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Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms.

Schwartz K, Syed AK, Stephenson RE, Rickard AH, Boles BR - PLoS Pathog. (2012)

Bottom Line: Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress.Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not.This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

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Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.
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ppat-1002744-g006: Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.

Mentions: We assayed synthetic PSMs peptides for their capacity to form fibers in vitro. To minimize the prevalence of polymeric “seed” complexes, all synthetic peptides were treated with HFIP/TFA and dried in vacuo prior to assay [34]. Incubation of the seven previously identified PSM peptides (αPSMs1–4, βPSMs1–2, and δ-toxin) demonstrated their capacity to self-assemble into fibers (Fig. 6B). We used the amyloid-specific dye thioflavin T (ThT) to observe amyloid formation over time [34], [35]. When we assayed the PSMs for polymerization in the presence of ThT at room temperature, we observed a robust increase in normalized fluorescence (Fig. 6A). Greater peptide concentration increased ThT fluorescence and showed rapid binding kinetics similar to an amyloid-nucleator system (Fig. 6A) [35], [36], [37]. PSM fibers exposed to ThT exhibited an emission spike near 490 nm that is also observed in other amyloid fibrils (Fig. 6C) [36], [38], [39]. Incubation of PSM fibers with the dye Congo red (CR) resulted in a characteristic absorbance “red shift”, indicative of cross β structure conserved in all amyloid fibers (Fig. 6D) [40]. Furthermore, PSM fibers isolated from solution through centrifugation displayed increased β-sheet content (Fig. 6E), which is consistent with data published from other bacterial amyloids [13], [16]. These in vitro observations compliment our genetic and physiological data, further supporting the notion that PSMs can form amyloid fibrils.


Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms.

Schwartz K, Syed AK, Stephenson RE, Rickard AH, Boles BR - PLoS Pathog. (2012)

Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369951&req=5

ppat-1002744-g006: Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.
Mentions: We assayed synthetic PSMs peptides for their capacity to form fibers in vitro. To minimize the prevalence of polymeric “seed” complexes, all synthetic peptides were treated with HFIP/TFA and dried in vacuo prior to assay [34]. Incubation of the seven previously identified PSM peptides (αPSMs1–4, βPSMs1–2, and δ-toxin) demonstrated their capacity to self-assemble into fibers (Fig. 6B). We used the amyloid-specific dye thioflavin T (ThT) to observe amyloid formation over time [34], [35]. When we assayed the PSMs for polymerization in the presence of ThT at room temperature, we observed a robust increase in normalized fluorescence (Fig. 6A). Greater peptide concentration increased ThT fluorescence and showed rapid binding kinetics similar to an amyloid-nucleator system (Fig. 6A) [35], [36], [37]. PSM fibers exposed to ThT exhibited an emission spike near 490 nm that is also observed in other amyloid fibrils (Fig. 6C) [36], [38], [39]. Incubation of PSM fibers with the dye Congo red (CR) resulted in a characteristic absorbance “red shift”, indicative of cross β structure conserved in all amyloid fibers (Fig. 6D) [40]. Furthermore, PSM fibers isolated from solution through centrifugation displayed increased β-sheet content (Fig. 6E), which is consistent with data published from other bacterial amyloids [13], [16]. These in vitro observations compliment our genetic and physiological data, further supporting the notion that PSMs can form amyloid fibrils.

Bottom Line: Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress.Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not.This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

Show MeSH
Related in: MedlinePlus