Recombinant Treponema pallidum Flagellar motor switch protein FliM (fliM)

What is the structural composition of T. pallidum FliM?

FliM is a flagellar motor switch protein that forms part of the complex responsible for controlling the direction of flagellar rotation in T. pallidum. Research has identified distinct functional regions within the FliM protein: the N-terminal region binds to the phosphorylated chemotaxis protein CheY (CheY-P), the middle region interacts with FliG, and the C-terminal region binds to FliN . This modular structure allows FliM to function as an intermediary in signal transduction pathways controlling flagellar rotation and bacterial motility.

How does FliM interact with other flagellar proteins in T. pallidum?

FliM forms a complex with FliG and FliN proteins to create the flagellar switch complex. Affinity blotting experiments have provided direct evidence of interaction between FliM and FliN, FliM and FliG, and FliM and CheY-P . In vivo studies confirm that FliM and FliN form a complex with a stoichiometry of 1:4 and function as a unit in bacterial flagellar systems . The interaction between these proteins is critical for transmitting chemotactic signals to the flagellar rotor, ultimately controlling the direction of bacterial movement.

What is the relationship between FliM and bacterial motility in T. pallidum?

FliM plays a crucial role in controlling the direction of flagellar rotation in T. pallidum, which directly impacts bacterial motility. As part of the switch complex, FliM responds to the binding of phosphorylated CheY, resulting in conformational changes that are propagated to FliG, inducing clockwise rotation of flagellar motors . This rotation change causes the bacterium to tumble, allowing it to reorient and navigate toward favorable environments. This mechanism is essential for T. pallidum's ability to disseminate throughout the host during infection.

What is the stoichiometry and turnover dynamics of FliM in bacterial flagellar motors?

Studies in E. coli have revealed that approximately 30 FliM molecules exist per motor, organized into two discrete populations . One population remains tightly associated with the motor structure, while the other undergoes stochastic turnover. This turnover depends on the presence of active CheY, suggesting a potential role in motor switching mechanisms . Additionally, motors that rotate exclusively in the counterclockwise (CCW) direction contain more FliM molecules than those rotating clockwise (CW), indicating dynamic regulation of FliM stoichiometry based on motor function.

How does FliM stoichiometry vary with flagellar rotation direction?

Research using fluorescent protein labeling has demonstrated that the stoichiometry of flagellar switch proteins varies depending on rotation direction. In E. coli, motors that rotate only counterclockwise (CCW) contain approximately 144±26 FliN molecules, while those rotating clockwise (CW) contain approximately 114±17 FliN molecules . The ratio of CCW-to-CW FliN copy numbers is approximately 1.26, very close to the previously reported ratio of 1.29 for FliM . This correlation reinforces the model of FliM-FliN functioning as a unit and suggests that protein exchange is a regulated process that responds to motor activity.

What experimental evidence supports the model of FliM-FliN exchange as a functional unit?

Multiple lines of evidence support the model that FliM and FliN exchange as a functional unit:

• Stoichiometric analysis shows a consistent 1:4 ratio of FliM:FliN in the switch complex

• Similar ratios of CCW-to-CW copy numbers for both FliM (1.29) and FliN (1.26)

• Exchange of FliN molecules occurs on a time scale similar to that of FliM

• Dependence of both FliM and FliN exchange on rotation direction

These findings suggest that, under physiological conditions, the switch complex is not a static structure but a dynamic assembly that can respond and adapt to changing environments by modulating protein exchange rates .

How does CheY phosphorylation influence FliM dynamics in the flagellar motor?

The phosphorylation state of CheY significantly impacts FliM dynamics in the flagellar motor. When CheY becomes phosphorylated (CheY-P), it binds to the N-terminal region of FliM, inducing conformational changes that are propagated to FliG . This interaction not only influences the rotation direction of the flagellar motor but also affects the turnover rate of FliM molecules within the motor complex. Studies have shown that the stochastic turnover of FliM molecules depends on the presence of active CheY, suggesting that protein exchange may be part of the mechanism for motor switching and adaptation .

How is FliM expressed in T. pallidum compared to other flagellar proteins?

Expression analysis of T. pallidum flagellar proteins reveals variation in transcript levels depending on growth conditions. While specific data on FliM expression levels compared to other flagellar proteins is limited in the search results, research on T. pallidum gene expression during in vitro culture versus rabbit infection shows that many flagellar genes maintain relatively stable expression patterns across different environments . This suggests that FliM, as a crucial component of the flagellar apparatus, likely maintains consistent expression to ensure proper motility function during infection.

What systems are used for recombinant production of T. pallidum FliM?

Recombinant T. pallidum FliM can be produced using various expression systems, including:

• E. coli expression systems

• Yeast expression systems

• Baculovirus expression systems

• Mammalian cell expression systems

The choice of expression system depends on research objectives, required protein yield, and downstream applications. E. coli systems are commonly used for initial protein characterization due to their simplicity and high yield, while mammalian or baculovirus systems may be preferred when proper protein folding and post-translational modifications are critical for functional studies.

What purification strategies are most effective for recombinant T. pallidum FliM?

Effective purification of recombinant T. pallidum FliM typically involves a multi-step approach:

• Affinity chromatography using His-tag or other fusion tags

• Size exclusion chromatography to separate oligomeric states

• Ion exchange chromatography for removing contaminants

• Endotoxin removal for applications requiring high purity

Optimal purification protocols should be tailored to the specific expression system used and the intended application of the purified protein. For structural studies, additional purification steps may be necessary to achieve the high homogeneity required for crystallization or cryo-EM analysis.

What is the potential of recombinant T. pallidum FliM as a diagnostic antigen for syphilis?

Recombinant T. pallidum FliM has emerged as a promising candidate for syphilis serological diagnostics. Recent reviews have identified novel recombinant antigens, including surface-exposed proteins, adhesins, and flagellar proteins like FliM, as potential candidates for improved syphilis diagnostics . These proteins may enhance the diagnostic capabilities for both early and late syphilis stages and potentially help differentiate between active infection and previously cured syphilis. The inclusion of FliM in an expanded T. pallidum antigen panel represents an opportunity to improve the sensitivity and specificity of syphilis serological tests.

How does FliM compare to other T. pallidum recombinant proteins in diagnostic performance?

While specific performance metrics for FliM are not detailed in the search results, comparative studies of other T. pallidum recombinant proteins provide a framework for understanding potential diagnostic performance. For example, recombinant proteins TmpA, TpN17, and TpN47 have shown varying diagnostic potential:

For ELISA-based diagnostics:

These metrics provide a baseline for evaluating FliM's potential diagnostic performance .

What immune responses does T. pallidum FliM elicit during natural infection?

The immune response to T. pallidum FliM during natural infection remains an area requiring further investigation. While the search results do not provide specific data on FliM immunogenicity during syphilis infection, research on other T. pallidum proteins suggests that the location and accessibility of proteins significantly influence their immunogenicity. Proteins that are surface-exposed tend to elicit stronger antibody responses compared to those that are periplasmic or cytoplasmic . Understanding the immune response to FliM during natural infection would provide valuable insights for diagnostic and vaccine development efforts.

What fluorescent labeling techniques are effective for studying FliM dynamics?

Fluorescent labeling has proven effective for studying FliM dynamics in bacterial flagellar motors. High-resolution fluorescence microscopy using genomically encoded YPet (yellow fluorescent protein) derivatives of FliM expressed at physiological levels has successfully enabled measurement of FliM stoichiometry and turnover in functioning flagellar motors . Similarly, CyPet (cyan fluorescent protein) labeling has been used to investigate FliN dynamics . These techniques allow for real-time observation of protein dynamics in living cells, providing insights into the dynamic nature of the flagellar motor complex under various conditions.

How can researchers quantify FliM-protein interactions in the switch complex?

Several methodologies are available for quantifying FliM-protein interactions:

• Affinity blotting: Provides direct evidence of protein-protein interactions, as demonstrated for interactions between FliM and FliN, FliM and FliG, and FliM and CheY-P

• Co-immunoprecipitation: Enables detection of protein complexes under native conditions

• Fluorescence resonance energy transfer (FRET): Allows for real-time monitoring of protein interactions in living cells

• Surface plasmon resonance: Provides quantitative binding kinetics for purified proteins

• Bacterial two-hybrid systems: Useful for screening potential interaction partners

These methods can be complemented with deletion and truncation mutant analyses to map specific binding regions within the FliM protein, as has been done to identify distinct regions of FliM that bind to different partner proteins .

What in vitro and in vivo models are suitable for studying T. pallidum FliM function?

• Rabbit infection model: Traditionally used for T. pallidum propagation and studying gene expression patterns during infection

• Long-term in vitro culture systems: Recently developed systems using Sf1Ep cells and specialized medium (TpCM-2) allow for comparing gene expression between in vitro and in vivo conditions

• Heterologous expression in model organisms: Expression of T. pallidum FliM in genetically tractable organisms like E. coli or B. subtilis can provide insights into conserved functions

• Cell-free protein synthesis systems: Useful for studying protein-protein interactions and biochemical properties of FliM

Each model system has advantages and limitations, and the choice depends on the specific research questions being addressed.

How might FliM contribute to T. pallidum pathogenesis and tissue invasion?

While direct evidence linking FliM to T. pallidum pathogenesis is not presented in the search results, flagellar motility is likely crucial for the spirochete's rapid dissemination during infection. Research on other T. pallidum proteins, such as the adhesin Tp0751, has demonstrated roles in vascular adhesion and endothelial cell interactions . Future research could investigate whether FliM-mediated motility facilitates these adhesion processes or enables T. pallidum to penetrate tissue barriers. Understanding the relationship between flagellar function and T. pallidum's invasive capabilities could reveal new therapeutic targets for preventing disseminated syphilis.

What are the challenges in developing T. pallidum FliM as a vaccine candidate?

Development of FliM as a vaccine candidate would face several challenges:

• Protein accessibility: If FliM is primarily periplasmic rather than surface-exposed, it might not elicit protective antibodies

• Cross-reactivity: Potential epitope conservation with flagellar proteins from commensal bacteria could lead to autoimmune responses

• Protein expression levels: Low expression levels in T. pallidum could limit its effectiveness as an immunogen

• Functional redundancy: T. pallidum might utilize multiple motility mechanisms, reducing the impact of targeting FliM alone

Research on other T. pallidum antigens, such as Tp0751, has shown mixed results in vaccine development efforts , highlighting the complexity of developing effective syphilis vaccines.

How could structural biology approaches advance our understanding of T. pallidum FliM?

Structural biology approaches could significantly advance our understanding of T. pallidum FliM by:

• Determining the three-dimensional structure of FliM and its complexes with FliG, FliN, and CheY-P

• Identifying conformational changes that occur during signal transduction

• Revealing the molecular basis of protein turnover and exchange within the motor complex

• Identifying potential binding sites for small molecule inhibitors

Techniques such as X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy could be employed to achieve these goals. The resulting structural insights would enhance our understanding of flagellar motor function and potentially guide the development of novel diagnostics or therapeutics targeting T. pallidum motility.