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Fluoride triggers lysis in Streptococcus mutans by inhibition of the Clp protease complex, leading to an unabated competence cascade.
| Proceedings of the National Academy of Sciences of the United States of America (PNAS) | Banerjee A, Pickelner S, Smith F, Tenuta LMA, Stockbridge RB. | 122(50):e2511884122.
Bacteria Proteolysis Apoptosis Cell Study

Abstract

Original abstract online at
https://www.pnas.org/doi/pdf/10.1073/pnas.2511884122?download=true

Fluoride has long been known to possess antimicrobial properties. For many bacteria, the toxic effects of fluoride are reversible. However, fluoride has also been shown to trigger lysis and cell death in many other diverse bacterial species, including dental pathogens. The underlying molecular mechanisms responsible for fluoride-induced cell lysis have not been established. Using Streptococcus mutans as a model, we show that fluoride elicits an uncontrolled stress response characterized by upregulation of competence pathways and extensive cell wall degradation. While controlled and limited autolysis under stress is an adaptive response, we show that expression of the competence-associated alternative sigma factor ComX is prolonged under fluoride stress, relative to other stressors. Using in vitro and in vivo analyses, we show that fluoride disrupts the typical tight temporal control of ComX by specifically inhibiting assembly and activity of Clp ATPases responsible for its proteolytic degradation. Unchecked, ComX upregulates bacteriocins and autolysins, while simultaneously suppressing immunity peptide expression via a 6S RNA-mediated mechanism. Thus, fluoride subverts cellular mechanisms to turn off competence pathways that are induced under cellular stress, causing irreversible damage to the cell wall and ultimately cell death. Phenylalanine partially restores Clp protease assembly and activity, providing a rationale for the frequent presence of a gene encoding chorismate mutase in fluoride-responsive operons. Together, our findings reveal the molecular mechanism of fluoride-dependent lysis in bacteria, fifty years after this phenomenon was first reported. These pathways could be exploited to potentiate the antimicrobial effects of oral fluoride.

Significance

It has been known for decades that fluoride has antimicrobial properties, but the molecular mechanisms that underlie fluoride-induced bacterial lysis remain unknown. Using Streptococcus mutans, the main pathogen responsible for dental caries, we show that the culprit is a runaway stress response. While transient induction of competence pathways is a common bacterial response to stress, fluoride prevents cells from turning off these cascades by disrupting proteolysis of competence-associated sigma factors. These findings provide a mechanistic foundation to understand how fluoride impacts bacteria. These findings could inform applications in dentistry, where fluoride is often used for caries control, as well as organofluorine bioengineering.

Data, Materials, and Software Availability

Proteomics analysis of fluoride treated samples described in Fig. 3 are available as Dataset S1. All study data are included in the article and/or supporting information.

Acknowledgments

This work was supported by NIH grants RM1 DE034220 (NIH/NIDCR) to R.B.S. and L.M.A.T. Orbitrap Fusion Lumos mass spectrometry in the University of Michigan Proteomics Core facility was supported by the Office of the Director, NIH under Award Number S10OD021619. Metabolomics measurements were performed by the University of Michigan Metabolomics Core (University of Michigan Medical School, Biomedical Research Core Facilities).

Author contributions

A.B., L.M.A.T., and R.B.S. designed research; A.B., S.P., and F.S. performed research; A.B. and R.B.S. analyzed data; and A.B., L.M.A.T., and R.B.S. wrote the paper.

Competing interests

R.B.S., L.M.A.T., and A.B. are listed as inventors on a pending patent filed by the University of Michigan describing inhibitors of Streptococcus mutans fluoride export.

Supporting Information

Appendix 01 (PDF)
Dataset S01 (XLSX)

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