Bacterial infections account for a significant proportion of global mortality, representing the second-leading cause of death and responsible for one in eight fatalities globally¹. This stark statistic highlights the urgency for an enhanced understanding of bacterial behavior, particularly chemotaxis. Chemotaxis, the directed movement of bacteria towards favorable environments, is intimately linked with pathogenicity. Our group has traditionally been focused on bioenergetics, studying energy transformation within cells. This shift towards chemotaxis may seem unexpected, but recent insights into metabolite signaling underpin it.

Fumarate, a metabolite processed by Complex II in the Krebs cycle and oxidative phosphorylation, has been found to influence flagellar orientation, favoring a clockwise (CW) state^2,3,4. Other metabolites and regulatory proteins, such as CheY and YcgR, have been shown to alter or immobilize the flagellar state^5,6.

The intriguing question arises: How do various metabolites function as signals to induce similar states in the flagella? The answer lies in the switch complex, also known as the C-ring, comprised of the FliG, FliM, and FliN proteins. Our study was guided by the hypothesis that understanding the assembly of the switch complex in different states will unlock broader knowledge of bacterial chemotaxis and, consequently, virulence.

Movie 1: Organization of the counterclockwise (CCW) and CW C-ring, showing the C-terminal FliF in blue, FliG in red, FliM in yellow, and three FliN subunits in shades of purple.

With the massive 6 MDa C-ring complex, cryo-electron microscopy (cryoEM) was the clear choice for its ability to visualize structures at near-atomic resolution. Following the purification of the complex, the inherent instability of the sample necessitated expedited preparation for cryoEM. This required a stringent yet efficient screening process to preserve the structural integrity required for precise analysis.

 Movie 2: Shows the C-ring at the base with FliG in red, FliM in yellow, and FliN in shades of purple; the MS-ring in blue; the MotAB in brown; the LP-ring in pink; and the rod in gray. The movie depicts the MotAB rotating the C-ring, the MS-ring, and the rod in the CCW direction. Then, the MotAB moves inward to rotate the flagellar assembly in the CW direction.

The cryoEM studies revealed the complex's arrangement in CW and counterclockwise (CCW) states. The structural differences between the CW and CCW states offer a mechanistic view of how the C-ring functions as a transmission switch, conveying torque and directional signals to the flagellar apparatus. We also discovered the symmetry mismatch between the C-ring and the MS-ring (in blue), which sits on top of the C-ring and is composed of multiple copies of FliF protein.

Movie 3: Showing flow of torque in CCW C-ring by employing multiple MotAB (in brown) interacting with the torque helix of the C-ring's FliG protein, in response to the environmental condition or load.

By delineating the structural basis of flagellar movement, we gain potential leverage points for therapeutic intervention. Understanding how bacteria orient themselves may lead to innovative strategies to curtail their pathogenic capabilities. This research enhances our understanding of bacterial chemotaxis, enriching our broader comprehension of bioenergetics and the interplay between cellular energy processes and bacterial motility.

The workflow and insights from our studies provide a foundation for novel strategies aimed at infection control. These findings enhance our ability to understand and potentially disrupt the processes critical for bacterial infection, laying the groundwork for improved methods to thwart bacterial virulence and impede the development of resistance.

References

1. Ikuta, Kevin S., et al. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet 400.10369, 2221-2248, doi:10.1016/S0140-6736(22)02185-7 (2022).

2. Cohen-Ben-Lulu, G. N. et al. The bacterial flagellar switch complex is getting more complex. EMBO J 27, 1134-1144, doi:10.1038/emboj.2008.48 (2008).

3. Koganitsky, A., Tworowski, D., Dadosh, T., Cecchini, G. & Eisenbach, M. A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase. J Mol Biol 431, 3662-3676, doi:10.1016/j.jmb.2019.08.001 (2019).

4. Zarbiv, G. et al. Energy complexes are apparently associated with the switch-motor complex of bacterial flagella. J Mol Biol 416, 192-207, doi:10.1016/j.jmb.2011.12.027 (2012).

5. Welch, M., Oosawa, K., Aizawa, S. & Eisenbach, M. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci U S A 90, 8787-8791, doi:10.1073/pnas.90.19.8787 (1993).

6. Fang, X. & Gomelsky, M. A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility. Mol Microbiol 76, 1295-1305, doi:10.1111/j.1365-2958.2010.07179.x (2010).

Link to the paper: https://www.nature.com/articles/s41564-024-01674-1

This blog post was written with minimal assistance from AI-based tools such as ChatGPT and Grammarly to ensure clarity and coherence.