Functional amyloid formation in streptococcus mutans

Amyloid was identified in the context of pathology but does not always represent aberrant protein folding. Functional amyloid is now recognized in all kingdoms of life. Amyloid aggregates possess evolutionarily conserved cross -sheet quaternary structures with common biophysical properties enabling their detection and study. Microorganisms are now known to produce purposeful amyloid within biofilm environments, but, considering their prevalence, little is known regarding molecular events influencing amyloid formation. Dental caries is a biofilm-dependent disease caused by dysbiosis and overgrowth of acidogenic and aciduric bacteria, particularly Streptococcus mutans. Amyloid is observed in vivo within dental plaque. Our group was first to discover oral amyloids, and we have identified four amyloid-forming proteins in S. mutans. P1 (AgI/II), WapA, and Cnm are sortase-localized adhesins and virulence factors. The fourth protein, Smu_63, negatively regulates biofilm cell density and genetic competence. Extensive tertiary and quaternary structural characterization of P1 is in hand, with characterization of the other proteins underway. Our X-ray fiber diffraction evidence proved a classical stacked -sheet amyloid structure for S. mutans amyloids, and our work revealed a new paradigm for multiple streptococcal and staphylococcal amyloids in that naturally occurring adhesin truncation products play two key roles within the organisms' biofilm life cycles. First, in monomeric form by promoting adherence to cognate ligands via quaternary interactions involving the cell surface-linked parent proteins, and second in amyloid form by quenching adhesive function and apparently facilitating detachment of aging biofilm cultures. The left-handed Z-conformer of extracellular DNA was recently associated with bacterial biofilm stability whereas right-handed B-DNA is associated with detachment. Of interest, the amyloid but not the monomeric form of neuropathologic A drives conversion of Z- to B-DNA. Also, membrane lipids impact amyloidogenesis by an unknown mechanism. Cardiolipin-rich mitochondrial membranes modulate amyloidogeneis of Parkinson and Huntingtin Disease-associated -synuclein and Htt, respectively. Cardiolipin is a prevalent anionic lipid in S. mutans cytoplasmic membranes and extracellular membrane vesicles, particularly under stress conditions. In this application we will define reciprocal mechanistic influences of amyloid-forming proteins on B- and Z-forms of DNA in vitro and in vivo in mono- and multi-species biofilms (Aim 1), determine the impact of membrane lipid composition on amyloid levels during biofilm progression and assess mechanistic interactions of specific lipids of interest on amyloidogenesis of known virulence-associated proteins (Aim 2), and continue to use state-of-the-art methods including solution and solid-state NMR spectroscopy to identify and characterize structural transitions reflective of monomer to amyloid conversion and determine changes in amyloid signatures for each protein upon exposure to different DNA conformers, amyloid-modulatory lipids, and other amyloidogenic proteins (Aim 3). Project Number: 1R01DE034692-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: L. Jeannine Brady (+1 co-PI) | Institution: UNIVERSITY OF FLORIDA, GAINESVILLE, FL | Award Amount: $574,966 | Activity Code: R01 | Study Section: Oral, Dental and Craniofacial Sciences Study Section[ODCS] View on NIH RePORTER: https://reporter.nih.gov/project-details/11293904