Recombinant Coxiella burnetii Cell division protein FtsB (ftsB), partial

What is the role of FtsB in Coxiella burnetii cell division?

FtsB is a critical component of the divisome complex in C. burnetii, functioning as part of the FtsQLB complex that plays a regulatory role in bacterial cell division. Based on studies of cell division in other bacteria, FtsB likely participates in the later stages of divisome assembly following the recruitment of FtsZ, FtsA, and ZipA to the division site . The FtsQLB complex is hypothesized to inhibit peptidoglycan synthesis until FtsN recruitment occurs, thereby controlling the timing of septal peptidoglycan synthesis .

In C. burnetii, as in other bacteria, FtsB likely forms a trimeric complex with FtsQ and FtsL that bridges the cytoplasmic and periplasmic components of the divisome. This complex is essential for proper divisome assembly and function, making it critical for successful bacterial replication within host cells.

How does FtsB expression relate to C. burnetii growth phases?

The expression of FtsB in C. burnetii likely follows temporal patterns associated with the bacterium's unique biphasic life cycle. During the metabolically active large cell variant (LCV) phase when the bacterium is actively replicating, FtsB expression would be expected to increase to support cell division. In contrast, during the small cell variant (SCV) phase when the bacterium is metabolically inactive, FtsB expression would likely decrease.

Researchers investigating FtsB expression should consider these growth phase transitions when designing experiments, particularly when cultivating C. burnetii in axenic media or cell culture systems. Timing of sample collection is critical to capturing the appropriate expression levels of cell division proteins like FtsB.

What are the optimal conditions for expressing recombinant C. burnetii FtsB?

When expressing recombinant C. burnetii FtsB, researchers should consider the following methodological approaches:

• Expression system selection: E. coli BL21(DE3) strains are commonly used for expression of bacterial recombinant proteins. For membrane-associated proteins like FtsB, C41(DE3) or C43(DE3) strains may provide better expression due to their tolerance for membrane protein expression.

• Codon optimization: C. burnetii has a different codon usage bias compared to E. coli. Codon optimization of the ftsB gene sequence for E. coli expression can significantly improve protein yields.

• Fusion tags: Consider using fusion tags that enhance solubility (such as MBP or SUMO) or facilitate purification (His6, GST). These approaches mirror those successfully used for other C. burnetii proteins like GroEL, YbgF, OmpH, and UPF0422 .

• Induction conditions: Use lower temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) for induction to promote proper folding and reduce inclusion body formation.

• Extraction conditions: Since FtsB is a membrane-associated protein, use mild detergents (DDM, LDAO) for extraction while maintaining protein structure and function.

What purification strategies yield the highest quality recombinant C. burnetii FtsB?

Purification of recombinant C. burnetii FtsB requires a multi-step approach:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged FtsB, similar to the approach used for other C. burnetii proteins .

• Secondary purification: Ion exchange chromatography can separate FtsB from proteins with similar affinity for IMAC.

• Size exclusion chromatography: This critical final step separates monomeric, properly folded FtsB from aggregates and provides buffer exchange into a stabilizing formulation.

• Quality control: Assess protein purity by SDS-PAGE and Western blot analysis using anti-His antibodies or specific antibodies against FtsB, similar to verification methods used for other C. burnetii recombinant proteins .

• Functional validation: Verify protein function through binding assays with known interaction partners (FtsQ and FtsL) or through complementation studies in bacterial systems.

How can researchers investigate FtsB interactions with other divisome components?

To investigate FtsB interactions with other divisome components in C. burnetii:

• Co-immunoprecipitation assays: Use tagged FtsB to pull down interaction partners from C. burnetii lysates, followed by mass spectrometry identification.

• Bacterial two-hybrid systems: Adapt bacterial two-hybrid assays to detect specific interactions between FtsB and other divisome proteins such as FtsQ, FtsL, and FtsW.

• Fluorescence microscopy: Use fluorescently tagged FtsB to visualize its localization during different stages of C. burnetii cell division in infected cells or axenic cultures.

• Cross-linking studies: Apply chemical cross-linking followed by mass spectrometry to capture transient interactions within the divisome complex.

• Surface plasmon resonance: Measure binding kinetics between purified FtsB and other divisome components to determine affinity constants and interaction dynamics.

These approaches build upon methodologies that have been successful in studying other C. burnetii proteins and cell division proteins in other bacterial systems .

What are the challenges in studying FtsB in the context of C. burnetii's intracellular lifestyle?

Studying FtsB in C. burnetii presents several unique challenges:

• Biosafety considerations: As a BSL-3 pathogen, work with virulent C. burnetii requires specialized containment facilities. Recent developments of attenuated strains with known genetic mutations have made some research more accessible .

• Growth requirements: C. burnetii's slow growth and obligate intracellular lifestyle make genetic manipulation challenging. Axenic culture systems have improved this, but still require specialized media and conditions.

• Temporal regulation: The biphasic lifecycle of C. burnetii means FtsB expression and function may vary significantly between developmental stages.

• Limited genetic tools: Though improving, genetic manipulation tools for C. burnetii remain relatively limited compared to model organisms.

• Structural studies: Membrane proteins like FtsB are inherently challenging for structural determination due to their hydrophobicity and requirement for a lipid environment.

Researchers can address these challenges by using the recently developed safer forms of C. burnetii for scientific investigation , which retain relevant biological properties while reducing biosafety risks.

How can researchers validate the specificity of antibodies against C. burnetii FtsB?

Validating antibodies against C. burnetii FtsB requires a multi-faceted approach:

• Western blot analysis: Test antibodies against both recombinant FtsB and native C. burnetii lysates, looking for specific bands at the expected molecular weight.

• Immunoprecipitation: Verify that the antibody can specifically pull down FtsB from C. burnetii lysates.

• Immunofluorescence microscopy: Confirm that the antibody localizes to the expected septum location during cell division in fixed C. burnetii cells.

• Pre-absorption controls: Pre-absorb antibodies with recombinant FtsB to demonstrate loss of specific signal in subsequent assays.

• Knockout/knockdown controls: Where possible, test antibodies in FtsB-depleted or knockout strains to confirm specificity.

This approach mirrors validation methods used for antibodies against other C. burnetii proteins like GroEL, YbgF, OmpH, and UPF0422, which have shown utility in serodiagnostic applications .

What bioinformatic approaches can help predict FtsB function in C. burnetii?

Several bioinformatic strategies can help predict FtsB function in C. burnetii:

• Sequence analysis: Multiple sequence alignment with FtsB homologs from other bacteria can identify conserved domains and functional residues.

• Structural prediction: Use homology modeling and ab initio structure prediction to generate models of C. burnetii FtsB structure.

• Protein-protein interaction prediction: Computational methods can predict potential interaction interfaces between FtsB and other divisome components.

• Evolutionary analysis: Phylogenetic analysis can reveal evolutionary relationships between FtsB proteins across bacterial species and identify C. burnetii-specific adaptations.

• Gene neighborhood analysis: Examining the genomic context of ftsB in C. burnetii can provide insights into functional relationships with other genes.

These computational approaches are particularly valuable given the experimental challenges of working with C. burnetii and can help guide targeted experimental investigations.

Could C. burnetii FtsB serve as a target for new antimicrobial strategies?

The FtsB protein presents several characteristics that make it a potential antimicrobial target:

• Essential function: As part of the essential divisome complex, FtsB is likely critical for C. burnetii replication and survival.

• Surface accessibility: Parts of FtsB are likely exposed at the cell membrane, potentially making them accessible to antibodies or small molecules.

• Structural uniqueness: Any C. burnetii-specific structural features of FtsB could provide specificity for targeted therapeutics.

• Interaction disruption: Small molecules that disrupt the interaction between FtsB and other divisome components could inhibit bacterial cell division.

• Limited host homology: As a prokaryotic cell division protein, FtsB has no direct homologs in human cells, reducing the risk of off-target effects.

Researchers investigating FtsB as an antimicrobial target should consider the distinct phases of C. burnetii infection and ensure that interventions target the metabolically active forms of the bacterium .

How might FtsB contribute to C. burnetii virulence and pathogenesis?

The relationship between FtsB function and C. burnetii virulence may involve several aspects:

• Growth rate regulation: Proper FtsB function is necessary for efficient bacterial replication, which directly impacts the progression of infection.

• Morphological transitions: FtsB may play a role in the transition between SCV and LCV forms, which is critical for the C. burnetii life cycle during infection.

• Response to stress: Cell division processes, including FtsB function, may be regulated in response to host-imposed stresses.

• Antigenic variation: As a bacterial protein, FtsB could potentially be recognized by the host immune system, making it a target for immune evasion strategies.

• Interaction with host factors: While primarily involved in bacterial cell division, FtsB or the divisome complex might interact with host factors during intracellular growth.

Understanding these relationships could provide insights into the differences between acute and chronic Q fever infections, which differ in their virulence characteristics .

What emerging technologies could advance the study of C. burnetii FtsB?

Several emerging technologies hold promise for advancing C. burnetii FtsB research:

• Cryo-electron microscopy: Could enable visualization of the complete C. burnetii divisome structure at near-atomic resolution.

• Super-resolution microscopy: Techniques like PALM and STORM could reveal the dynamic localization and interactions of FtsB during C. burnetii cell division.

• CRISPR interference: CRISPRi systems adapted for C. burnetii could enable conditional knockdown of ftsB to study its function without generating lethal mutations.

• Microfluidics: Single-cell analysis in microfluidic devices could capture the heterogeneity in FtsB expression and function across a C. burnetii population.

• Synthetic biology approaches: Reconstitution of minimal divisome complexes containing FtsB could allow for controlled study of its interactions and function.

These technologies could help overcome current limitations in studying this challenging intracellular pathogen and its cell division machinery.

How does lipopolysaccharide variation in C. burnetii impact cell division and FtsB function?

The relationship between lipopolysaccharide (LPS) variation and cell division in C. burnetii presents an intriguing research direction:

• Phase variation effects: C. burnetii undergoes LPS phase variation between virulent phase I and avirulent phase II forms. This variation may impact cell envelope properties and potentially affect divisome assembly and function .

• Membrane fluidity: LPS composition influences membrane fluidity and organization, which could affect the localization and function of membrane-associated divisome proteins like FtsB.

• Spatial coordination: LPS synthesis and cell division must be spatially and temporally coordinated. FtsB, as part of the divisome, may interact with LPS synthesis machinery.

• Stress response: Changes in LPS in response to environmental stresses might be coordinated with alterations in cell division rates mediated through FtsB and other divisome components.

• Evolutionary adaptations: C. burnetii's unique lifestyle may have led to co-evolution of its LPS structures and cell division machinery, potentially resulting in pathogen-specific features of FtsB function.

This research direction connects two critical aspects of C. burnetii biology—its unique LPS, which is a major virulence determinant, and its cell division machinery, which enables bacterial replication and persistence .