenicity and specificity:

Diagnostic potential assessment framework:

• Immunoreactivity evaluation:

  • Screening against sera from acute and chronic Q fever patients

  • Comparison with established seroreactive antigens (e.g., Com1, GroEL, YbgF)

  • Assessment of sensitivity and specificity using ROC curve analysis

  • Similar approaches to those used for evaluating CBU_1718, CBU_0092, and other proteins

• Cross-reactivity testing:

  • Evaluation against sera from patients with other bacterial infections

  • Analysis of conserved epitopes with other bacterial species

  • Development of specific antibody detection assays

  • Consideration of potential false positives from related pathogens

• Clinical validation:

  • Testing with well-characterized patient cohorts

  • Temporal antibody response profiling

  • Correlation with disease stage and severity

  • Combination with other biomarkers for improved diagnostic accuracy

Seroreactivity comparison table based on similar C. burnetii proteins:

While specific data for CBU_0114 is not available, the framework used to evaluate these other C. burnetii proteins would be applicable .

How might studying CBU_0114 contribute to novel therapeutic approaches for Q fever?

Understanding effector protein function can inform therapeutic strategies:

Therapeutic exploitation pathways:

• Target-based drug discovery:

  • Identification of druggable pockets in protein structure

  • Virtual screening for small molecule inhibitors

  • Structure-activity relationship studies

  • Disruption of protein-protein interactions with host targets

• Vaccine development:

  • Evaluation as subunit vaccine component

  • Analysis of protective immune responses

  • Comparison with current vaccine approaches (e.g., Q-Vax)

  • Combination with other immunogenic proteins

  • Consideration of results from previous recombinant protein vaccine trials

• Host-directed therapy:

  • Identification of critical host pathways modulated by CBU_0114

  • Targeting host factors required for effector function

  • Modulation of host immune responses to enhance bacterial clearance

  • Similar to approaches studying C. burnetii interaction with proteasome activity

• Delivery system development:

  • Exploitation of C. burnetii secretion signals for targeted delivery

  • Development of attenuated vaccine strains with modified effector profiles

  • Utilization of bacterial membrane vesicles for drug delivery

  • Leveraging knowledge of phase variation for intervention strategies