Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms

Yarmola, Elena G.; Ishkov, Ivan P.; Cologna, Nicholas M. di; Menashe, Megan; Whitener, Robert L.; Long, Joanna; Abranches, Jacqueline; Hagen, Stephen J.; Brady, L. Jeannine

doi:10.1128/spectrum.01661-22

Cited by 7 publications

(5 citation statements)

References 84 publications

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“…Besides P1 and WapA polypeptides, S. mutans expresses another two amyloidogenic proteins, the secreted protein Smu.63 and the serotype-restricted collagen-and lamininbinding protein Cnm, which are, respectively, required for sucrose-independent biofilm formation [23,28,41] and cardiovascular virulence [25]. Using BLASTp analysis, we confirmed that S. sanguinis genomes harbor genes encoding for P1 homologues (52% identity with S. mutans P1) but not for WapA, Smu63c or Cnm.…”

Section: Discussionmentioning

confidence: 73%

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion

Franco,

Alves,

Naveed

et al. 2023

IJMS

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Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

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Section: Discussionmentioning

confidence: 73%

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion

Franco,

Alves,

Naveed

et al. 2023

IJMS

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“…Upon mechanical stirring, both C123 and full-length P1 form insoluble aggregates that exhibit classic amyloid properties (Oli et al 2012;Besingi et al 2017;Barran-Berdon et al 2020). Recently we demonstrated that S. mutans amyloid aggregates are localized to the non-adherent fraction of aging biofilms, and that amyloid formation diminishes S. mutans adhesion (Yarmola et al 2022). Thus, the C-terminal derivative of P1 plays a major regulatory role not only in initial adhesion and biofilm genesis through quaternary interactions of its monomeric form with other P1 domains, but also in biofilm maturation and detachment through the formation of functional amyloid.…”

Section: Biological Contextmentioning

confidence: 98%

“…The conservation of a distinct β-sheet binding surface on C3 for A3VP1 and MAb interactions that is distal from the C3/C2 interface coupled with a shift for the HSE triad at this C3/C2 interface on binding suggests that protein-protein interactions lead to allosteric modulation of the internal C123 structure. Given the role of monomeric C123 binding to intact P1 on the S. mutans cell wall surface in regulating cellular adhesion and the observation that its transition from a monomeric to amyloid form correlates with biofilm detachment (Heim et al 2015;Yarmola et al 2022), we our currently pursuing NMR experiments to structurally characterize both forms of the protein and environmental triggers of dynamics or structural transitions. These CSPs provide guidance for specific residues that can be isotopically enriched for querying changes in C123 structure via lower resolution solid-state NMR measurements within the context of intact cell walls and/or functional amyloid formation (Tang et al 2016).…”

Section: Interactions Of C3 With A3vp1 and Mab 6-8cmentioning

confidence: 99%

Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1

Peng,

Nogueira,

Rivière

et al. 2023

Preprint

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Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1’s naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional β-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.

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“…Moreover, approximately 20% of S. mutans clinical isolates express the collagen- and laminin-binding adhesin Cnm (Sato et al ., 2004; Nomura et al ., 2009; Nakano et al ., 2010; Abranches et al ., 2011; Avilés-Reyes et al ., 2014b), an important virulence factor associated with dental caries severity and recurrence (Miller et al ., 2015; Esberg et al ., 2017; Garcia et al ., 2021) that also contributes to extra-oral infections such as cerebral microbleeds, hemorrhagic stroke and infective endocarditis (Abranches et al ., 2011; Nomura et al ., 2013; Miller et al ., 2015; Avilés-Reyes et al ., 2017; Inenaga et al ., 2018; Hosoki et al ., 2020; Nomura et al ., 2020; Arora et al ., 2021). In addition to binding to collagen, Cnm, WapA and P1 have also been characterized as functional amyloids that help model the biofilm structure (Oli et al ., 2012; Besingi et al ., 2017; di Cologna et al ., 2021; Yarmola et al ., 2022). In addition to sharing functional and structural similarities, Cnm and WapA were both shown to be post-translationally modified through glycosylation (Avilés-Reyes et al ., 2017; Avilés-Reyes et al ., 2018).…”

Section: Introductionmentioning

confidence: 99%

Post-translational modification by the Pgf glycosylation machinery modulatesStreptococcus mutansphysiology and virulence

Cologna

Andresen²,

Samaddar

et al. 2022

Preprint

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Streptococcus mutans is a keystone pathogen of dental caries, and the ability to form biofilms is essential for its pathogenicity. We identified a glycosylation machinery (Pgf) in S. mutans that post-translationally modifies two surface-associated adhesins, Cnm and WapA. The four pgf genes (pgfS, pgfM1, pgfE, and pgfM2) are part of the S. mutans core genome and we hypothesized that the scope of Pgf goes beyond Cnm and WapA. By inactivating each pgf gene individually or creating a quadruple pgf mutant in S. mutans OMZ175, we showed that the Pgf machinery is important for biofilm formation. Compared to OMZ175, differences in surface charge, membrane stability, and genetic competence were also observed for most mutants. Importantly, in silico analyses and tunicamycin MIC assays suggest a functional redundancy between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Using a rat oral colonization model, we showed a 10-fold reduction in recovered CFUs for the pgf quadruple mutant compared to OMZ175. Finally, using Cnm as a model, we showed by glycoproteomics analyses that Cnm is heavily modified with N-acetyl hexosamine in OMZ175 whereas phosphorylations were observed for the pgfS mutant. Our findings indicate that the Pgf machinery participates in important aspects of S. mutans pathobiology.

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Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms

Cited by 7 publications

References 84 publications

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion

Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1

Post-translational modification by the Pgf glycosylation machinery modulatesStreptococcus mutansphysiology and virulence

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