Recombinant Bartonella henselae Trigger factor (tig)

What is the molecular structure and function of B. henselae trigger factor?

Trigger factor in B. henselae is a ribosome-associated molecular chaperone that assists in co-translational protein folding. This multidomain protein typically consists of three domains: an N-terminal ribosome-binding domain, a central peptidyl-prolyl isomerase (PPIase) domain, and a C-terminal chaperone domain. The protein interacts with nascent polypeptides as they emerge from the ribosome, preventing premature folding and aggregation. While specific structural details of B. henselae trigger factor remain to be fully characterized, comparative analysis with homologous proteins suggests it likely adopts a similar structure to other bacterial trigger factors, with potential adaptations related to B. henselae's intracellular lifestyle .

How does the tig gene expression change during B. henselae host cell infection?

During host cell infection, B. henselae undergoes significant transcriptional reprogramming. The tig gene expression may vary between extracellular and intracellular environments, similar to other genes involved in stress response and protein homeostasis. Recent transcriptomic analyses comparing intracellular and extracellular B. henselae phenotypes have revealed differential expression patterns that may include changes in tig expression . The bacterium's adaptive response during host cell infection is centrally coordinated by the BatR/BatS two-component system, which is activated at physiological blood pH (7.4) and regulates many genes essential for host adaptation . While not specifically identified in the provided research, tig expression might be influenced by this regulatory system given its importance in protein quality control during stress conditions encountered within host cells.

What genomic context surrounds the tig gene in B. henselae?

The genomic organization around tig in B. henselae provides insights into potential co-regulation with other genes. In many bacteria, tig is located in proximity to genes involved in translation and protein quality control. Genomic analysis should examine whether tig is part of an operon or independently regulated, which would influence experimental approaches for studying its expression. Comparative genomic analysis with other Bartonella species and related α-proteobacteria would reveal conservation patterns and potential horizontal gene transfer events that might have influenced tig evolution in B. henselae.

How might trigger factor contribute to B. henselae's intracellular survival mechanisms?

Trigger factor likely plays a critical role in B. henselae's adaptation to the intracellular environment by ensuring proper folding of stress response proteins and virulence factors. When B. henselae transitions from extracellular to intracellular environments, it encounters significant stresses including oxidative stress, nutrient limitation, and pH changes that necessitate substantial proteome remodeling . As a chaperone, trigger factor would be essential during this transition to maintain protein homeostasis.

The intracellular survival of B. henselae involves inhibition of phagosome-lysosome fusion and replication within host cells . This process requires the precise timing of multiple virulence factors, many of which may depend on trigger factor for proper folding. Experimental approaches to investigate this relationship could include:

What is the relationship between trigger factor and the VirB/D4 type IV secretion system in B. henselae?

The VirB/D4 type IV secretion system (T4SS) is essential for B. henselae host adaptation, serving as a critical virulence determinant . As a molecular chaperone, trigger factor might be involved in the proper folding and assembly of T4SS components or its effectors (Beps). Research has demonstrated that the expression of the VirB/D4 T4SS requires both the BatR/BatS two-component system and the alternative sigma factor RpoH1, which is controlled by stringent response components SpoT and DksA .

A methodological approach to investigate the relationship between trigger factor and T4SS could include:

• Immunoprecipitation studies to identify potential interactions between trigger factor and T4SS components

• Comparative proteomics of wild-type and tig-mutant strains to assess T4SS assembly

• Functional assays measuring T4SS-dependent effector translocation in tig-deficient backgrounds

• Transcriptomic analysis to determine if tig expression correlates with T4SS gene expression during infection

Current data suggests that VirB/D4 T4SS is needed at early stages of mammalian host colonization , which might coincide with peak trigger factor activity as the bacterium adapts to the new environment.

How does trigger factor contribute to B. henselae's stress response during host adaptation?

B. henselae encounters various stresses during host adaptation, including pH changes, nutrient limitation, oxidative stress, and temperature shifts. The BatR/BatS pH-dependent signaling distinguishes between arthropod and mammalian host environments, while stringent response signaling modulates bacterial responses between early and late colonization stages . Trigger factor likely plays a crucial role in maintaining protein homeostasis during these transitions.

The stress response in B. henselae involves substantial transcriptional reprogramming. The stringent response components SpoT and DksA regulate the alternative sigma factor RpoH1, which controls expression of the VirB/D4 T4SS . As trigger factor is often part of the general stress response in bacteria, it may be co-regulated with these pathways to ensure proper protein folding during stress conditions.

Research methodologies to investigate this role could include:

• Quantitative proteomics under various stress conditions (pH, temperature, oxidative stress)

• ChIP-seq analysis to identify transcription factors binding to the tig promoter

• Comparative stress survival assays between wild-type and tig-mutant strains

• Transcriptional reporter fusions to monitor tig expression during host cell infection

What expression systems yield optimal quantities of functional recombinant B. henselae trigger factor?

For the efficient production of recombinant B. henselae trigger factor, several expression systems can be considered:

The most common approach utilizes E. coli BL21(DE3) with a pET vector system, incorporating an N-terminal His-tag for purification. Expression should be optimized at lower temperatures (18-20°C) to enhance proper folding. Addition of molecular chaperones (GroEL/ES) to the expression system may improve folding and solubility. For functional analysis, removal of the affinity tag using specific proteases (TEV or thrombin) should be considered to eliminate potential interference with activity.

What purification strategies yield the highest activity of recombinant B. henselae trigger factor?

A multi-step purification strategy is recommended to obtain highly pure and active trigger factor:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged constructs

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose) to separate based on charge differences

• Polishing step: Size exclusion chromatography to remove aggregates and obtain homogeneous protein

Critical factors affecting purification success include:

• Buffer composition: Typically 20-50 mM Tris or phosphate buffer, pH 7.5-8.0, with 100-300 mM NaCl

• Presence of reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent disulfide bond formation

• Addition of glycerol (5-10%) to enhance stability

• Temperature control (4°C recommended for all steps)

Activity assessment should be performed after each purification step using functional assays such as protein refolding assistance or peptidyl-prolyl isomerase activity measurements.

What functional assays can verify the activity of purified recombinant B. henselae trigger factor?

Multiple complementary approaches should be employed to verify the functional activity of purified trigger factor:

• Peptidyl-prolyl isomerase (PPIase) activity:

  • Measure using synthetic tetrapeptides containing proline

  • Spectrophotometric detection of cis-trans isomerization rates

  • Comparison with commercial PPIases as positive controls

• Protein refolding assistance:

  • Measure enhancement of refolding yield of model substrates (e.g., citrate synthase, luciferase)

  • Monitor prevention of aggregation by light scattering

  • Compare refolding kinetics with and without trigger factor

• Ribosome binding:

  • Co-sedimentation assays with purified ribosomes

  • Fluorescence anisotropy with labeled trigger factor

  • Surface plasmon resonance to determine binding constants

• Thermal stability assessment:

  • Differential scanning fluorimetry (Thermofluor assay)

  • Circular dichroism spectroscopy to monitor secondary structure

  • Analysis of temperature-dependent activity profiles

How can trigger factor be used to study B. henselae adaptation between arthropod vectors and mammalian hosts?

Trigger factor could serve as a molecular probe to understand B. henselae's adaptation between different host environments. The bacterium initiates its infection cycle in a mammalian host after replication in the midgut of arthropod vectors . This transition involves substantial physiological adaptations, potentially mediated in part by trigger factor's chaperone activity.

Research approaches could include:

• Comparative expression analysis of tig between bacteria isolated from arthropod vectors versus mammalian hosts

• Identification of trigger factor client proteins specific to each host environment using crosslinking mass spectrometry

• Development of fluorescently tagged trigger factor to visualize its subcellular localization during host transition

• Creation of conditional tig mutants to assess survival defects in specific host environments

The BatR/BatS pH-dependent signaling system distinguishes between arthropod and mammalian host environments, while stringent response signaling modulates bacterial responses between early and late colonization stages . Investigating whether trigger factor activity is regulated by these systems would provide insights into its role in host adaptation.

How might trigger factor contribute to B. henselae's interaction with endothelial cells?

B. henselae exhibits a unique interaction with vascular endothelium, which is considered the primary blood-seeding niche for Bartonella colonization . This interaction leads to characteristic vasoproliferative lesions through stimulation of angiogenesis. Trigger factor may be involved in ensuring proper folding of adhesins and other surface proteins critical for endothelial cell attachment and invasion.

The invasion of B. henselae into human endothelial cells occurs through two distinct pathways: as single bacteria through endocytosis or as bacterial aggregates in the form of invasomes . Both pathways likely require properly folded surface proteins, potentially dependent on trigger factor chaperoning activity.

Research strategies could include:

• Proteomics analysis of B. henselae surface proteins dependent on trigger factor for proper folding

• Assessment of endothelial cell adhesion and invasion efficiency in tig-depleted bacteria

• Investigation of trigger factor's potential role in BadA folding, a major adhesin essential for attachment to extracellular matrix proteins

• Evaluation of angiogenic factor production in endothelial cells infected with wild-type versus tig-modified B. henselae

What insights can trigger factor provide about B. henselae's bacteriophage interactions?

Upregulation of phage-associated genes has been observed in intracellular B. henselae compared to planktonic bacteria, suggesting increased phage activity within host cells . Previous studies have shown the presence of bacteriophage-like particles in B. henselae, with potential implications for bacterial growth and pathogenesis .

Trigger factor, as a molecular chaperone, might play an underappreciated role in phage protein folding or in modulating bacterial responses to phage activity. Research questions could explore:

• Whether trigger factor expression correlates with phage gene expression in intracellular bacteria

• If trigger factor interacts with phage proteins based on co-immunoprecipitation studies

• The potential role of trigger factor in modulating lytic versus lysogenic phases of phage life cycles

• Whether bacteriophage proteins are clients of trigger factor chaperone activity

This represents an unexplored area that could provide novel insights into the complex relationship between B. henselae, its bacteriophages, and host interaction.

How does B. henselae trigger factor compare with homologs from other α-proteobacteria?

Comparative analysis of trigger factor across α-proteobacteria can provide evolutionary insights and identify unique features of the B. henselae protein. Important comparisons include:

Methodological approaches for comparative studies should include:

• Sequence alignment and phylogenetic analysis

• Structural modeling and comparison

• Complementation studies in heterologous systems

• Domain swapping experiments to identify functionally important regions

These comparisons could reveal adaptations of B. henselae trigger factor that support its unique intracellular lifestyle and host interactions.

Could trigger factor be involved in B. henselae's stringent response pathway?

The stringent response is a bacterial stress response that coordinates adaptation to nutrient limitation. In B. henselae, this response involves SpoT and DksA, which control the alternative sigma factor RpoH1 . This regulatory pathway is critical for modulating the expression of virulence factors, particularly differentiating between early and late stages of host colonization.

As a molecular chaperone involved in protein quality control, trigger factor might intersect with the stringent response pathway in several ways:

• Ensuring proper folding of stringent response proteins like SpoT

• Being transcriptionally regulated by RpoH1, similar to other stress-response proteins

• Contributing to survival during the nutrient-limited conditions that trigger the stringent response

• Assisting in the folding of proteins needed during different stages of host colonization

Research approaches to investigate this relationship could include:

• Analysis of tig promoter for RpoH1 binding sites

• Transcriptomic and proteomic analysis of tig expression during stringent response activation

• Biochemical interaction studies between trigger factor and stringent response components

• Phenotypic analysis of tig mutants under stringent response-inducing conditions

Understanding this potential relationship would provide insights into how B. henselae coordinates protein quality control with its adaptive responses during infection.

What are the key unsolved questions about B. henselae trigger factor?

Despite advances in understanding B. henselae pathogenesis, several fundamental questions about its trigger factor remain unanswered:

• The precise substrate specificity of B. henselae trigger factor and whether it differs from other bacterial homologs

• The regulatory mechanisms controlling tig expression during different stages of infection

• Whether trigger factor is essential for B. henselae virulence and host adaptation

• The structural features that might distinguish B. henselae trigger factor from other bacterial trigger factors

• The potential of trigger factor as a target for antimicrobial development

Future research should address these questions through integrated approaches combining genetics, biochemistry, structural biology, and infection models.

What emerging technologies could advance our understanding of trigger factor function in B. henselae?

Several cutting-edge technologies show promise for unraveling trigger factor's role in B. henselae:

• CRISPR interference for conditional knockdown in difficult-to-transform bacteria

• Single-cell proteomics to capture cell-to-cell variability in trigger factor activity

• Cryo-electron microscopy for high-resolution structural analysis

• Proximity labeling approaches to identify the trigger factor interactome in vivo

• Advanced animal models with tissue-specific reporters to track B. henselae infection dynamics