Recombinant Burkholderia pseudomallei Cell division protein FtsQ (ftsQ), partial

What is the function of FtsQ in Burkholderia pseudomallei cell division?

FtsQ is a critical cell division protein in B. pseudomallei that participates in the divisome complex, a multi-protein assembly responsible for bacterial cell division. The protein contains three domains: a short cytoplasmic N-terminal domain, a single transmembrane segment, and a periplasmic C-terminal domain that interacts with other cell division proteins. As part of the bacterial divisome, FtsQ helps coordinate septal peptidoglycan synthesis at the cell division site and mediates protein-protein interactions essential for cell division progression .

Methodologically, researchers have studied FtsQ function through:

• Gene deletion studies demonstrating its essentiality

• Fluorescence localization showing mid-cell accumulation during division

• Bacterial two-hybrid assays revealing interaction networks with other divisome proteins

• Complementation studies with recombinant constructs to rescue division defects

How is recombinant B. pseudomallei FtsQ typically expressed and purified?

Expression of recombinant B. pseudomallei FtsQ typically employs E. coli-based expression systems using vectors such as pET series plasmids with IPTG-inducible promoters. Due to challenges with full-length membrane protein expression, many researchers work with partial constructs focusing on the periplasmic domain.

A standard purification protocol includes:

• Transformation into expression hosts (typically BL21 or derivatives)

• Culture growth at 37°C until OD600 reaches 0.8

• Induction with 500 μM IPTG at decreased temperature (16-25°C)

• Cell lysis using methods similar to those described for B. pseudomallei flagellin

• Purification via affinity chromatography using His-tag or GST-tag approaches

• Quality assessment via SDS-PAGE and Western blot analysis

Optimization is often required to prevent inclusion body formation, with strategies including:

• Lower induction temperatures (16°C)

• Reduced IPTG concentrations (0.1-0.5 mM)

• Co-expression with chaperone proteins

• Use of solubility-enhancing fusion partners

What are the structural characteristics of B. pseudomallei FtsQ?

While the specific crystal structure of B. pseudomallei FtsQ has not been determined, structural predictions based on homologous proteins indicate:

• A conserved POTRA domain in the periplasmic region

• β-strand-rich C-terminal domain involved in protein-protein interactions

• Structural similarity to FtsQ proteins from other Gram-negative bacteria

Researchers typically use a combination of approaches to characterize FtsQ structural properties:

• Secondary structure prediction algorithms

• Homology modeling based on solved structures from related bacteria

• Circular dichroism spectroscopy to assess secondary structure content

• Limited proteolysis to identify domain boundaries

• Structural assessment under various buffer conditions to maintain stability

How does B. pseudomallei FtsQ interact with host immune responses during infection?

While FtsQ's primary role is in bacterial cell division, research indicates that bacterial cell division proteins can be recognized by the host immune system. B. pseudomallei FtsQ may interact with the host immune system in several ways:

• As a pathogen-associated molecular pattern (PAMP) recognized by pattern recognition receptors (PRRs)

• Through antibody recognition during adaptive immune responses

• As a potential vaccine candidate due to its conservation and essentiality

Analysis of immune responses to cell division proteins requires:

• Expression of soluble domains for immunological studies

• Assessment of serum from melioidosis patients for anti-FtsQ antibodies

• Evaluation of cytokine production in immune cell cultures stimulated with recombinant FtsQ

• Animal models to evaluate immunogenicity and protection

Parallels can be drawn with studies of B. pseudomallei flagellin, which demonstrated that recombinant flagellin proteins induce strong immune responses via TLR5 activation . Similar studies with FtsQ could reveal whether it also engages specific immune receptors.

What genetic and structural variations exist in FtsQ across B. pseudomallei strains and related Burkholderia species?

B. pseudomallei demonstrates considerable genomic diversity across isolates due to its high recombination rates. Analysis of FtsQ conservation requires:

• Comparative genomic analysis across multiple sequenced strains

• Assessment of selection pressures using dN/dS ratios

• Identification of conserved functional domains versus variable regions

Based on similar analyses of other B. pseudomallei proteins, we would expect:

The high recombination rates observed in B. pseudomallei (r/m = 4.5 in Clade A, r/m = 8.5 in Clade B, and r/m = 6 in Clade C) suggest potential diversity in non-essential regions of FtsQ while maintaining functional conservation in critical domains.

How can B. pseudomallei FtsQ be targeted for antimicrobial development?

As an essential cell division protein, FtsQ represents a potential target for novel antimicrobials against B. pseudomallei, which is intrinsically resistant to many antibiotics. Research approaches include:

• Structure-based drug design targeting the essential protein-protein interaction sites

• High-throughput screening of compound libraries against purified recombinant FtsQ

• Fragment-based approaches to identify small molecules that disrupt FtsQ interactions

Methodological considerations:

• Development of assays measuring FtsQ interaction with partner proteins

• Use of bacterial two-hybrid or FRET-based systems to screen for inhibitors

• Establishment of minimum inhibitory concentration (MIC) testing protocols

• Assessment of specificity against B. pseudomallei versus mammalian cells

The use of purified recombinant protein allows for detailed binding studies, as demonstrated with other B. pseudomallei virulence factors .

What methodological challenges exist when working with recombinant B. pseudomallei proteins in biosafety contexts?

B. pseudomallei is classified as a Tier 1 select agent by the CDC due to its potential as a bioterrorism agent , creating unique challenges for recombinant protein research:

• Requirement for Biosafety Level 3 (BSL-3) facilities for handling live bacteria

• Strict regulatory compliance for possession and transfer

• Limited availability of genetic tools that comply with select agent guidelines

Researchers overcome these challenges through:

• Working with recombinant proteins in E. coli expression systems (BSL-1/BSL-2)

• Using avirulent surrogate organisms like B. thailandensis

• Implementing select-agent-compliant genetic manipulation systems

• Working with genome-synthesized constructs rather than template DNA from viable organisms

Experimental design considerations include decontamination procedures, biosecurity protocols, and appropriate documentation to comply with regulatory requirements.

How should experiments be designed to assess the structural interactions of FtsQ with other divisome proteins?

To examine FtsQ interactions with other divisome components:

• In vitro protein-protein interaction studies:

  • Surface plasmon resonance (SPR) with immobilized recombinant FtsQ

  • Isothermal titration calorimetry (ITC) to measure binding kinetics

  • Pull-down assays with tagged FtsQ constructs

• Bacterial two-hybrid (BTH) analysis:

  • Fusing FtsQ and potential partners to complementary adenylate cyclase fragments

  • Measuring interaction through reporter gene expression

  • Systematic testing of truncated constructs to map interaction domains

• Microscopy-based approaches:

  • Fluorescence localization studies in B. thailandensis (as a BSL-2 surrogate)

  • FRET analysis with fluorescently tagged proteins

  • Superresolution microscopy to track co-localization during cell division stages

Successfully mapping these interactions requires careful construct design to ensure proper folding of the recombinant proteins and proper interpretation of negative results, which may result from technical issues rather than lack of interaction.

What are the optimal conditions for expressing soluble domains of B. pseudomallei FtsQ for structural studies?

Based on experiences with other B. pseudomallei recombinant proteins , optimal expression conditions typically include:

The periplasmic domain (typically amino acids ~60-270) is most amenable to soluble expression. Inclusion of the native signal sequence should be avoided, as it can cause targeting issues in E. coli.

For structural studies, additional considerations include:

• Buffer optimization screening (pH 6.5-8.0, various salt concentrations)

• Stabilizing additives (5-10% glycerol, low concentrations of detergents for hydrophobic regions)

• Assessment of protein monodispersity by dynamic light scattering

How can researchers validate the functional significance of specific FtsQ domains through mutagenesis studies?

To validate domain function through site-directed mutagenesis:

• Design stage:

  • Identify conserved residues through multiple sequence alignment

  • Focus on residues predicted to be at protein-protein interfaces

  • Create both alanine substitutions and charge reversals

• Functional complementation:

  • Express mutant constructs in conditionally lethal ftsQ depletion strains

  • Quantify division defects (filamentation, growth rates)

  • Image cells to assess septum formation

• Protein interaction assessment:

  • Compare wild-type and mutant protein interaction profiles

  • Use bacterial two-hybrid or co-immunoprecipitation approaches

  • Measure binding affinities of purified components

• In vivo localization:

  • Create fluorescent protein fusions with mutant variants

  • Track recruitment to division sites

  • Correlate localization defects with functional impacts

Such experiments should include comprehensive controls, including expression level verification to ensure observed phenotypes are not due to protein instability or altered expression.

What strategies can overcome inclusion body formation when expressing recombinant B. pseudomallei FtsQ?

Inclusion body formation is common with membrane-associated proteins like FtsQ. Effective strategies include:

• Expression optimization:

  • Reduce expression temperature to 16°C

  • Lower IPTG concentration (0.05-0.1 mM)

  • Use enriched media like Terrific Broth

  • Express only the soluble periplasmic domain

• Fusion tags that enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

  • NusA

• Co-expression approaches:

  • Molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Rare tRNA-encoding plasmids for codon optimization

• Refolding strategies if inclusion bodies persist:

  • Solubilization in 8M urea or 6M guanidine HCl

  • Gradual removal of denaturant by dialysis

  • On-column refolding during affinity purification

The approach used successfully for B. pseudomallei flagellin protein involving B-PER extraction followed by affinity chromatography may serve as a starting point, with modifications for the membrane-associated nature of FtsQ.

How can researchers address variability in immune responses when using recombinant B. pseudomallei proteins in immunological studies?

Variability in immune responses to recombinant proteins can compromise experimental reproducibility. Based on studies with B. pseudomallei flagellin , researchers should:

• Ensure protein quality:

  • Verify absence of contaminating LPS using Limulus Amebocyte Lysate assays

  • Confirm proper folding using circular dichroism

  • Assess aggregation state by size-exclusion chromatography

• Standardize experimental conditions:

  • Use consistent cell numbers and protein concentrations

  • Implement standard curves with known stimulants (LPS, flagellin)

  • Include biological replicates from multiple donors

• Account for donor variability:

  • Screen for TLR polymorphisms that may affect responses

  • Consider previous exposure to related organisms

  • Group data by relevant clinical variables (diabetes status, previous melioidosis)

• Use appropriate controls:

  • Include heat-denatured protein controls

  • Test proteinase K-treated samples to confirm protein-dependent effects

  • Compare responses with those from unrelated bacterial proteins

As observed with B. pseudomallei flagellin, individual variation in cytokine responses can be significant and should be accounted for in experimental design and analysis .

What are the recommended approaches for designing antigenic epitopes from B. pseudomallei FtsQ for diagnostic or vaccine development?

When identifying potential antigenic regions of FtsQ:

• Epitope prediction:

  • Use algorithms to identify B-cell epitopes (e.g., BepiPred, ABCpred)

  • Predict T-cell epitopes using MHC binding prediction tools

  • Focus on surface-exposed regions based on structural models

• Experimental validation:

  • Synthesize peptide arrays covering FtsQ sequence

  • Screen with sera from melioidosis patients versus controls

  • Test for T-cell reactivity using ELISPOT or intracellular cytokine staining

• Cross-reactivity assessment:

  • Compare sequences with homologs from related bacteria

  • Test against sera from patients with other infections

  • Evaluate conservation across B. pseudomallei isolates

Based on experiences with B. pseudomallei flagellin , focusing on unique regions rather than conserved domains can improve specificity. The truncated approach used for flagellin (removing conserved N and C termini) increased specificity from 82.5% to 96.3% and could serve as a model for FtsQ epitope design.

How might CRISPR-Cas9 technologies be applied to study FtsQ function in B. pseudomallei?

CRISPR-Cas9 offers new possibilities for genetic manipulation of B. pseudomallei, which has traditionally been challenging due to select agent restrictions . Potential applications include:

• Precise genome editing:

  • Introduction of point mutations to study specific residues

  • Creation of fluorescent protein fusions at the native locus

  • Generation of conditional depletion strains

• CRISPR interference (CRISPRi):

  • Catalytically inactive Cas9 (dCas9) to repress FtsQ expression

  • Titratable repression to identify threshold levels required for function

  • Time-resolved studies by inducing repression at different growth stages

• CRISPR activation (CRISPRa):

  • Upregulation of FtsQ to study effects of overexpression

  • Simultaneous modulation of multiple divisome components

Implementation considerations include:

• Select-agent-compliant plasmid systems

• Optimized guide RNA design for the GC-rich B. pseudomallei genome

• Delivery methods compatible with biosafety restrictions

These approaches could overcome the limitations of traditional genetic techniques while maintaining compliance with select agent regulations.

What potential exists for developing cross-protective vaccines targeting conserved epitopes in cell division proteins across Burkholderia species?

Cell division proteins like FtsQ are essential and often well-conserved across bacterial species, suggesting potential as vaccine candidates. Research directions include:

• Comparative analysis:

  • Identify regions of FtsQ conserved across B. pseudomallei, B. mallei, and B. thailandensis

  • Map conservation against predicted surface exposure

  • Prioritize regions unique to pathogenic Burkholderia

• Multivalent approaches:

  • Combine conserved epitopes from multiple cell division proteins

  • Design chimeric proteins incorporating multiple antigens

  • Evaluate synergy with established vaccine candidates like flagellin

• Delivery platforms:

  • Evaluate recombinant protein formulations with various adjuvants

  • Test DNA vaccine approaches encoding optimized constructs

  • Explore outer membrane vesicle presentation of FtsQ epitopes

Similar to the approach using truncated flagellin , focusing on Burkholderia-specific regions while avoiding conserved domains shared with commensal bacteria could improve specificity and reduce potential cross-reactivity.

How can systems biology approaches integrate FtsQ function into broader understanding of B. pseudomallei pathogenesis?

Systems biology offers opportunities to connect cell division processes with virulence and host-pathogen interactions:

• Interactome mapping:

  • Identify the complete set of FtsQ protein-protein interactions

  • Map connections between cell division and virulence networks

  • Discover potential moonlighting functions beyond division

• Transcriptional regulation:

  • Characterize expression patterns of FtsQ under different conditions

  • Identify regulatory networks governing expression during infection

  • Compare expression in different host cell types

• Integration with host response data:

  • Correlate FtsQ expression with host immune signatures

  • Identify potential synchronization between bacterial division and host cell processes

  • Build predictive models of division dynamics during infection

• Metabolic connections:

  • Link cell division to metabolic adaptations during infection

  • Identify metabolic requirements for proper FtsQ function

  • Connect to known metabolic shifts observed in intracellular B. pseudomallei

These approaches would build upon existing knowledge of B. pseudomallei genomics and recombination to create a more integrated understanding of how core cellular processes contribute to pathogenesis.