Recombinant Bartonella quintana Chaperone protein DnaK (dnaK), partial

What is Bartonella quintana DnaK and what is its role in bacterial physiology?

Bartonella quintana DnaK is a heat shock protein 70 (Hsp70) family member that functions as a molecular chaperone. According to proteomic analyses, B. quintana DnaK has a molecular weight of approximately 68.2 kDa and an isoelectric point of 4.6 . It is primarily localized in the cytoplasm with a predicted hydrophobicity index of -0.381 .

DnaK plays a critical role in bacterial survival by:

• Assisting in proper protein folding under normal and stress conditions

• Preventing protein aggregation during heat shock

• Facilitating protein transport across membranes

• Contributing to bacterial adaptation to different host environments

As a chaperone, DnaK interacts with nascent polypeptide chains and helps maintain protein homeostasis, which is essential for B. quintana as it transitions between human hosts and arthropod vectors.

How does B. quintana DnaK compare structurally to DnaK proteins in other bacterial species?

While the search results don't provide a direct structural comparison of B. quintana DnaK with other bacterial species, typical DnaK proteins consist of two major domains:

• N-terminal nucleotide-binding domain (NBD) with ATPase activity

• C-terminal substrate-binding domain (SBD) that recognizes and binds client proteins

B. quintana DnaK appears to be well-conserved functionally, as demonstrated by identification in proteomic studies where it was classified with high confidence (E-value of 0) in peptide mass fingerprinting analyses . The conservation of DnaK across bacterial species suggests structural similarity, though species-specific variations likely exist that could affect substrate specificity or interaction with co-chaperones.

Why has partial recombinant DnaK become a focus for researchers studying Bartonella infections?

Partial recombinant DnaK has gained research interest for several important reasons:

• Diagnostic potential: DnaK is recognized by sera from B. quintana-infected patients, making it a candidate for serological diagnostics

• Vaccine development: Studies on similar proteins from related organisms (e.g., Coxiella-like endosymbionts) have shown that partial DnaK proteins can be considered vaccine candidates due to their immunogenicity

• Host-pathogen interactions: DnaK may play a role in B. quintana's ability to adapt to different hosts, including humans and recently discovered non-human primate reservoirs like Japanese macaques

• Epitope preservation: Even partial DnaK proteins can contain sufficient epitopes to elicit immune responses, as demonstrated by immunoreactivity studies

What expression systems are most effective for producing recombinant B. quintana DnaK?

Escherichia coli is the most commonly used expression system for recombinant B. quintana proteins. Based on the literature, the following approaches are effective:

Expression vector selection:

• pET expression systems with T7 promoters provide high-level expression

• Vectors introducing affinity tags (His-tag, GST) facilitate downstream purification

E. coli strain considerations:

• BL21(DE3) and its derivatives are suitable for DnaK expression

• Strains lacking the lon and ompT proteases help minimize protein degradation

Expression conditions:

• Induction with 0.5-1.0 mM IPTG when culture reaches OD600 of 0.6-0.8

• Post-induction growth at 25-30°C rather than 37°C to enhance solubility

• Harvesting cells 3-6 hours post-induction for optimal yield

Similar approaches have been successfully used to express partial DnaK coding sequences from related organisms, as demonstrated in studies of Coxiella-like endosymbionts .

What are the most effective purification strategies for recombinant B. quintana DnaK?

Purification of recombinant B. quintana DnaK typically employs a multi-step approach:

Step 1: Initial capture

• Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

• Glutathione affinity chromatography for GST-fusion proteins

Step 2: Intermediate purification

• Ion exchange chromatography (typically anion exchange due to DnaK's pI of 4.6)

• Cleavage of fusion tags if necessary (TEV or PreScission protease)

Step 3: Polishing

• Size exclusion chromatography to remove aggregates and obtain homogeneous protein

• Endotoxin removal if the protein will be used for immunological studies

Quality control:

• SDS-PAGE analysis to confirm purity and molecular weight (~68.2 kDa)

• Western blotting with anti-DnaK antibodies to confirm identity

• Mass spectrometry for definitive identification and analysis of post-translational modifications

How can researchers optimize the solubility of recombinant B. quintana DnaK during expression?

Optimization of recombinant B. quintana DnaK solubility requires addressing several factors:

Expression temperature modulation:

• Lower temperatures (15-25°C) slow protein synthesis, allowing more time for proper folding

• Extended expression times at lower temperatures often yield more soluble protein

Media and additive optimization:

• Supplementing growth media with osmolytes (glycerol, sorbitol)

• Adding molecular chaperone inducers (ethanol, benzyl alcohol) at low concentrations

• Using specialized media formulations (Terrific Broth, Auto-induction media)

Co-expression strategies:

• Co-express with bacterial chaperones (GroEL/GroES, DnaJ, GrpE)

• Use of specialized E. coli strains that overexpress rare tRNAs for codon optimization

Construct design considerations:

• Expression of functional domains separately if full-length protein proves insoluble

• Creation of truncated constructs focusing on well-folded domains

• Using solubility-enhancing fusion partners (SUMO, MBP, TrxA)

What methods are most effective for analyzing the structure-function relationship of B. quintana DnaK?

Effective methods for structure-function analysis of B. quintana DnaK include:

Computational approaches:

• Homology modeling based on existing DnaK structures

• Molecular dynamics simulations to understand conformational changes

• Protein-protein docking to predict interactions with co-chaperones and substrates

Experimental structural biology:

• X-ray crystallography to determine high-resolution 3D structure

• NMR spectroscopy for solution structure and dynamics

• Cryo-EM for visualization of DnaK-substrate complexes

Functional assays:

• ATPase activity measurements using colorimetric phosphate detection

• Substrate binding assays using fluorescence anisotropy

• Protein aggregation prevention assays with model substrates

• Thermal stability assessments via differential scanning fluorimetry

Mutagenesis studies:

• Site-directed mutagenesis of key residues identified through structural analysis

• Creation of domain-swap chimeras with other bacterial DnaK proteins

• Deletion mapping to identify minimal functional domains

How can epitope mapping of B. quintana DnaK contribute to vaccine and diagnostic development?

Epitope mapping of B. quintana DnaK provides critical information for both vaccine and diagnostic development:

For vaccine development:

• Identification of immunodominant B-cell epitopes for antibody production

• Mapping of T-cell epitopes that stimulate cellular immunity

• Selection of conserved epitopes across Bartonella species for broader protection

• Exclusion of epitopes with potential cross-reactivity to human proteins

The Immune Epitope Database and Analysis Resource (IEDB-AR) can be used to predict epitopes, as demonstrated in similar studies of partial DnaK proteins . Some predicted HLA-A and B alleles of MHC-I and HLA-DR alleles of MHC-II have shown similarity to T-cell responses in patients with related infections .

For diagnostic development:

• Identification of species-specific epitopes to distinguish B. quintana from related bacteria

• Selection of epitopes targeted early in infection for acute diagnosis

• Mapping of epitopes consistently recognized across patient populations

Studies have shown that B. quintana proteins, particularly those in the 35-37 kDa range, can be strongly recognized by sera from infected patients, suggesting potential diagnostic value .

What role does ATP binding and hydrolysis play in the chaperone function of B. quintana DnaK?

ATP binding and hydrolysis are fundamental to DnaK's chaperone function through a regulated cycle:

ATP-bound state:

• The nucleotide-binding domain (NBD) and substrate-binding domain (SBD) are coupled

• The substrate-binding pocket is in an open conformation

• Fast substrate binding and release kinetics

• Low affinity for substrate proteins

ADP-bound state:

• The NBD and SBD are uncoupled

• The substrate-binding pocket adopts a closed conformation

• Slow substrate binding and release kinetics

• High affinity for substrate proteins

ATP hydrolysis cycle regulation:

• Co-chaperone DnaJ stimulates ATP hydrolysis

• Nucleotide exchange factor GrpE facilitates ADP release and ATP rebinding

• This cycle enables DnaK to bind unfolded proteins, prevent aggregation, and assist in folding

Targeting the ATPase activity of B. quintana DnaK could be a potential therapeutic strategy, as disruption of this cycle would impair bacterial protein homeostasis.

How can recombinant B. quintana DnaK be used to develop serological diagnostics for B. quintana infections?

Recombinant B. quintana DnaK holds significant potential for serological diagnostics through several approaches:

ELISA-based detection systems:

• Coating plates with purified recombinant DnaK protein

• Detecting patient antibodies using labeled secondary antibodies

• Establishing appropriate cut-off values through ROC curve analysis with confirmed positive and negative samples

Immunoblot assays:

• Using recombinant DnaK in Western blots to detect specific antibodies

• Developing line immunoassays with multiple recombinant antigens

Multiplex serological platforms:

• Incorporating DnaK alongside other immunoreactive B. quintana proteins

• Combining with antigens from related pathogens for differential diagnosis

Serological studies have demonstrated that sera from B. quintana-infected patients recognize specific bacterial proteins, with several immunoreactive proteins identified through proteomic and immunoblot analyses . The table below shows the immune response pattern observed in patients infected with B. quintana:

Data adapted from patient serological profiles

What are the challenges in distinguishing B. quintana DnaK from related bacterial species in diagnostic applications?

Developing species-specific diagnostics using DnaK faces several challenges:

Sequence conservation issues:

• DnaK is highly conserved across bacterial species, potentially leading to cross-reactivity

• Closely related species like B. henselae share significant homology with B. quintana DnaK

Cross-reactivity management strategies:

• Focus on variable regions within the DnaK sequence

• Use epitope mapping to identify B. quintana-specific epitopes

• Employ competitive binding assays to enhance specificity

• Implement absorption steps with heterologous antigens

Differential diagnostic approaches:

• Combine DnaK with more species-specific antigens like the 35 kDa protein observed in B. quintana but not B. henselae

• Use PCR-based detection methods targeting the ribC gene for species differentiation

• Implement multispacer typing (MST) methods for definitive speciation

Studies have shown that while cross-reactivity exists between B. quintana and B. henselae in serological tests, species-specific proteins can be identified through comprehensive proteomic analyses .

How can recombinant B. quintana DnaK be used to study host-pathogen interactions?

Recombinant B. quintana DnaK provides several approaches to investigate host-pathogen interactions:

Cell culture models:

• Incubation with human endothelial cells to study adhesion and invasion

• Exposure to erythrocytes to examine binding and entry mechanisms

• Co-culture with immune cells to assess immunomodulatory effects

Receptor identification:

• Protein-protein interaction studies using pull-down assays

• Surface plasmon resonance to measure binding kinetics to host proteins

• Cross-linking studies followed by mass spectrometry to identify binding partners

Immunomodulation studies:

• Assessment of cytokine profiles induced by DnaK in human cells

• Evaluation of DnaK's effect on antigen-presenting cell function

• Analysis of T-cell activation patterns in response to DnaK presentation

In vivo models:

• Using recombinant DnaK to evaluate tissue tropism in animal models

• Tracking immune responses to DnaK during experimental infection

• Testing protective efficacy of anti-DnaK antibodies against challenge

Such studies are particularly relevant given B. quintana's adaptation to different hosts, including humans and non-human primates like Japanese macaques .

How do genomic variations in dnaK affect the evolution and host adaptation of B. quintana?

Analysis of genomic variations in dnaK provides insights into B. quintana evolution and host adaptation:

Evolutionary mechanisms:

• Bartonella gene transfer agent (BaGTA) mediates high-frequency genome-wide recombination in Bartonella species

• This recombination may help maintain genome integrity and counter Muller's ratchet (accumulation of deleterious mutations)

• Variations in dnaK could reflect adaptation to different host environments

Host adaptation implications:

• Comparison of dnaK sequences across B. quintana strains from different hosts (humans vs. macaques)

• Assessment of positive selection signatures in specific domains

• Correlation of variants with host range and virulence characteristics

Recent research has identified B. quintana in Japanese macaques, expanding the known host range beyond humans . Comparative genomic analysis revealed that while the general genomic features are similar between human and macaque strains, there are significant differences including:

• A large chromosomal inversion (~0.68 Mb) in the Japanese macaque strain

• Absence of certain genes in the macaque strain, suggesting host-specific adaptation

• Average nucleotide identity values indicating closer relationship between macaque strains than to human strains

What methodologies are most effective for studying DnaK's role in B. quintana stress response and pathogenesis?

Effective methodologies for studying DnaK's role in stress response and pathogenesis include:

Gene manipulation approaches:

• Creation of conditional knockdowns (as complete knockouts may be lethal)

• CRISPR interference to modulate expression levels

• Site-directed mutagenesis to create ATPase-deficient mutants

Stress response characterization:

• Transcriptomic analysis under various stress conditions (heat, oxidative, pH)

• Proteomic analysis of DnaK-associated proteins during stress

• Metabolomic assessment of changes induced by DnaK modulation

Pathogenesis models:

• Insect cell infection models mimicking vector environment

• Human cell line infections to study persistence mechanisms

• Creation of fluorescently tagged DnaK to visualize localization during infection

Systems biology integration:

• Multi-omics integration to build comprehensive models of DnaK function

• Network analysis of DnaK interactions during different infection stages

• Computational prediction of essential pathways involving DnaK

These approaches can help elucidate how DnaK contributes to B. quintana's remarkable ability to persist in human erythrocytes and endothelial cells, causing chronic bacteremia and potentially serious complications like endocarditis .

How can structural information about B. quintana DnaK be leveraged for drug and vaccine development?

Structural information about B. quintana DnaK provides multiple avenues for therapeutic development:

Structure-based drug design:

• Identification of druggable pockets unique to bacterial DnaK

• Virtual screening of compound libraries against these targets

• Design of peptidomimetics that interfere with substrate binding

• Development of allosteric modulators of ATPase activity

Vaccine design strategies:

• Structure-guided epitope selection focusing on surface-exposed, immunogenic regions

• Rational design of DnaK-derived peptide vaccines

• Development of structurally stabilized immunogens preserving key epitopes

• Creation of chimeric proteins combining multiple B. quintana antigens

Therapeutic antibody development:

• Structural analysis of neutralizing epitopes

• Design of recombinant antibodies targeting functional domains

• Creation of bispecific antibodies targeting DnaK and other virulence factors

Immunoepitope analysis of partial DnaK proteins has already identified potential B-cell and T-cell epitopes that could inform vaccine development . These approaches are particularly relevant given the increasing incidence of B. quintana endocarditis cases, which can be challenging to diagnose and treat .