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. 2024 Oct 21;24(43):13574–13582. doi: 10.1021/acs.nanolett.4c03166

Structure-Guided Bacteria Specificity and Wide Activity Spectrum of Endotoxin-Responsive Peptide Nanonets

Nhan Dai Thien Tram †, Jan Kazimierz Marzinek ‡, Louis Perrin ‡, Devika Mukherjee †, Vanitha Selvarajan †, Peter John Bond ‡,§, Pui Lai Rachel Ee †,*
PMCID: PMC11528433  PMID: 39431594

Abstract

graphic file with name nl4c03166_0006.jpg

Peptide nanonets offer a promising avenue for constructing anti-infective biomaterials. Our group recently reported innovative designs of synthetic BTT nanonets that fibrillate selectively in response to bacterial endotoxins. Herein, we delved deeper into the molecular interactions between our peptides and these bacteria-specific biomolecules, which is an aspect critically missing from major works in the field. Using microscopic and biophysical techniques, we identified phosphate moieties in endotoxins as being the most essential to the initiation of peptide fibrillation. This was strongly supported by molecular dynamics simulations in an outer membrane environment with variable states of phosphorylation. To support the claim over bacterial specificity, we demonstrated a lack of nanonet formation in the presence of various phosphate-containing biomolecules native to human biology. The structural importance of phosphate moieties among pathogenic strains strongly indicates a wide clinical spectrum of our peptides, which was experimentally verified.

Keywords: antimicrobial peptide, nanonets, broad spectrum, bacterial specificity, antibiotic resistance, self-assembly

Nanonets serve as important immune defense elements by entrapping pathogens.1 Immobilized microbes are rendered unable to adhere and colonize host tissues, which typically form the first stage of infectious pathogenesis. Several works have experimented with synthetic mimetics of amyloid nanonets as anti-infective biomaterials.2−4 Our group recently reported the successful designs of synthetic β-hairpin antimicrobial peptides (AMPs) called BTT peptides that were capable of amyloid fibrillation selectively in the presence of bacteria.5 We identified lipopolysaccharide (LPS) and lipoteichoic acid (LTA), characteristic components in the cell envelope of Gram-negative and Gram-positive bacteria, respectively, as major nucleators of amyloid fibrillation. The amyloid formation process was observed to follow lag-free kinetics, indicating that the bacterial membrane served as an amyloid-nucleating site for gathering peptide monomers. Bacteria-responsiveness and multifunctional trap-and-kill activity set our peptide nanonets apart from other designs in this growing field. There is a critical need to explore in greater detail the molecular interactions between our peptides and bacterial LPS and LTA, as we observed that similar mechanistic investigation was mostly missing from other notable works on bacteria-trapping peptide nanofibrils.2,4,6,7 Such insights can inform designs of follow-up constructs and have implications for the overall clinical utility, hence further advancing the development of anti-infective nanonets.

To that end, we employed microscopic and biophysical techniques to evaluate the influence of environmental factors on the fibrillation capacity and aggregate morphology of BTT2-4A, a potent candidate AMP identified in our prior work.5 Collectively, experimental results indicated phosphate moieties in bacterial endotoxins as the key mediator of amyloid induction. Atomic-resolution molecular dynamics (MD) simulations of BTT peptides interacting with a physiologically realistic bacterial outer membrane (OM) environment in variable states of phosphorylation were then performed to provide mechanistic insights at the molecular level. They highlighted the importance of interactions of a peptide β-sheet with phosphate groups in the LPS layer. To illustrate bacterial specificity, we showed that a range of phosphate-containing biomolecules of significance to human biology lacked an amyloid-inducing functionality. The structural importance of phosphate moieties to nanonet formation supports the wide clinical spectrum of our peptides, given the prevalence and essentiality of membrane phosphates to viability and antibiotic resistance mechanisms among pathogenic strains.8−12

Ionic Species in Aqueous Buffer Are Essential to Peptide Fibrillation

Salts in aqueous solutions are known to influence the fibrillation process of amyloidogenic proteins in nature.13 To evaluate the importance of inorganic ions, we used dynamic light scattering (DLS) to monitor the hydrodynamic radius (RH) of the BTT2-4A aggregates over 3 h. In PBS buffer, the presence of LPS or LTA caused a rapid increase in RH within the first hour, reaching 12 μm (Figure 1A). BTT2-4A aggregate size was substantially lower in distilled water, thus indicative of the importance of the ions in PBS buffer to peptide fibrillation. Although LTA caused a clear increase in size, RH only reached 2 μm, which was far smaller than in buffer alone (Figure 1C). In the presence of LPS, however, BTT2-4A formed small aggregates (RH ∼ 200–300 nm), which indicated an absence of fibrillation.

Figure 1.

Figure 1

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Fibrillation by BTT2-4A in aqueous solution. (A, C) Hydrodynamic radius of BTT2-4A aggregates (20 μM) was monitored using DLS when incubated with 5 μg/mL LPS or LTA in (A) PBS buffer or (C) distilled water. Experiments were repeated three times. Results are expressed as mean ± SEM and curve-fitted. (B) TEM images of BTT2-4A aggregates (20 μM) when incubated with 5 μg/mL LPS or LTA in PBS buffer or water. Control images without an LPS or LTA are also provided. Scale bar: 200 nm. Length measurements were performed using ImageJ, with at least 15 discrete instances for an image. (D) Hydrodynamic radius of BTT2-4A aggregates (20 μM) was measured using DLS in the presence of 5 μg/mL LTA in aqueous solution of various acidic pH, adjusted using HCl. Experiments were repeated three times. Data points at pH 2–7 were curve-fitted. (E) TEM images of BTT2-4A aggregates (20 μM) when incubated with 5 μg/mL LTA at pH 4 or pH 2. Scale bar = 200 nm.

The morphology of the aggregates was then visualized using transmission electron microscopy (TEM). When incubated with LPS or LTA in PBS buffer, BTT2-4A formed expansive nanonets (Figure 1B). When PBS was replaced with distilled water, we observed drastic changes in the aggregate morphology. In the presence of LPS, BTT2-4A formed nanosized amorphous aggregates (∼30 nm, Figure 1B), which were clearly smaller than BTT2-4A aggregates in PBS buffer alone. In water alone, peptide aggregation was negligible. We postulate that LPS in water provided amyloid nucleation but failed to facilitate fibril elongation, suggesting that the latter requires a certain level of ionic strength. In contrast, LTA in water could induce BTT2-4A to form extensive nanofibrils, despite more needle-like and spaced-out appearances than in PBS (Figure 1B). Secondary structures of BTT2-4A in water were evaluated by using circular dichroism (CD) spectroscopy. When induced by LPS, the CD spectrum was characteristic of β-sheet structures, with a maximum at 196 nm and a minimum at 218 nm (Figure S1A). Meanwhile, the LTA-induced CD spectrum displayed a minimum at 198 nm, indicative of random-coiled structures. Previously, BTT2-4A was observed to exhibit high β-sheet and hairpin turn content after exposure to LTA.5 This conformational difference in monomers might explain the discrepancies in morphology of LTA-induced nanofibrils in PBS versus water (Figure 1B).

We next investigated the effect of the pH level on the self-assembly kinetics. To prevent buffer ions from confounding the results, distilled water was used as the base for the pH adjustment. Since LPS-induced fibrillation was shown to be ineffective in distilled water, only LTA-induced fibrils were studied. As we lowered the pH level from 7.0 to 2.0, there was minimal correlation with the aggregate hydrodynamic size, with RH values within the 2–3 μm range after 3 h (Figure 1D). Below pH 2.0, a reduction in BTT2-4A aggregate size became apparent. At pH 1.2, the self-assembly kinetic curve exhibited a noticeable lag phase within the first hour of incubation, in agreement with reports that environmental factors can influence the lag phase of amyloidogenesis.14 Using TEM, the morphology of LTA-induced BTT2-4A aggregates was visualized. At pH 4.0, the nanofibrils appeared cluttered (Figure 1E), resembling peptide self-assemblies in PBS buffer more than those in distilled water at neutral pH (Figure 1B). At pH 2.0, BTT2-4A formed clumpy aggregates instead of nanofibrils (Figure 1E).

Inner Core Oligosaccharide (OS) Is Essential to the Amyloid-Inducing Ability of LPS

LPS molecules in the OM commonly consist of O-antigen, outer and inner core OS, and lipid A.15 The components display varying chemical profiles and interstrain variability.16−19 To identify the chemical moieties essential to LPS function as an amyloid nucleator, we tested various LPS truncates extracted from E. coli, where Ra lacks the O-antigen and Rd lacks both the O-antigen and outer core OS (Figure 2A). The extent of amyloid formation by BTT2-4A was evaluated using K114 dye, whose emission intensity increases upon binding to amyloids.20 Ra or Rd variants triggered BTT2-4A amyloid formation comparable to intact LPS (∼1.0 au, Figure 2B). In contrast, lipid A induced a far lower K114 fluorescence similar to the LPS-free background signal (<0.5 au). Structural comparison between Rd and lipid A underlined the importance of inner core OS, which is truncated in the latter. Using TEM, Ra and Rd LPS in PBS were observed to induce cluttered BTT2-4A nanofibrils (Figure 2C), which resembled the effect of intact LPS (Figure 1B). While still able to induce extensive peptide self-assembly, the aggregate morphology appeared more sheet-like and less compact (Figure 2C), thereby further supporting the essentiality of inner core OS to peptide–LPS interactions. Using CD spectroscopy, LPS, Ra, and Rd were all found to induce a β-sheet conformation in BTT2-4A (Figure S1B). The spectra also exhibited a gradual decrease in signals at signature peaks in the order LPS > Ra > Rd, suggesting that the absence of O-antigen and subsequently outer core OS affected the peptide structure despite negligible effects on fibril morphology. Meanwhile, the CD spectrum induced by lipid A was not of recognizable shape (Figure S1B), in agreement with TEM observation of altered morphology (Figure 2C).

Figure 2.

Figure 2

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Fibrillation of BTT2-4A in the presence of truncated LPS mutants. (A) Schematic outlining the LPS segments and truncated mutants. (B) K114 fluorescence of 20 μM BTT2-4A was measured after being incubated with 5 μg/mL of LPS or its truncated mutants for 30 min. Experiments were repeated three times. The fluorescence intensities (λEX = 360 nm, λEM = 550 nm) are expressed as mean ± SEM, after being normalized against the signal of positive control PAPf39. (C) TEM images of 20 μM BTT2-4A aggregates in the presence of 5 μg/mL of Ra, Rd, or lipid A (in PBS buffer). Scale bar = 0.5 μm. (D–G) MD simulation of interactions between phosphorylated and dephosphorylated LPS Rd membrane and BTT2-4A peptides, arranged as parallel beta-sheets. (D) The root-mean-square deviation (RMSD) of backbone atoms for the 4 central peptides with respect to the first frame, and (E) percentage of structured residues of BTT2-4A molecules monitored on dephosphorylated (blue) versus phosphorylated (orange) OM systems over 1000 ns. (F) Side and (G) top view snapshots of six BTT2-4A monomers before and after 1000 ns with respect to OM. Peptides are shown in cartoon representation with yellow corresponding to beta-sheet and cyan/white to unstructured regions. Lys and Leu residues are shown as licorice in blue and green, respectively. Lipid membrane is shown as lines with lipid tails in cyan, oxygens in red, and nitrogens in blue. Lipid phosphates are shown as orange spheres.

With comparative analysis of chemical structures, polyanionicity seems to set the inner core OS apart from the other LPS components. At neutral pH, it is negatively charged in most bacteria strains, which is constitutively missing from outer core OS and O-antigen.21 Sugar units in the latter components are commonly modified with acetyl, methyl, or glycosyl functional groups, all of which are uncharged. In comparison, oligosaccharides in inner core OS carry phosphates, which might engage in electrostatic interactions with the positively charged BTT2-4A. This postulation is in line with the capability of lipid A, natively phosphorylated in the E. coli F583 strain, to also promote peptide self-assembly (Figure 1B). This proposed significance of phosphate moieties in LPS is further supported by the distinction in behavior between LPS-induced and LTA-induced amyloidogenesis in distilled water (Figure 1). While sharing many chemical moieties with LPS (i.e., phosphate groups, saccharide units, fatty acid chains),22 each LTA molecule carries distinctly more phosphate groups in its long chain of glycerol and ribitol. This might explain the more robust fibrillation-inducing ability of LTA even in the absence of ionic species in the media. The drastic change in kinetics of BTT2-4A aggregation at acidic pH < 2 provides other supporting evidence (Figure 1D). As phosphate groups in LTA become increasingly protonated as the solution pH approaches 1.0 (i.e., pKa of phosphate monoester), the number of charged groups available for amyloid-inducing interactions with BTT2-4A decreases, hence compromising peptide self-assembly.

We next explored the essential role of the OM phosphate groups using explicitly solvated all-atom MD simulations. For each system, six β-hairpins arranged in a parallel β-sheet orientation were initially positioned near the outer surface of an OM model, itself composed of divalent cation cross-linked LPS Rd in the outer leaflet and phospholipids in the inner leaflet. Two OM systems were studied, containing either phosphorylated or dephosphorylated inner core OS. We observed drastic differences in the time-dependent behavior of BTT2-4A peptides when placed above a phosphorylated compared to dephosphorylated inner core OM. Snapshots of BTT2-4A monomers before and after 1000 ns of simulation show a stable peptide assembly adsorbed to the surface of the phosphorylated membrane (Figure 2F,G). The backbone root-mean-square deviation (RMSD) of the four central peptides in the assembled sheet compared to the initial coordinates remained low, and the overall structured residue content remained as high as ∼80–85% (Figure 2D,E), indicating a high stability. In contrast, the BTT2-4A assembly positioned onto the dephosphorylated OM rapidly dissociated, resulting in a drastic decrease in structured residues and increase in RMSD within 100 ns. Similar behavior was displayed by BTT2, another in-house nanonet-forming peptide,5 on phosphorylated versus dephosphorylated LPS Rd (Figure S2).

Phosphate Moieties of Lipid A also Play an Important Role in Inducing Peptide Fibrillation

To explore the significance of phosphate moieties in lipid A, we used alkaline phosphatase (ALP) to catalyze lipid A dephosphorylation.23 The K114 assay showed a lower extent of BTT2-4A amyloid formation with ALP-treated LPS compared to untreated LPS (Figure 3A). The generality of the LPS dephosphorylation effect on peptide amyloids was demonstrated by testing on BTT1-3A, another fibrillating BTT peptide,5 which showed similar results to BTT2-4A (Figure 3C). To visualize the effect of lipid A dephosphorylation, we performed confocal microscopy with FITC-labeled peptides (green fluorescence). Compared to untreated LPS, ALP-treated LPS induced visibly smaller BTT2-4A and BTT1-3A aggregates (Figure 3B). We next tested the effect of ALP pretreatment on LTA activity using the K114 assay and confocal microscopy. Similar to LPS, dephosphorylation lowered the amyloid-inducing capacity of LTA (Figure S3). Overall, the results support the importance of phosphate groups in both Gram-negative and Gram-positive endotoxins.

Figure 3.

Figure 3

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Fibrillation of peptides in the presence of dephosphorylated LPS. (A, C) K114 fluorescence of 20 μM (A) BTT2-4A or (C) BTT1-3A measured in the presence of 5 μg/mL LPS, with or without ALP pretreatment. Experiments were repeated three times. The fluorescence intensities (λEX = 360 nm, λEM = 550 nm) are expressed as mean ± SEM, after being normalized against the signal in the absence of ALP pretreatment. (B) Confocal microscopy images of 64 μM FITC-BTT2-4A and FITC-BTT1-3A in the presence of 16 μg/mL LPS with or without ALP pretreatment. Magnification = 100×. Scale bar: 10 μm.

To gain molecular insights into the importance of lipid A phosphates, we performed MD simulations using a similar membrane model but with lipid A as the OM outer leaflet, either phosphorylated or dephosphorylated. Like our observations with the LPS Rd membrane, BTT2-4A (Figure 4) and BTT2 (Figure S4) assemblies remained stably arranged on phosphorylated lipid A over 1000 ns but rapidly dissociated on the dephosphorylated counterpart. This observation was independent of the initial orientation of peptide molecules, with either Lys or Leu residues facing the membrane surface. To demonstrate the robustness of the observed stability, simulations were additionally performed for peptide sheets initially in an antiparallel arrangement. After 1000 ns, these fibrillating peptides clearly maintained a stable self-assembly (Figure S5A,B), with >80% of structured residues (Figure S5C). BTT4, a nonfibrillating peptide from the BTT series,24 was simulated as a negative control. The percentage of structured BTT4 residues gradually dropped to 40%, and the simulation with phosphorylated OM lipid A revealed the poorest assembly stability (Figure S5B).

Figure 4.

Figure 4

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MD simulation of interactions between phosphorylated and dephosphorylated lipid A outer membrane and BTT2-4A peptides, arranged as parallel beta-sheets. (A) Side and (B) top view snapshots of six BTT2-4A monomers before and after 1000 ns of simulations with respect to OM. In the case of dephosphorylated lipid A OM, the peptides were initially placed with either Lys or Leu residues facing the membrane surface. Peptides are shown in cartoon representation with yellow corresponding to beta-sheet and cyan/white to unstructured regions. Lys and Leu residues are shown as licorice in blue and green, respectively. Lipid membrane is shown as lines with lipid tails in cyan, oxygens in red, and nitrogens in blue. Lipid phosphates are shown as orange spheres. (C) The percentage of structured peptide residues and (D) number of clusters of BTT2-4A molecules were monitored over the simulation time of 1000 ns.

We also simulated the peptide assemblies in aqueous solution alone as an additional negative control (Figure S6). Regardless of parallel or antiparallel starting arrangements, all peptides drastically dissociated from one another, in agreement with our previous finding of random-coiled peptide structures in aqueous buffer.5

Bacterial Specificity of BTT2-4A Fibrillation

Considering the anti-infective application of our peptide nanonets, we next evaluated whether peptide–phosphate interactions were specific to bacterial endotoxins. Using DLS, we first tested the effect of common anions on human body fluids. In 200 mM, the RH of BTT2-4A aggregates remained at 400–500 nm after 1 h of incubation (Figure S7A), which decreased further at lower NaCl concentrations (<200 nm, Figure S7C). In 10 mM Na2HPO4 NaCl, RH values were even smaller (<100 nm, Figure S7B). Both phosphate and chloride ions at supraphysiological concentrations did not induce nonspecific peptide fibrillation. To identify the ions in PBS buffer required for LPS-triggered fibrillation, we incubated BTT2-4A and LPS in a 10 mM buffer solution made of NaH2PO4 and Na2HPO4 (1:2.5 molar ratio) to simulate major ions in PBS (Figure S7D). An apparent lack of fibrillation suggests that the omitted chloride anions were essential for the process.

Apart from LPS and LTA, phosphates can be found in biomolecules expressed by bacteria or humans, such as phospholipids in cellular membranes, prompting additional investigation. We tested DOPG (bacteria) and DOPC (human) small unilamellar vesicles (SUVs) as mimetics of cell membranes.25 The RH of the BTT2-4A aggregates remained largely unchanged over 1 h of incubation with the SUVs (Figure S8A). In the absence of BTT2-4A, control RH of DOPG and DOPC SUVs averaged at 484 and 103 nm (Figure S8B), hence unlikely to confound the estimated RH of peptide aggregates. Confocal microscopy visualization of FITC-labeled BTT2-4A showed small amorphous aggregates (Figure 5E,F), instead of expansive nanonets. Overall, the results support a lack of phospholipid-triggered fibrillation, which starkly differs from the behavior of the pathological α-synuclein.26

Figure 5.

Figure 5

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Specificity and spectrum of the peptide fibrillation process. (A, F) Aggregation of peptides in the presence of physiologically relevant phosphate- or sulfate-containing biomolecules. Confocal microscopy images of FITC-labeled BTT2-4A were taken after 1 h of incubation with (A) 0.03 μg/mL salmon DNA, (B) 30 μg/mL salmon DNA, (C) 50 μM polyP, (D) 100 μg/mL heparan sulfate, (E) DOPG SUVs, or (F) DOPC SUVs. Magnification = 100×. Scale bar = 10 μm. (G, I) SEM images of (G) P. aeruginosa ATCC 9027, (H) K. pneumoniae ATCC 700603, and (I) drug-resistant clinical isolate K. pneumoniae M7 either untreated or treated with 20 μM BTT2-4A for 30 min. Magnification = 15,000×. Scale bar = 1 μm.

We next tested extracellular DNA as a physiologically pertinent phosphate carrier. Bacteria can release DNA into the surroundings for several functions.27 Human serum also contains circulated extracellular DNA,28 but at a far lower concentration range.27 Therefore, we tested salmon DNA as a representative nucleic acid over a wide range of concentrations. DLS and K114 assays showed amyloid fibrillation at 5 and 30 μg/mL of DNA, but only amorphous aggregates (RH ∼ 2 μm) at 30 ng/mL DNA (Figure S9). These results suggest that DNA levels in bacterial cultures, but not normal human serum, could induce amyloidogenesis. Confocal images of FITC-labeled BTT2-4A, however, offered contradictory observations of minimal peptide aggregates at 30 μg/mL of DNA (Figure 5B), which was even smaller at 30 ng/mL (Figure 5A). To reconcile the results, we propose that DNA, while incapable of inducing fibrillation, can influence peptide–dye interactions culminating in an overestimation of amyloid content by the K114 assay. Inorganic polyphosphate (polyP) is a ubiquitous biomolecule that comprises multiple covalently linked phosphate groups. polyP is mostly a cytosolic component in human cells, but membrane-associated or secreted in bacteria.29,30 At both 50 and 250 μM polyP, low K114 signal (<0.5 au) and small RH (<400 nm) indicate a lack of BTT2-4A fibrillation (Figure S10), which is supported by confocal observation of small FITC-BTT2-4A aggregates after incubation with polyP (Figure 5C). Amyloidogenic properties of BTT peptides substantially differ from naturally occurring amyloidogenic proteins, whose core β-sheet structures can be nucleated by polyP.31 To expand the scope of specificity investigation beyond phosphates, we examined heparan sulfate as a representative sulfate-containing polymer. Prevalent in humans,32 it has been associated with the generation of amyloid plaques in neurodegenerative diseases.33,34 Using DLS and confocal microscopy, we showed that BTT2-4A only formed small amorphous aggregates when incubated with 100 μg/mL heparan sulfate (Figures 5D and S11), indicating a lack of amyloid fibrillation.

Overall, we demonstrated that biologically significant phosphate- and sulfate-containing biomolecules did not induce amyloid fibrillation. Of interest, some of the tested amyloid inducers were found to trigger amyloid formation by natural peptides,26,33,34 thus reaffirming the clinical advantage of our peptide design. These findings are in line with our previous findings that BTT2-4A displayed no systemic toxicity after 24 h of treatment in mice.5 Amyloid-inducing interactions appeared to be driven not only by electrostatic attractions but also by other chemical features unique to bacterial endotoxins. This assessment concurs with the observation that the truncation of O-antigen and outer core OS had an impact on peptide conformation within the nanonets (Figure S1B).

Implications on the Antibacterial Spectrum of BTT Nanonets

Among Gram-negative pathogens, LPS phosphates are known to play major roles in cell viability,35 OM stabilization,8 virulence, and immunogenicity.36 Mutations in pathogenic strains can result in dephosphorylated LPS, which underlies their pathogenicity.11,12,37,38 However, partial dephosphorylation is more prevalent, with phosphates at the inner core OS typically left intact. Meanwhile, Klebsiella pneumoniae strains constitutively missing phosphate moieties at inner core OS are mostly nonpathogenic isolates from the environment,10 thus not relevant to anti-infective applications. E. coli and Salmonella strains can exhibit a deep-rough LPS phenotype where the inner core heptose is truncated alongside the attached phosphates, but this mutation can lower bacterial fitness by increasing vulnerability to hydrophobic drugs and macrophage phagocytosis.10 To the best of our knowledge, complete removal of phosphates from both inner core OS and lipid A has not been reported in human pathogens. In addition, there are minimal records on Gram-positive pathogenic strains with dephosphorylated LTA. Combining the critical role of phosphate moieties in facilitating fibrillation-inducing capacity and their essentiality and prevalence among pathogenic strains, we postulate that anti-infective potentials of BTT peptide nanonets can cover a wide spectrum of human pathogens.

P. aeruginosa ATCC 9027 and K. pneumoniae ATCC 700603 were observed using SEM to trigger the formation of bacteria-trapping BTT2-4A nanonets after 30 min of incubation (Figure 5G,H). This illustrates the robust applicability of BTT peptide nanonets to various Gram-negative pathogens. In a prior work, we demonstrated that an E. coli clinical isolate with mutation at the mcr-1 gene, in which the negative charge of phosphate at lipid A but not inner core OS was masked by chemical modification, retained its ability to induce nanonet formation.5 Herein, we further showed that the mcr-1-positive K. pneumoniae M7 clinical isolate induced an extent of peptide fibrillation similar to the ATCC strain (Figure 5I). These observations support the idea that bacterial LPS mutants retaining phosphate moieties at either inner core OS or lipid A are adequately robust to enable peptide fibrillation, thereby accentuating the wide clinical spectrum of our peptide nanonets.

In this work, we identified phosphate groups on bacterial endotoxins as the key chemical moieties of the bacterial OM responsible for initiating amyloid fibrillation by BTT peptides. This conclusion was systematically supported by a collection of biochemical assays, microscopy, and MD simulations. To illustrate the bacterial specificity of the process, we showed that phosphate ions in solution and several phosphate- and sulfate-containing biomolecules of significance to both bacteria and human cells did not induce amyloid fibrillation. Notably, the aggregation profile of BTT peptides vastly differed from that of amyloidogenic proteins in nature. By analyzing the importance of endotoxin phosphates in the context of their essentiality to the viability and functions of most human pathogens, the activity spectrum of BTT peptide nanonets is assessed to be adequately broad for anti-infective applications.

Acknowledgments

The research work described here was conducted with facilities provided by the National University of Singapore. We would like to acknowledge the financial support provided by MOE AcRF grants (A-000-4347-00-00, A-8002484-00-00, and A-8001963-00-00) awarded to P.L.R.E. We would like to acknowledge the assistance of the Electron Microscopy Unit and the Confocal Microscopy Unit (Yong Loo Lin School of Medicine, National University of Singapore) in sample preparations. We acknowledge support from BII (A*STAR) core funds. The computational work for this article was partially performed on resources of the National Supercomputing Centre, Singapore (https://www.nscc.sg). Figures 2A, 3A, and the TOC graphic were created in BioRender. Ee, R. (2024) BioRender.com/f52y694.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.4c03166.

  • Materials; experimental methods; peptide sequences; and supplementary results including CD spectra, MD simulations, DLS, K114, and confocal images (PDF)

The authors declare no competing financial interest.

Supplementary Material

nl4c03166_si_001.pdf (1.1MB, pdf)

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