Recombinant Coxiella burnetii Putative ankyrin repeat protein CBU_0781 (CBU_0781)

What is CBU_0781 (AnkG) in Coxiella burnetii?

CBU_0781, also known as AnkG, is a type IV secretion system (T4SS) effector protein produced by Coxiella burnetii, the causative agent of Q fever. It functions as an anti-apoptotic factor that helps ensure host cell survival during infection. The protein contains ankyrin repeat domains, which are structural motifs that typically mediate protein-protein interactions. AnkG has been experimentally verified as a substrate of the Dot/Icm secretion system that C. burnetii uses to inject virulence factors into host cells . Research has demonstrated that AnkG is essential for efficient intracellular replication of C. burnetii, highlighting its importance in the pathogen's lifecycle .

What molecular interactions has CBU_0781 been shown to form?

AnkG (CBU_0781) has been demonstrated to bind to multiple host cell components through co-immunoprecipitation and RNA-immunoprecipitation studies. Specifically, AnkG binds to:

• The host cell DExD box RNA helicase 21 (DDX21)

• The host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex

• Several host cell RNAs, including the regulatory 7SK RNA

These interactions are functionally significant as the 7SK snRNP complex regulates the positive transcription elongation factor b (P-TEFb). The binding of AnkG to DDX21 appears to be mediated by salt bridges and occurs independently of its binding to the 7SK snRNP complex, suggesting multiple distinct molecular mechanisms at work .

Why is CBU_0781 considered significant in C. burnetii pathogenesis?

CBU_0781 (AnkG) is significant in C. burnetii pathogenesis for several key reasons:

• It interferes with host cell 7SK snRNP complex function, leading to significant changes in host cell transcription

• It ensures host cell survival by inhibiting apoptosis

• Its activity is essential for efficient intracellular replication of C. burnetii

• It represents one of the approximately 150 virulence factors that C. burnetii injects into host cells via its T4SS

This multifunctional role makes AnkG a critical component in the pathogen's ability to establish and maintain infection within host cells . Furthermore, as an immunoreactive protein, AnkG has potential applications in serodiagnostic tests for Q fever .

How was CBU_0781 identified as a T4SS effector protein?

CBU_0781 (AnkG) was identified as a T4SS effector through several complementary approaches:

• Bioinformatic analysis: The presence of eukaryotic-like domains, specifically ankyrin repeat domains, which are uncommon in prokaryotes but common in proteins that interact with eukaryotic systems

• PmrA response element identification: Researchers examined the promoter regions of C. burnetii genes for PmrA consensus sequences (TTAA-N6-TTAA), which regulate many T4SS components and substrates. Several ankyrin repeat domain-containing proteins, including AnkB, AnkJ, and AnkM, were found to contain similar PmrA responding elements .

• Experimental verification: Direct experimental evidence demonstrating the Dot/Icm-dependent translocation of AnkG from the bacterial cell into the host cytosol. This was achieved using a shuttle vector system developed for C. burnetii genetic manipulation .

The identification process illustrates how combining bioinformatic predictions with experimental validation can successfully identify bacterial effector proteins.

What expression systems have been used for studying recombinant CBU_0781?

Researchers have employed several expression systems to study recombinant CBU_0781, with methodologies adapted for both in vitro and in vivo analyses:

The development of the pKM230 shuttle vector, derived from RSF1010, represented a significant advance as it enabled the stable expression of recombinant proteins directly in C. burnetii. This system includes an mCherry fluorescent marker that allows visualization of transformed bacteria approximately 3-4 weeks after infection of mammalian cells . The successful rescue of this shuttle vector by transformation of E. coli with total C. burnetii DNA from infected cells has confirmed its stability and utility for genetic studies of C. burnetii .

What structural features characterize the CBU_0781 protein?

CBU_0781 (AnkG) contains several key structural features that contribute to its function as a bacterial effector protein:

• Ankyrin repeat domains: These 33-residue motifs form helix-turn-helix structures that mediate protein-protein interactions. The presence of these eukaryotic-like domains is characteristic of many bacterial effectors that interact with host proteins .

• Protein-protein interaction motifs: Beyond the ankyrin repeats, AnkG likely contains additional structural elements that enable its specific interactions with host factors such as DDX21.

• RNA-binding regions: The ability of AnkG to bind to the 7SK RNA and potentially other host RNAs suggests the presence of RNA-binding domains within its structure .

• Secretion signal: Although not specifically described in the search results, T4SS effectors typically contain C-terminal signals that target them for secretion through the Dot/Icm system.

The detailed three-dimensional structure of AnkG has not been fully characterized, but understanding these structural elements is crucial for elucidating the molecular mechanisms underlying its various functions.

How does CBU_0781 manipulate host cell transcription through 7SK snRNP interaction?

CBU_0781 (AnkG) exerts a sophisticated effect on host cell transcription through its interaction with the 7SK small nuclear ribonucleoprotein (snRNP) complex. This interaction represents a critical mechanism by which C. burnetii modifies the host cell environment to promote its own survival and replication.

The 7SK snRNP complex normally functions as an important regulator of the positive transcription elongation factor b (P-TEFb) . P-TEFb is responsible for phosphorylating RNA polymerase II, thereby facilitating the transition from transcription initiation to elongation for many genes. By targeting this regulatory complex, AnkG can potentially influence the expression of numerous host genes simultaneously.

The molecular mechanism appears to involve:

• Direct binding of AnkG to the 7SK RNA component of the snRNP complex

• Interference with the normal regulatory function of the 7SK snRNP

• Alteration of P-TEFb activity, leading to changes in transcriptional elongation

• Resulting shifts in host cell gene expression patterns that favor bacterial survival

Research has demonstrated that this manipulation of host transcription by AnkG leads to significant changes in host cell gene expression and is essential for ensuring host cell survival during infection . This represents a sophisticated example of how bacterial pathogens can hijack fundamental host cell processes to create a favorable environment for their replication.

What is the relationship between the anti-apoptotic activity of CBU_0781 and its molecular interactions?

The anti-apoptotic activity of CBU_0781 (AnkG) appears to be intricately linked to its molecular interactions with host cell components, particularly the 7SK snRNP complex and DDX21. This relationship illuminates how a bacterial effector can prevent host cell death through sophisticated manipulation of cellular machinery.

The current understanding suggests several potential mechanisms:

• Transcriptional regulation via 7SK snRNP: By interfering with the function of the 7SK snRNP complex, AnkG may alter the expression of pro- and anti-apoptotic genes, shifting the balance toward cell survival .

• DDX21 modulation: The interaction with DDX21, a DExD box RNA helicase, may affect RNA processing pathways that influence cell death decisions. This interaction appears to be mediated by salt bridges and occurs independently of AnkG's binding to 7SK snRNP .

• Integration of multiple signals: The ability of AnkG to bind both protein factors (DDX21) and RNA complexes (7SK snRNP) suggests it may function at the intersection of different cellular pathways to comprehensively block apoptotic signaling.

Research has established that AnkG's anti-apoptotic activity is essential for efficient intracellular replication of C. burnetii . This highlights how preventing host cell death is a critical aspect of the pathogen's survival strategy, ensuring that the intracellular niche remains intact throughout the bacterial replication cycle.

How do PmrA response elements regulate the expression of CBU_0781?

The regulation of CBU_0781 (AnkG) expression through PmrA response elements represents an important mechanism by which C. burnetii coordinates the production of its T4SS effectors. PmrA is a response regulator that controls the expression of numerous genes, including those encoding components and substrates of the Dot/Icm secretion system.

The PmrA-mediated regulation involves several elements:

• PmrA consensus sequences: The canonical PmrA response element consists of the motif TTAA-N6-TTAA in the promoter region of target genes. Researchers examining the promoter regions of C. burnetii T4SS-related genes identified similar elements in several ankyrin repeat domain-containing proteins, including AnkB, AnkJ, and AnkM .

• Variations in the consensus sequence: Interestingly, the putative PmrA responding elements upstream of some ankyrin proteins (ankJ and ankM) showed variations from the strict consensus, with one nucleotide differing from the expected sequence. This observation aligns with the understanding that PmrA responding elements do not have strict sequence requirements in certain positions .

• Genome-wide regulation: Using a PmrA-binding motif model constructed from sequence information of verified PmrA-responsive genes, researchers identified 126 C. burnetii open reading frames potentially regulated by this system .

This regulatory mechanism allows C. burnetii to coordinate the expression of multiple virulence factors, ensuring they are produced at appropriate times during infection. The presence of PmrA response elements in the promoters of various ankyrin repeat-containing proteins, likely including AnkG, suggests these effectors form part of a coordinated virulence program controlled by this regulatory system.

What methodologies are effective for studying CBU_0781 translocation into host cells?

Studying the translocation of CBU_0781 (AnkG) from C. burnetii into host cells requires specialized methodologies that can detect this protein transfer event. Based on the research approaches described in the literature, several effective techniques include:

• Fluorescent protein fusion systems: Developing constructs where AnkG is fused to fluorescent reporter proteins (such as mCherry) allows visualization of its localization during infection. The shuttle vector pKM230, which includes an mCherry marker, has been successfully used for this purpose .

• Immunofluorescence microscopy: Using antibodies specific to AnkG to detect its presence in host cells after infection can provide evidence of translocation.

• Fractionation studies: Biochemical separation of host and bacterial components followed by Western blotting can detect AnkG in host cell fractions.

• β-lactamase (TEM) fusion assays: This approach involves fusing AnkG to TEM β-lactamase, which can cleave a fluorescent substrate in the host cytosol, allowing quantitative measurement of translocation efficiency.

• Genetic complementation: The ability of C. burnetii dot/icm genes to complement corresponding L. pneumophila mutants has been demonstrated, suggesting a functional conservation that can be exploited to study effector translocation .

These methodologies have been instrumental in establishing that AnkG is indeed secreted in a Dot/Icm-dependent manner, confirming its status as a bona fide T4SS effector protein .

How can protein-protein interactions of CBU_0781 be experimentally validated?

Validating the protein-protein interactions of CBU_0781 (AnkG) requires multiple complementary approaches to ensure reliability and biological relevance. The following methodologies have proven effective:

Research on AnkG has successfully employed co-immunoprecipitation and RNA-immunoprecipitation to demonstrate its binding to DDX21 and the 7SK snRNP complex . These studies revealed that AnkG's interaction with DDX21 is likely mediated by salt bridges and occurs independently of its binding to the 7SK snRNP complex, highlighting the importance of using multiple methods to characterize distinct interaction mechanisms .

Furthermore, characterizing the specific domains of AnkG responsible for these interactions would typically involve creating truncated or mutated versions of the protein to identify the minimal binding regions required for each interaction partner.

What assays can measure the anti-apoptotic activity of CBU_0781?

The anti-apoptotic activity of CBU_0781 (AnkG) can be quantified using various complementary assays that measure different aspects of programmed cell death. The following methodological approaches provide a comprehensive assessment:

• Cell viability assays:

  • MTT/MTS/WST-1 assays that measure metabolic activity

  • Trypan blue exclusion for membrane integrity

  • Calcein-AM/Ethidium homodimer live/dead staining

• Apoptosis-specific markers:

  • Annexin V/Propidium Iodide flow cytometry to distinguish early apoptosis from late apoptosis/necrosis

  • TUNEL assay to detect DNA fragmentation

  • JC-1 staining to assess mitochondrial membrane potential

• Biochemical assays:

  • Caspase activity assays (particularly caspase-3/7)

  • Cytochrome c release from mitochondria

  • PARP cleavage detection by Western blot

• Genetic approaches:

  • AnkG knockout/complementation studies in C. burnetii

  • Heterologous expression of AnkG in model cell systems

  • RNA interference to knock down specific host targets of AnkG

• Imaging techniques:

  • Time-lapse microscopy of cells expressing fluorescent apoptosis reporters

  • High-content imaging to quantify nuclear morphological changes

Research has demonstrated that AnkG's anti-apoptotic activity is essential for efficient intracellular replication of C. burnetii . By employing multiple assays that assess different stages of the apoptotic pathway, researchers can gain comprehensive insights into how AnkG interfaces with host cell death machinery and promotes pathogen survival.

How should contradictory results regarding CBU_0781 function be approached?

When confronting contradictory results regarding CBU_0781 (AnkG) function, researchers should employ a systematic approach to reconcile discrepancies and advance understanding:

• Evaluate methodological differences:

  • Expression systems used (bacterial versus mammalian)

  • Cell types and infection models

  • Protein tagging approaches that might affect function

  • Timing of observations during infection

• Consider context-dependent effects:

  • AnkG may have different functions depending on cellular location

  • Its activity might vary across infection stages

  • Interactions could be cell-type specific

  • Other C. burnetii effectors may modulate AnkG function

• Examine protein variants:

  • Strain-specific polymorphisms in AnkG

  • Natural isoforms or post-translational modifications

  • Experimental constructs that may lack critical domains

• Design critical experiments:

  • Direct comparisons under identical conditions

  • Domain mapping to identify functional regions

  • Use of multiple complementary techniques

  • Genetic approaches (knockout/complementation)

• Integrate with broader knowledge:

  • Consider AnkG in the context of other bacterial effectors with similar domains

  • Examine homologs in related pathogens like Legionella pneumophila

  • Evaluate consistency with known host pathways affected during infection

The complexity of AnkG's interactions with both DDX21 and the 7SK snRNP complex, which appear to occur through independent mechanisms , exemplifies how apparent contradictions may actually reflect the multifunctional nature of bacterial effectors. Thorough investigation can transform seeming contradictions into deeper insights about protein function.

What are the challenges in purifying recombinant CBU_0781 for structural studies?

Purifying recombinant CBU_0781 (AnkG) for structural studies presents several technical challenges that researchers must overcome:

• Expression optimization:

  • Bacterial expression systems may yield insoluble protein due to improper folding

  • Eukaryotic expression systems may be necessary but provide lower yields

  • Codon optimization for the expression host is often required

  • Expression temperature, inducer concentration, and timing require optimization

• Solubility considerations:

  • AnkG may form inclusion bodies in bacterial systems

  • Fusion tags (MBP, SUMO, GST) may improve solubility but need removal for structural studies

  • Specialized solubilization and refolding protocols may be necessary

  • Buffer optimization to maintain stability during purification

• Purification complexity:

  • Multi-step purification is typically required to achieve structural biology-grade purity

  • Ankyrin repeat proteins can aggregate during concentration steps

  • Removal of nucleic acids (particularly when studying an RNA-binding protein)

  • Maintaining stability during the extended timeframes required for structural studies

• Structural analysis challenges:

  • Flexibility of ankyrin repeat domains may complicate crystallization

  • Size limitations for certain NMR approaches

  • Protein-protein or protein-RNA complexes add another layer of complexity

  • Domain boundaries must be precisely determined for truncation constructs

How can the molecular mechanisms of CBU_0781 be translated into potential therapeutic targets?

Translating knowledge about CBU_0781 (AnkG) molecular mechanisms into therapeutic targets requires a strategic research approach that bridges basic science with drug discovery principles:

• Target identification and validation:

  • Determine which AnkG interactions are essential for C. burnetii survival

  • Identify the specific binding interfaces between AnkG and host factors (DDX21, 7SK snRNP)

  • Evaluate whether targeting these interactions would affect normal host cell function

  • Create cell-based models to validate the importance of specific interactions

• Structural characterization:

  • Obtain high-resolution structures of AnkG alone and in complex with binding partners

  • Identify "druggable" pockets at interaction interfaces

  • Map the minimal binding domains required for each interaction

  • Use computational approaches to predict potential binding sites

• Screening approaches:

  • Develop high-throughput assays to measure AnkG-host protein interactions

  • Screen compound libraries for molecules that disrupt these interactions

  • Consider fragment-based approaches for difficult-to-drug interfaces

  • Explore peptide mimetics based on binding site structural information

• Translational research considerations:

  • Evaluate compounds for cellular permeability and toxicity

  • Determine effects on bacterial replication in cellular models

  • Assess potential for resistance development

  • Design animal models to validate efficacy in vivo

The characterization of AnkG as an essential factor for C. burnetii intracellular replication suggests that targeting this protein or its interactions could provide an effective therapeutic approach. The fact that AnkG interferes with host cell 7SK snRNP complex function indicates that disrupting this interaction might restore normal host cell processes and compromise bacterial survival.

Furthermore, AnkG's immunoreactive properties suggest potential applications in both diagnostics and vaccine development, expanding the translational potential of research on this protein.