Multifunctional peptide nanonets and methods of use thereof

Abstract

Disclosed is a synthetic β-hairpin antimicrobial peptide (AMP) comprising (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component to initiate amyloid nucleation; and (b) a structural module comprising a first side strand and a second side strand for β-sheet formation and molecular stacking during fibrillation; wherein the synthetic β-hairpin AMP comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the synthetic β-hairpin AMP self-assembles into nanonets in the presence of the bacterial membrane component or a bacterium. Also disclosed is a pharmaceutical or adjuvant composition comprising the AMP. Also disclosed is a method for treating a condition associated with a bacterial infection, or an immune-related disease, or for delaying the development of antibiotic resistance in a subject in need thereof using the AMP or composition.

Description

MULTIFUNCTIONAL PEPTIDE NANONETS AND METHODS OF USETHEREOFFIELD OF THE INVENTION

[0001] The present invention generally relates to synthetic nanonet- forming peptides and methods of use thereof. In particular, the present invention relates to synthetic nanonet-forming peptides and methods of their anti-infection and anti-inflammatory uses.BACKGROUND

[0002] The development pipeline for antibiotics especially those with novel mechanism of actions have been facing problems, for example when used against Gramnegative infections. Gram- negative pathogens are recognized by the WHO to have high clinical priority. As opposed to traditional antibiotics aimed directly at killing bacteria or curtailing their growth, current anti-virulence strategies are focused on suppressing hostpathogen interactions to prevent bacterial pathogenesis. As such, anti- virulence agents are expected to exert minimal selective pressure on the pathogens, hence facing a lower likelihood of resistance development by the microbes. For example, anti-adhesion agents, as a sub-category of anti-virulence compounds, prevent host adhesion and invasion by targeting either the expression or function of bacterial adhesins. However, since functional homology between virulence factors is closely related to phylogenetic distance, the activity spectrum of anti-adhesion agents is generally narrow. This potentially limits their utility against polymicrobial co-infections. In addition, adhesion to the epithelial or endothelial linings may occur non- specifically without involving binding between adhesins and ligands on host surfaces. In such scenarios, adhesins are no longer viable targets for anti-adhesion strategies.

[0003] Bacterial entrapment using net-like nanostructures, generally referred to as nanonets, is an alternative anti-adhesion approach. The mechanism of action does not involve the incapacitation of virulence factors and this fundamental difference means that the activity spectrum does not face similar limitations. In nature, trap-and-kill is a ubiquitous immune defense mechanism. In response to the presence of pathogens, peptides or nucleic acid building blocks are released from host cells and promptly self- assemble in solution to form nanofibrils, which then cross-link to generate nanonets. These nanoscale constructs can ensnare the microbial intruders, effectively restrain theirspread and undermine their ability to evade local microbicides. Of note, the human body adopts this strategy at various body sites such as small intestine, urinary tract, and blood vessels. Inspired by this evolutionarily conserved immune strategy, nanonets-forming peptides found in nature or synthetic nanonets-forming peptides have been used. However, for nanonets-forming peptides found in nature, initiation of aggregation is controlled at the cellular level in response to host detection of pathogens. Once secreted in solution, self-assembly of the peptide occurs spontaneously even in the absence of bacteria. For most synthetic peptides reported in literature, they also indiscriminately selfassemble in solution. This may cause the problem of random self-assembly after the peptide is administered into the body, before reaching the infection site. Furthermore, for nanonets-forming peptides found in nature, most of them lack intrinsic antimicrobial activity and are reliant on other antimicrobial components of host immune defense for bacterial killing. For most synthetic peptides reported in literature, they also lack killing effect, and have been reported for trapping effect only. Further, despite previous attempts to mimic naturally occurring nanonets, most synthetic peptide-based materials were observed to only form disjointed short nanofibrils, which are restricted to the bacterial surfaces and incapable of physically immobilizing the bacteria.

[0004] Another problem associated with anti-virulence strategies is that following antibiotic-induced lysis and cell death, gram-negative bacteria release membrane -bound lipopolysaccharides (LPS) into the bloodstream. Unchecked systemic inflammatory responses to endotoxemia can result in collateral damage to host tissues and disruptions to physiological processes, potentially leading to septic shock with a high mortality rate. Thus far, efforts to develop target- specific therapies against sepsis has been thwarted by the dynamic interplay among structurally diverse pro-inflammatory and antiinflammatory agents of both host and microbial origins. In general, antibodies targeting individual cytokines have not yielded promising outcomes in treating septic patients in clinical trials. More recent works have explored the feasibility of simultaneously capturing multiple key inflammatory mediators by exploiting the common abundance of negative charges on LPS and human pro-inflammatory cytokines, with synthetic cationic nanoconstructs of diverse materials and morphologies reported. However, none of the synthetic nanonet peptides reported in literature have been shown to have antiinflammatory bioactivity, which can complement bacterial killing activity in treating infections. Conventional target- specific strategies to inactivate particular inflammationmediators have largely failed to improve the prognosis of septic patients in clinical trials. This is due to the complex mix of pro- and anti-inflammatory cytokines during cytokine storm.

[0005] A further problem associated with traditional antibacterial treatment using antibiotics is resistance development over prolonged antibiotic treatment. Most antibiotic adjuvants in clinical practices help increase potency of the active pharmaceutical ingredients (APIs) through synergistic interactions. However, they do not show ability to protect the API against resistance development over prolonged antibiotic treatment. Most synergistic adjuvants work by directly targeting the resistance mechanism in the bacteria (e.g. membrane efflux proteins, antibiotic -degrading enzyme) such as the use of clavulanic acid as an adjuvant in Augmentin. However, this traditional approach only works against a limited subset of bacteria strains, and is vulnerable to eventual biomolecular changes in bacteria acquired through spontaneous mutations.

[0006] Thus, there is a need for a novel peptide which can overcome or ameliorate at least one of the above-described problems, i.e. can (1) self-assemble to nanonets only in response to the presence of bacteria, (2) possesses dual (trap and kill) antimicrobial function, (3) have anti-inflammatory activity to complement bacterial killing activity; and (4) delay the development of resistance among pathogenic bacteria against clinically relevant classes of antibiotics.SUMMARY

[0007] The present disclosure describes a series of synthetic P-hairpin antimicrobial peptides (AMPs) that are capable of self-assembling into nanonets in response to the presence of bacteria, hence conferring specificity. These nanonets can entrap bacteria and kill them, entrap endotoxins and pro-inflammatory cytokines, and delay the onset of antibiotic -resistance.

[0008] In one aspect, the present disclosure refers to a synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component to initiate amyloid nucleation; and (b) a structural module comprising a first side strand and a second side strand for P-sheet formation and molecular stacking during fibrillation; wherein the synthetic P-hairpin AMP comprises the sequence of SEQ ID NO:1 or SEQ ID NO: 2; and wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of the bacterial membrane component or a bacterium.

[0009] In another aspect, the present disclosure refers to a pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP as disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0010] In another aspect, the present disclosure refers to a method for treating a condition associated with a bacterial infection, or an immune -related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, comprising administering the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to (a) trap and kill the bacterium; and / or (b) trap an endotoxin and a pro- inflammatory cytokine; and / or (c) trap and reduce motility of the bacterium.

[0011] In another aspect, the present disclosure refers to use of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, in the manufacture of a medicament for treating a condition associated with a bacterial infection, or an immune-related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to(a) trap and kill the bacterium; and / or(b) trap an endotoxin and a pro-inflammatory cytokine; and / or(c) trap and reduce motility of the bacterium.

[0012] In another aspect, the present disclosure refers to a kit comprising the pharmaceutical or adjuvant composition of as disclosed herein, and a dispenser and / or applicator.

[0013] Advantageously, formation of nanonets by the synthetic P-hairpin AMP as disclosed herein is selectively responsive to the presence of bacteria cells, and biomolecules unique to the bacteria membranes (for example, endotoxin, lipopolysaccharide (LPS), or lipoteichoic acid (LT A)). Thus, the synthetic P-hairpin AMP as disclosed herein confer greater spatiotemporal control over the process compared to nanonets-forming peptides found in nature or synthetic nanonets -forming peptidescurrently reported in literature. This property minimizes random self-assembly after the administration of peptides into the body, before they reach the infection site.

[0014] Advantageously, the nanonets formed by the synthetic P-hairpin AMP as disclosed herein can simultaneously trap and kill bacteria. Dual antimicrobial function enables manipulation of the antimicrobial profile of the peptides to meet the clinical needs through rational design of the peptide sequence. It also enables more complete killing before entrapped microbes can escape from the mesh.

[0015] Advantageously, the synthetic P-hairpin AMP as disclosed herein demonstrates selective activity against Gram- negative bacterial pathogens, including clinical strains which are resistant to last-line antibiotics (e.g. colistin).

[0016] Advantageously, the synthetic P-hairpin AMP as disclosed herein additionally demonstrates anti-inflammatory activity. Mechanism of anti-inflammatory activity involves selective trapping of bacterial endotoxin LPS and pro-inflammatory cytokines through electrostatic interactions. Anti-inflammatory cytokines were not trapped by the nanonets.

[0017] Advantageously, the synthetic P-hairpin AMP as disclosed herein form nanonets which can delay the development of resistance among pathogenic bacteria against clinically relevant classes of antibiotics. Mechanism of action involves general reduction in bacterial motility. Motility reduction mechanism of the disclosed peptide nanonets is mainly achieved through physical entrapment of the bacteria cells, which is applicable to a wide range of pathogens and can be more challenging for bacteria to mount resistance against.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0019] Figure 1 illustrates the general structure and sequence of the P-hairpin peptides and the mechanistic pathways of bacteria-induced formation of peptide nanonets.Figure la shows the general structure and sequence of the P-hairpin peptides. Striated black lines between the side strands represent hydrogen bonds between the backbone amides. Sequences of the reverse turns are outlined to the right, with ‘DP’ denoting D- Proline. Orange codes for the position replaced by Ala via Ala scanning.Figure lb is a schematic drawing showing proposed mechanistic pathways of bacteria- induced formation of peptide nanonets.

[0020] Figure 2 illustrates BTT2 nanonets formation triggered by Gram-positive and Gram-negative bacteria.Figure 2a-2b show SEM images of E. coli ATCC 25922 and Figure 2f-2g show SEM images of S. aureus ATCC 29737. Figure 2a, 2f: SEM images after treatment with 20 pM of BTT2 for 30 min; Figure 2b, 2g: without any treatment. Magnification = 15,000x. Scale bar = 1 pm.Figure 2c: Confocal microscopy images of E. coli ATCC 25922; and Figure 2h: Confocal microscopy images of S. aureus ATCC 29737 after treatment with 64 pM of FITC-BTT2 for 1 h. The bacteria membrane was then stained with FM4-64;Figure 2d: Confocal microscopy images of 64 pM of FITC-BTT2 after being incubated for 1 h with 16 pg mL1of LPS. Magnification = 200x. Scale bar = 10 pm.Figure 2e: KI 14 fluorescence emission of selected BTT peptides after incubation for 30 min with LPS, LTA or PBS buffer alone. Experiments were repeated three times. The fluorescence intensities (A x = 360 nm, XEM = 550 nm) are expressed as mean ± SE, after being normalized against the signal of the positive control PAPf39.

[0021] Figure 3 illustrates the diameter of the peptide nanofibrils observed from SEM images after being incubated with different bacteria strains for 30 min.Figure 3a is a bar graph showing compiled average and standard deviation values.Figure 3b-3f are histograms showing distribution of fibril diameter when (b) BTT2 was incubated with E. coli ATCC 25922 (Figure 2a); BTT2-4A with (c) E. coli ATCC 25922 (Figure 12c), and (d) E. coli M2 (Figure 12d); and BTT1-3A with (e) E. coli ATCC 25922 (Figure l id), and (f) S. aureus ATCC 29737 (Figure l ie). Each histogram displays the median and interquartile range (IQR) compiled from 100 instances of diameter measurement across a microscopic field, using ImageJ. Caution was taken to consider only distinctive fibril strands and avoid highly cluttered fibrils.

[0022] Figure 4 are confocal microscopy images.Figure 4a is a confocal microscopy image of 64 pM FITC-BTT2 in Tris-Cl buffer.Figure 4b-4c are confocal microscopy images of E. coli ATCC 25922 after being incubated for 1 h with (b) Tris-Cl buffer alone, or (c) FITC-labelled peptides at 64 pM. Their membranes were stained with FM4-64. Magnification = 200x. Scale bar = 10 pm.

[0023] Figure 5 are SEM images of (Figure 5a) untreated E. coli M2; and (Figure 5b-5d) 20 pM BTT2 after 30 min incubation with (Figure 5b) MHB alone, (Figure 5c) 5 pg mL1LPS (in MHB), and (Figure 5d) 5 pg mL1LTA (in MHB). Magnification = 15,000x. Scale bar = 1 pm.

[0024] Figure 6 illustrates the secondary structures of selected peptides, studied using CD spectroscopy.50 pM of (Figure 6a) BTT1, and (Figure 6b) BTT2 and their Ala scanning analogs were tested in PBS buffer alone, or in the presence of 100 pg mL1LPS. (Figure 6c) 50 pM of fibrillating peptides were tested in the presence of 100 pg mL1LTA.

[0025] Figure 7 illustrates the secondary structure of BTT2 studied using CD spectroscopy.Figure 7a illustrates CD spectra of BTT2 (10 / 20 / 50 pM, in PBS), obtained in the presence of 50 / 100 / 200 pg mL1of LPS. The percentage of (Figure 7b) parallel P-sheet and (Figure 7c) turn secondary structures adopted by the peptide was estimated by using the web-based tool BeStSel (http: / / bestsel.elte.hu / index.php) to analyze the respective spectra.

[0026] Figure 8 illustrates the kinetics of BTT2 amyloido genesis. Extent of amyloid- like structure was monitored using KI 14 dye. 20 pM of BTT2 was incubated with 5 pg mL'1of LPS for 3 h at room temperature. Experiments were repeated three times. The fluorescence intensities (ki.x = 360 nm, ZEM = 550 nm) are expressed as mean ± SE, after being normalized against the signal of the positive control PAPf39.

[0027] Figure 9 are confocal microscopy images of S. aureus ATCC 29737 after being incubated for 1 h with (Figure 9a) Tris-Cl buffer alone, (Figure 9b) FITC-labelled BTT1, or (Figure 9c) FITC-labelled BTT3. Their membranes were stained with FM4-64. Magnification = 200x. Scale bar = 10 pm.

[0028] Figure 10 illustrates the importance of the side strand and turn sequences to BTT2 amyloid formation. (Figure 10a) KI 14 fluorescence of 20 pM of BTT2-As and BTT2-At after being incubated for 30 min in the presence of 5 pg mL'1LPS, 5 pg mL'1LTA, or PBS buffer alone. Experiments were repeated three times. KI 14 fluorescence intensity is expressed as mean ± SE, after being normalized against the signal of the positive control PAPf39. (Figure 10b) CD spectra of 50 pM of BTT2-As and BTT2-At were obtained in the presence of 100 pg mL'1LPS. (Figure 10c, lOd) The hydrodynamicradius of 20 pM of (Figure 10c) BTT2-At, and (Figure lOd) BTT2 aggregates was monitored using DLS in the presence of 5 pg mL1LPS (in PBS).

[0029] Figure 11 illustrates modulation of peptide fibrillating capacity.(Figure 11 a) KI 14 fluorescence emission of Ala scanning analogues of BTT1. Each peptide was incubated for 30 min in the presence of LPS, LTA or PBS buffer alone. Experiments were repeated three times. The fluorescence intensities (Xi.x = 360 nm, XEM = 550 nm) are expressed as mean ± SE, after being normalized against the signal of the positive control PAPf39.SEM images of (Figure 11b) E. coli ATCC 25922, and (Figure 11c) S. aureus ATCC 29737 after being incubated with 20 pM of BTT1-3A for 30 min. Magnification = 15,000x. Scale bar = 1 pm.Agglutination curves when (Figure lid) E. coli ATCC 25922, and (Figure lie) S. aureus ATCC 29737 were treated with BTT1-3A at different concentrations. Experiments were repeated three times, with data expressed as mean ± SE, after being normalized against the ODeoo at 0 h.(Figure Ilf) MIC values against E. coli ATCC 25922 and S. aureus ATCC 29737. Experiments were repeated three times. Colistin was used as the positive control against E. coli, and vancomycin against S. aureus.

[0030] Figure 12 illustrates modulation of antimicrobial activity against drugresistant strains. (Figure 12a) KI 14 fluorescence emission of Ala scanning analogs of BTT2. Each peptide was incubated for 30 min in the presence of LPS, LTA or PBS buffer alone. Experiments were repeated three times. The fluorescence intensities (A x = 360 nm, XEM = 550 nm) are expressed as mean ± SE, after being normalized against the signal of the positive control PAPf39. (Figure 12b) MIC screening was performed against mcr- 7 -positive clinical isolates. Experiments were repeated three times. Colistin was tested as the positive control.SEM images of (Figure 12c) E. coli ATCC 25922, and (Figure 12d) E. coli M2 after being incubated with 20 pM of BTT2-4A for 30 min. Magnification = 15,000x. Scale bar = 1 pm. (Figure 12e) Agglutination curves when E. coli strains were treated with BTT2- 4 A at different concentrations. Experiments were repeated three times, with data expressed as mean ± SE, after being normalized against the ODeoo at 0 h.

[0031] Figure 13 shows that MIC screening was performed against multiple mcr-1 clinical isolates. Experiments were repeated three times.

[0032] Figure 14 are confocal microscopy images of 64 pM of FITC-BTT2-4A after being incubated with (Figure 14a) E. coli ATCC 25922, or (Figure 14b) E. coli M2 for 1 h. The bacteria membrane was stained with FM4-64. Magnification = 200x. Scale bar = 10 pm.

[0033] Figure 15 are confocal microscopy images of 64 pM of FITC-BTT2-4A in (Figure 15a) Tris-Cl buffer alone, or (Figure 15b) 16 pg mL-1 LPS. Magnification = 200x. Scale bar = 10 pm.

[0034] Figure 16 showed trypsin stability of amyloid fibrils. The extent of amyloid- like structure was estimated using KI 14 dye. (Figure 16a) Experimental scheme describing the time points at which different components (20 pM peptide, 5 pg mL1LPS, or 15 nM trypsin) are added to the incubation mixture under various set-ups: (1) peptide is pre-incubated with trypsin before the addition of LPS; (2) peptide, trypsin, and LPS are added at the same time and co-incubated; (3) Peptide and LPS are pre-incubated to generate pre-formed fibrils before the addition of trypsin; and (4) buffer-induced aggregation. (Figure 16b) KI 14 signals of BTT2 under various set-ups. (Figure 16c-16e) KI 14 fluorescence of selected peptides (Figure 16c) pre-formed fibrils (Set-up 3), (Figure 16d) co-incubated with LPS and trypsin (Set-up 2), or (Figure 16e) preincubated with trypsin (Set-up 1). Experiments were repeated three times. The fluorescence intensities (A x = 360 nm, Z.EM = 550 nm) are expressed as mean ± SE, after being normalized against the signal of BTT2 after 30 min of incubation with LPS without trypsin exposure.

[0035] Figure 17 are agglutination curves when E. coli ATCC 25922 was treated with 40 pM of BTT1-3A or BTT2-4A in the presence of 15 nM trypsin. Experiments were repeated three times, with data expressed as mean ± SE, after being normalized against the ODeoo at 0 h.

[0036] Figure 18 are SEM images of E. coli ATCC25922 after treatment with 20 pM of BTT2-4A for 30 min in MHB in the presence of (Figure 18a) 10% FBS, or (Figure 18b) 50% FBS. For each sample slide, at least five separate positions were imaged. Magnification = 15,000 x. Scale bar = 1 pm.

[0037] Figure 19 are SEM images of E. coli M2 incubated for 30 min in ex vivo peritoneal fluid collected from C57BL / 6NTac mice in the presence of 20 pM of (Figure 19a) BTT1-3A and (Figure 19b) BTT2-4A, and (Figure 19c) without any peptide. For each sample slide, at least five separate positions were imaged. Images in (Figure 19b-19c) were taken from two different mice to illustrate reproducibility of the observations. Magnification = 15,000 x. Scale bar = 1 pm. (Figure 19d) SEM image of E. coli M2 after co-incubated with 20 pM of BTT1-3A for 30 min in ex vivo peritoneal lavage (with red blood cells) collected from C57BL / 6NTac mice. For each sample slide, at least five separate positions were imaged. Magnification = 3,000 x. Scale bar = 5 pm.

[0038] Figure 20 showed live / dead staining of bacteria entrapped by peptide nanonets. Confocal microscopy images of E. coli ATCC 25922 after being incubated with FITC-labelled peptides (green) for 1 h in PBS. The bacteria cells were then stained with a mixture of DAPI (blue) and PI (red). Magnification = 200x. Scale bar = 10 pm.

[0039] Figure 21 showed the development of resistance of E. coli M2 against BTT1 and colistin was monitored using serial passage over 14 days.

[0040] Figure 22 showed in vivo antimicrobial efficacy and biosafety of nanonet- forming peptides. (Figure 22a-b) Bacteria burden of C57BE / 6NTac mice (n = 4 or 5 mice per treatment group) induced with peritonitis. After given i.p. inoculation of E. coli M2 (109CFU mF1), mice were given injections of 2.25 mg kg1BTT1-3A, BTT2-4A, colistin, or saline (vehicle control). Peritoneal fluid and blood were collected post-mortem (Figure 22a) 2 h after a single i.p. injection, or (Figure 22b) 5 h after multiple-injection regimen (three i.p. injections 1 h apart). Statistical significance of differences between data groups were evaluated using one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test (ns: p > 0.05, *: p < 0.05, **: p < 0.01, p < 0.001). (Figure 22c- g) Blood biochemical analysis of mice (n = 3) at 24 h post-treatment with 2.5, 5, or 10 mg kg1of BTT1-3A and BTT2-4A, and saline control. Data are presented as mean ± SD. (Figure 22h) Kidney histology images after treatment saline and various concentrations of peptides.

[0041] Figure 23 showed organ bacteria burden in C57BL / 6 mice (n = 4 for saline control, n = 5 per treatment group) induced with peritonitis. After inoculated with 109CFU mL1of E. coli M2 (i.p. injection) for 30 min, the mice were given a single i.p. injection of 2.25 mg kg1of BTT1-3A, BTT2-4A, colistin, or saline (vehicle control). For negative control group, the mice were injected with saline. After 2 h of treatment, the mice were euthanized for organ collection, followed by tissue homogenization, and quantification of bacteria load by colony counting on LB agar plates.

[0042] Figure 24 are TEM images of 20 pM BTT2-4A aggregates when incubated in PBS buffer alone or in the presence of 5 pg / mL LPS. Scale bar = 2 pm.

[0043] Figure 25 shows that peptide nanonets entrap LPS via two different mechanisms. (Figure 25a) FITC-LPS trapping index of peptide was computed by measuring fluorescence emission of FITC-LPS in the supernatant (ki.x= 488 nm and XEM = 520 nm) after 1 h of incubating LPS (2 pg / mL) with peptide at different concentrations. (Figure 25b) Confocal microscopy images of 64 pM peptide incubated with 32 pg / mL FITC-LPS for 1 h. Magnification = 200x. Scale bar = 20 pm. (Figure 25c) FITC-LPS trapping index of pre-formed nanonets was determined after 1 h pre-incubation with LPS or LTA (2 pg / mL) followed by 1 h incubation with FITC-LPS (2 pg / mL). For (Figure 25a) and (Figure 25c), three repeats were conducted in duplicates and data were expressed as mean ± SE, and data points were fitted to a non-linear regression inhibitor concentration versus response model for computation of EC50 values. (Figure 25d) Proposed dual mechanism of LPS trapping by the peptide nanonets via scaffold integration and post-binding.

[0044] Figure 26 illustrates that vortexing release FITC-LPS from nanonets pellet back to solution. After 1 h of incubating LPS (2 ug / mL) with peptide at different concentrations, sample was centrifuged and fluorescence emission of FITC-LPS (XEX= 488 nm and XEM = 520 nm) in the supernatant was measured, before and after vortexing the centrifuged solutions. Results were normalized against peptide-free controls subjected to identical processing steps. Three independent experiments were performed, and data are presented as mean ± SE.

[0045] Figure 27 illustrates the extent of amyloid formation by fibrillating peptides. KI 14 fluorescence of peptide at different concentrations was measured (XEX = 360 nm and XEM = 550 nm) after 1 h of incubation with LPS (2 pg / mL), then normalized against the positive control (20 pM BTT2, 5 pg / mL LPS). Three independent experiments in duplicates were conducted and data are presented as mean ± SE.

[0046] Figure 28 illustrates the trapping of cytokines by LTA-induced nanonets. After 1 h of incubating LTA (5 ug / mL) and cytokine (2 ng / mL) with peptide at different concentrations, sample was centrifuged and the cytokine remaining in the supernatant was quantified using ELISA. Results were normalized against peptide-free controls subjected to identical processing steps. Three independent experiments were performed in duplicates and data are presented as mean ± SE. Statistical significance of differences between data groups were evaluated using ANOVA followed by Tukey post-hoc test (**: p < 0.01, ***: p < 0.001, ****; p < 0.0001).

[0047] Figure 29 illustrates that peptide nanonets nullify the colistin-inactivating effect of extracellular LPS. (Figure 29a) Proposed mechanism of fibrillating peptide rescuing the activity of colistin. (Figure 29b) MICs of BTT2-4A and colistin against different bacteria strains were determined either in the absence or presence of 100 pg / mL cell-free LPS. (Figure 29c) MICs of colistin were determined in the presence of free LPS (100 pg / mL) and varying sub-MIC concentrations of BTT2-4A. MIC assays were conducted in duplicates in three independent experiments.

[0048] Figure 30 illustrates that peptide nanonets display selective binding towards pro-inflammatory cytokines. (Figure 30a) Isoelectric point (PI) and net charge of the cytokines tested in this work. (Figure 30b) Cytokine trapping by LPS -induced nanonets. After 1 h of incubating LPS (5 pg / mL) and cytokine (2 ng / mL) with peptide at different concentrations, sample was centrifuged and the cytokine remaining in the supernatant was quantified using ELISA. Results were normalized against peptide-free controls subjected to identical processing steps. Three independent experiments were performed in duplicates and data are presented as mean ± SE. Statistical significance between samples were evaluated using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test (*: p < 0.05, **: p < 0.01).

[0049] Figure 31 illustrates that peptide nanonets suppress LPS-induced release of pro-inflammatory cytokines by RAW cells. (Figure 31a) Effect of peptide post-treatment on LPS-induced TNF-a and IL-6 production by RAW 264.7 cells at different LPS concentrations. After 1 h of incubating the cells with LPS, peptide was added followed by 23 h incubation before cytokine in the culture media was quantified. Results were normalized against the peptide-free negative control. Three independent experiments were conducted, and data are presented as mean ± SE. Statistical significance between samples were evaluated using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). (Figure 31b) Proposed anti-inflammatory mechanisms of the fibrillating peptides: (1) LPS trapping by the nanonets, (2) Cytokine trapping by the nanonets, and (3) Intracellular effect by soluble peptide molecules. (Figure 31c) Quantification of immune cells and cytokine production in bronchoalveolar lavage fluids (BALF) collected from mice after 4 h treatment (n = 5-6 per group). Mice were given LPS only or saline as controls. Data are presented as mean ± SD. Statistical significance between samples were evaluatedusing one-way analysis of variance (ANOVA) followed by Tukey post-hoc test, and p values are shown in figure.

[0050] Figure 32 illustrates the peptide pre-treatment effect on RAW cells. Effect of peptide pre-treatment on LPS-induced TNF-a and IL-6 production by RAW 264.7 cells at different LPS concentrations. After 1 h of incubating the cells with peptide, LPS was added followed by 24 h incubation before cytokine in the culture media was quantified. Results were normalized against the negative control in the absence of peptides. Three independent experiments were conducted and data are presented as mean ± SE.

[0051] Figure 33 illustrates lack of cytotoxicity at tested peptide concentrations. Cell viability of RAW 264.7 cells was determined 24 h after incubation with LPS and peptide at different concentrations. Three independent experiment were conducted and data are presented as mean ± SE.

[0052] Figure 34 illustrates: (Figure 34A) Heatmap displaying FICI values for combinations between BTT peptides and antibiotics against E. coli M2; FICI value of < 0.5 indicates synergy. (Figure 34B) Serial passage showing development of resistance over 18 days. (Figure 34C) Live-image fluorescence tracking of movement by individual cells. Motility of individual cells was quantified by MTrackJ. (Figure 34D) Soft agar assay to test bacteria motility, with diameter of spread quantified. (Figure 34B-D) Rifampicin was tested alone or in combination with sub-MIC concentrations of BTT peptides. Cultures at passge 18 of serial passage in (Figure 34B) were studied in (Figure 34C-D).

[0053] Figure 35 illustrates antimicrobial activity and synergism of BTT peptides with Rifampicin against bacteria strains after 18-days passage treated with different combinations of BTT peptides with Rifampicin. (Figure 35A) Scheme of serial passage of E. coli M2 treated with different combinational therapies for 18 days. P18_R, P18_R1, P18_R4, P18_R1-3A and P18_R2-4A are names of E. coli M2 clones after 18 days of serial passage with different treatments. (Figure 35B) Fold-change in FICI of Rifampicin with BTT peptides against Pl 8 clones of E. coli M2 compared with initial FICI on Day 0. (Figure 35C) Biofilm formation capacity of Pl 8 clones was quantified using crystal violet, then compared with the control E. coli M2 strain without any serial passage treatment.

[0054] Figure 36 illustrates (Figure 36A) Serial passage showing development of resistance in E. coli when different peptides at sub-lethal concentrations were combinedwith ceftazidime over 18 days. (Figure 36B) Soft agar assay to test bacteria motility, with diameter of spread quantified.

[0055] Figure 37 illustrates serial passage showing development of resistance in E. coli when different peptides at sub-lethal concentrations were combined with streptomycin over 10 days.

[0056] Figure 38 illustrates agglutination curves when (Figure 38a) E. coli ATCC 25922, and (Figure 38b) S. aureus ATCC 29737 were treated with 80 pM of BTT2-2A or 20 pM of BTT1; and (Figure 38c) E. coli M2 was treated with 40 pM of BTT1-3A. Experiments were repeated three times, with data expressed as mean ± SE, after being normalized against the OD600 at 0 h.DETAILED DESCRIPTION

[0057] This disclosure features the sequence design, physical and functional characterizations of synthetic nanonet-forming peptides. This includes demonstration of multiple functionalities of the peptides in both in vitro and in vivo context: 1) bacteria- trapping activity, 2) bactericidal activity, 3) anti-inflammatory activity, and 4) ability to delay the development of anti-antibiotic resistance. A series of 15 to 16 residue-long synthetic P-hairpin peptides is herein constructed with the ability to self-assemble into nanonets in response to the presence of bacteria, enabling spatiotemporal control over microbial killing. The bacterial membrane components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were shown to play a major role in determining the amyloid- nucleating capacity as triggered by Gram-positive and Gram-negative bacteria respectively. These nanonets displayed both trapping and killing functionalities, hence offering a direct improvement from the trap-only biomimetics in literature. By substituting a single turn residue of the non-amyloidogenic BTT1 peptide, the nanonet- forming BTT1-3A analog was produced with comparable antimicrobial potency. With the same sequence manipulation approach, BTT2-4A analog modified from BTT2 peptide showed improved antimicrobial potency against colistin-resistant clinical isolates. The peptide nanonets also demonstrated robust stability against proteolytic degradation, and promising in vivo efficacy and biosafety profile. Overall, these bacteria- responsive peptide nanonets are promising clinical anti-infective alternatives for circumventing antibiotic resistance.

[0058] In one aspect, the present disclosure refers to a synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component to initiate amyloid nucleation; and (b) a structural module comprising a first side strand and a second side strand for P-sheet formation and molecular stacking during fibrillation; wherein the synthetic P-hairpin AMP comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of the bacterial membrane component or a bacterium.

[0059] As used herein, the term “peptide” includes a polymer of amino acids linked through peptide bonds or modified peptide bonds, produced synthetically. The term "amino acid” can include D-amino acids.

[0060] The beta hairpin is a small protein structure motif in which two P-strands, linked by a turn or a short loop, fold to form hydrogen bonds with each other. The two P- strands that are adjacent in primary structure, oriented in an antiparallel direction (the N- terminus of one sheet is adjacent to the C-terminus of the next), and linked by a short loop of two to five amino acids. Beta hairpins can occur in isolation or as part of a series of hydrogen bonded strands that collectively comprise a beta sheet.

[0061] The hairpin turn of the synthetic P-hairpin AMP as disclosed herein acts as the "recognition module" for interacting with bacterial membrane components to initiate peptide conformational change and amyloid nucleation. In one example, the hairpin turn comprises three amino acids. In another example, the hairpin turn comprises four amino acids. In another example, the hairpin turn comprises the sequence of SEQ ID NO: 3. In another example, the hairpin turn comprises the sequence of SEQ ID NO: 4.• Sequence of the hairpin turn of BTT1-3A (SEQ ID NO: 3): LTA• Sequence of the hairpin turn of BTT2-4A (SEQ ID NO: 4): VpPA (‘p’: d- Proline).

[0062] The two side strands of the synthetic P-hairpin AMP as disclosed herein act as the ‘structural module’ for P-sheet formation and molecular stacking during the fibrillation process. In one example, the first side strand at the N-terminal of the AMP comprises the sequence of SEQ ID NO: 5, and the second side strand at the C terminal of the AMP comprises the sequence of SEQ ID NO: 6.• Sequence of the first side strand at the N-terminal of the synthetic P-hairpin AMP as disclosed herein (SEQ ID NO: 5): LKLKLK• Sequence of the second side strand at the C terminal of the synthetic P-hairpin AMP as disclosed herein (SEQ ID NO: 6): KLKLKL

[0063] In one example, the synthetic P-hairpin AMP as disclosed herein is BTT1-3A, which comprises the sequence of SEQ ID NO: 1 (LKLKLKLTAKLKLKL). In another example, the synthetic P-hairpin AMP as disclosed herein is BTT2-4A, which comprises the sequence of SEQ ID NO: 2 (LKLKLKVpPAKLKLKL) (‘p’: d-Proline).

[0064] Only when the synthetic P-hairpin AMP as disclosed herein in solution encounter a bacterial membrane component or a bacterium, amyloid nucleation is initiated. Amyloid fibrils undergo elongation as hairpin peptides stack at the tail-end of the fibril. Once the nanofibrils grow sufficiently long, they start to form physical crosslinks. Eventually, a mature interlocking network of fibrils entraps other bacteria in the surroundings. The mature interlocking network of fibrils is termed as "nanonets" to trap and promote bacterial agglutination. In one example, the bacterial membrane component is lipoteichoic acid (LTA) on the bacterial surface of a Gram-positive bacterium. In another example, the bacterial membrane component is lipopolysaccharide (LPS) on the bacterial surface of a Gram-positive bacterium. In one example, the bacterium is a Grampositive bacterium. In another example, the bacterium is a Gram-negative bacterium.

[0065] Advantageously, formation of nanonets is selectively responsive to the presence of bacteria cells, and biomolecules unique to the bacteria membranes (for example, endotoxin, lipopolysaccharide (LPS), or lipoteichoic acid (LTA)). Thus, the synthetic P-hairpin AMP as disclosed herein confer greater spatiotemporal control over the nanonets-forming process compared to nanonets-forming peptides found in nature or synthetic nanonets-forming peptides currently reported in literature. This property minimizes random self-assembly after the peptide being administered into the body, before reaching the infection site.

[0066] Synthetic peptide nanofibrils have been widely investigated for various biomedical applications, but remain relatively underexplored for anti-infective purposes. This disclosure is the first to offer microscopic evidence of extensively cross-linked synthetic nanonets suitable for anti-infective applications. Despite previous attempts to mimic naturally occurring nanonets, most synthetic peptide-based materials were observed to only form disjointed short nanofibrils, which are restricted to the bacterialsurfaces and incapable of physically immobilizing the bacteria, instead of expansive nanonets like our BTT peptides. This invention holds potential to advance the development of synthetic nanonets as anti-infective biomaterials that can help tackle the antibiotic resistance crisis.

[0067] The nanonets formed by the synthetic P-hairpin AMP as disclosed herein displayed both bacterial trapping and killing functionalities, hence offering a direct improvement from the trap-only biomimetics in literature. The synthetic P-hairpin AMP as disclosed herein display effective antimicrobial activity against a wide range of bacteria species because of the common occurrence and essentiality of LPS and LTA to the biology of Gram-negative (Zhang G, 2013) and Gram-positive bacteria (Percy, M.G. 2014) respectively. The presence of these membrane components enables the formation of trap-and-kill nanonets by the disclosed peptides. In one example, the synthetic P- hairpin AMP as disclosed herein can form nanonets to trap and kill a Gram-positive bacterium selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Eisteria monocytogenes. In another example, the synthetic P-hairpin AMP as disclosed herein can form nanonets to trap and kill a Gram-negative bacterium selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera. In another example, the synthetic P-hairpin AMP as disclosed herein can form nanonets which display potent antimicrobial and agglutinating activity against an antibioticresistant bacterium such as a clinical isolate. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Escherichia coli. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Klebsiella aerogenes. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Pseudomonas aeruginosa. In another example, the antibioticresistant bacterium is colistin- and / or carbapenem-resistant Acinetobacter baumannii. Inanother example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Klebsiella pneumonia. The synthetic P-hairpin AMP as disclosed herein are effective against clinical isolates with colistin and / or carbapenem resistance gene. The synthetic P-hairpin AMP as disclosed herein are effective against Gram-negative clinical isolates with only carbapenem resistance gene because their resulting mutations do not cause any relevant change to the bacterial membranes.

[0068] Advantageously, the nanonets formed by the synthetic P-hairpin AMP as disclosed herein can simultaneously trap and kill bacteria. Dual antimicrobial function enables manipulation of the antimicrobial profile of the peptides to meet the clinical needs through rational design of the peptide sequence. It also enables more complete killing before entrapped microbes can escape from the mesh.

[0069] In another aspect, the present disclosure refers to a pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP as disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0070] As used herein, "adjuvant composition" refers to a composition comprising the synthetic P-hairpin AMP as disclosed herein, which works synergistically with an antibiotic to treat a bacterial infection, or to delay the development of antibioticresistance.

[0071] As used herein, the term "pharmaceutically acceptable carrier or diluent" refers to molecular entities and compositions that are compatible with the synthetic P-hairpin AMP as disclosed herein, physiologically tolerable and do not typically produce an allergic, toxic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; glycine; hyaluronic acid; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol,propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0072] The pharmaceutical or adjuvant composition as disclosed herein may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In one example, the pharmaceutical or adjuvant composition as disclosed herein is for intravenous injection. In another example, the pharmaceutical or adjuvant composition as disclosed herein is for intraperitoneal injection. In another example, the pharmaceutical or adjuvant composition as disclosed herein is for intratracheal administration.

[0073] For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

[0074] Some examples of suitable carriers, diluents, excipients for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

[0075] Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and / or time delay agents. Suitable binders include gum acacia, gelatine, com starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include com starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginicacid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0076] Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

[0077] Suspensions for oral administration may further comprise dispersing agents and / or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl- pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or dioleate, -stearate or -laurate and the like.

[0078] The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

[0079] The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

[0080] Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and / or fungicidal agent and / or any other suitablepreservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

[0081] Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and / or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.

[0082] Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.

[0083] The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

[0084] The compositions may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.

[0085] In another aspect, the present disclosure refers to a method for treating a condition associated with a bacterial infection, or an immune -related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, comprising administering the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to (a) trap and kill the bacterium; and / or (b) trap an endotoxin and a pro- inflammatory cytokine; and / or (c) trap and reduce motility of the bacterium.

[0086] In another aspect, the present disclosure refers to use of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, in the manufacture of a medicament for treating a condition associated with a bacterial infection, or an immune-related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to(a) trap and kill the bacterium; and / or(b) trap an endotoxin and a pro-inflammatory cytokine; and / or(c) trap and reduce motility of the bacterium.

[0087] In another aspect, the present disclosure refers to the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, for use in treating a condition associated with a bacterial infection, or an immune -related disease, or for use in delaying the development of antibiotic resistance in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to(a) trap and kill the bacterium; and / or(b) trap an endotoxin and a pro-inflammatory cytokine; and / or(c) trap and reduce motility of the bacterium.

[0088] As used herein, the term "treating" and grammatical variations of that term, refer to administration of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to a subject as describedherein by any appropriate means as described herein. Such treatment includes any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

[0089] As used herein, the term "subject" refers to patients of human or other mammal and includes any individual in need of treatment of a condition, disorder or disease (such as a bacterial infection, or an immune -related disease) using the methods as disclosed herein. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs), laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters), companion animals (eg. cats, dogs) and captive wild animals (eg. foxes, deer, dingoes). In a particular example, the subject is a human.

[0090] As used herein, the term "administering" and variations thereof refers to contacting, applying, delivering or providing the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to the subject as disclosed herein, by appropriate means.

[0091] The first function of the multi-functional nanonets formed by the synthetic P- hairpin AMP as disclosed herein is bacterial trapping and killing, as discussed herein. In one example, the bacterial infection is caused by a Gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Eisteria monocytogenes. In another example, the bacterial infection is caused by a Gram-negative bacteria selected from the group consisting of Escherichia coll, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera. In some examples, the bacterial infection is caused by an antibiotic -resistant bacterium. In a particular example, the antibiotic -resistant bacteriumis colistin- and / or carbapenem- resistant Escherichia coli. In another example, the antibiotic -resistant bacterium is colistin- and / or carbapenem- resistant Klebsiella aerogenes. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Pseudomonas aeruginosa. In another example, the antibioticresistant bacterium is colistin- and / or carbapenem-resistant Acinetobacter baumannii. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Klebsiella pneumonia.

[0092] In some examples, the condition associated with a bacterial infection is selected form the group consisting of sepsis, septic arthritis, listeriosis, skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, abscesses, toxic shock syndrome (TSST-1), urinary tract infection (UTI), bloodstream infections, otitis media, sinusitis, meningitis, bacterial endocarditis, pharyngitis, cellulitis, impetigo, erysipelas, scarlet fever, necrotizing fasciitis, glomerulonephritis, rheumatic fever, myocarditis, arrhythmias, gastroenteritis, peritonitis, brucellosis, cat-scratch disease, cholera, Legionnaires' disease, pertussis, salmonella infections, shigellosis, tularemia, typhoid fever, acne, athlete's feet, onychomycosis, caries, periodontal disease, cold sore, rhinitis, and pink eye.

[0093] In certain examples, the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition as disclosed herein is administered to the patient after treatment of a bacteria infection, either with the composition of the invention or with an alternative therapy, to prevent further recurrence.

[0094] Advantageously, the synthetic P-hairpin AMP as disclosed herein can simultaneously trap and kill bacteria, enabling more complete killing before entrapped microbes can escape from the mesh. In addition, Gram-negative pathogens are recognized by the WHO to have high clinical priority, as the development pipeline for antibiotics against Gram-negative infections has been alarmingly lacking. The synthetic P-hairpin AMP as disclosed herein demonstrates selective activity against Gram-negative bacterial pathogens, including clinical strains which are resistant to last-line antibiotics (e.g. colistin).

[0095] The second function of the multi-functional nanonets formed by the synthetic P-hairpin AMP as disclosed herein is anti-inflammatory function, which complements its bacterial trapping and killing activity in treating infections. None of the synthetic nanonet peptides reported in literature have been shown to have anti-inflammatory bioactivity. Assuch, the synthetic P-hairpin AMP as disclosed herein can be used to treat an immune- related disease.

[0096] In one example, the immune-related disease is a bacterial infection-associated inflammatory disease. In one example, the bacterial infection-associated inflammatory disease is caused by endotoxins. Endotoxins (lipopolysaccharides, LPS) are agents of pathogenicity of Gram-negative bacteria, implicated in the development of Gramnegative shock. Endotoxin reacts with lipopolysaccharide-sensitive cells producing endogenous mediators such as tumour necrosis factor alpha (TNFa). Macrophages are cells mediating the toxic activities of LPS and TNFa is the primary mediator of the lethal action of endotoxin. During a Gram-negative bacterial infection, LPS released from bacteria cells can enter the bloodstream and cause septic complications with excessive host inflammatory responses. In one particular example, the bacterial infection-associated inflammatory disease is sepsis.

[0097] As discussed herein, the synthetic P-hairpin antimicrobial peptides (AMPs) as disclosed herein has the ability to self-assemble into amyloid-like nanonets in response to bacterial endotoxins. The side strands of the synthetic P-hairpin AMPs as disclosed herein carry three Leu-Lys repeat units, and the resulting polycationicity proved integral to facilitating interactions between the peptides and endotoxins. The disclosed fibrillating peptides could display anti-inflammatory functionality by capturing free endotoxins and pro-inflammatory cytokines in solution via electrostatic attractions. Endotoxin trapping can impede its engagement with host immune and endothelial cells, including activation of Toll-like receptor 4 (TLR4) and induction of nitric oxide production. By alleviating the damage of endotoxemia, the multifunctional nanonets formed by the synthetic P- hairpin AMPs as disclosed herein offer a holistic anti-infective therapy that mitigates both upstream and downstream ramifications of infectious pathogenesis.

[0098] In one example, the nanonets formed by the synthetic P-hairpin AMPs as disclosed herein trap an endotoxin LPS. Endotoxin trapping can impede its engagement with host immune and endothelial cells and ensuing unwanted immune response. In another example, after trapping and sequestering endotoxin such as LPS, antagonistic interactions between an antibiotic and extracellular LPS can be nullified, and the activity of the antibiotic can be restored.

[0099] In another example, the nanonets formed by the synthetic P-hairpin AMPs as disclosed herein trap a pro-inflammatory cytokine selected from the group consisting oftumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin- 1 a (IL- 1 a), interleukin- ip (IL-ip), interleukin- 12a (IL-12α), interleukin- 12P (IL-12P), interleukin- 18 (IL-18), and high mobility group box 1 (HMGB1). In another example, the nanonets formed by the synthetic P-hairpin AMPs as disclosed herein do not trap an anti-inflammatory cytokine selected from the group consisting of interleukin-4 (IL-4), interleukin- 10 (IL- 10), interleukin- 11 (IL-11), interleukin- 13 (IL-13), transforming growth factor beta 1 (TGF-pi), and interleukin- 18BP (IL-18BP). Pro-inflammatory cytokines (e.g. TNF-a, IL- 6, IL- la, IL-ip, IL- 12a, IL-12P, IL- 18 and HMGB1) mostly carry negative charges, whereas many anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13, TGF-pi and IL- 18BP) are positively charged. The apparent trapping selectivity of the nanonets formed by the synthetic P-hairpin AMPs as disclosed herein towards the pro-inflammatory cytokines is indicative of electrostatic interactions mediated by the cationic Lys residues on the peptides.

[0100] The third function of the multi-functional nanonets formed by the synthetic P- hairpin AMP as disclosed herein is delaying the development of antibiotic resistance in a subject with bacterial infection. Most antibiotic adjuvants in clinical practices help increase potency of the active pharmaceutical ingredients (API) through synergistic interactions. However, they do not show ability to protect the API against resistance development over prolonged antibiotic treatment.

[0101] The mechanism of delaying the development of antibiotic resistance is due to the nanonets formed by the synthetic P-hairpin AMP as disclosed herein trapping and reducing the motility of the bacteria. The motility reduction mechanism is mainly achieved through physical entrapment of the bacteria cells, which is applicable to a wide range of pathogens and can be more challenging for bacteria to mount resistance against. In one example, the synthetic P-hairpin AMP as disclosed herein may be co-administered with an antibiotic. The antibiotics may be of various classes. In another example, the antibiotic belongs to a class selected from the group consisting of aminoglycoside, macrolide, fluoroquinolone, P-lactam, glycopeptide, oxazolidinone, and ansamycin. In another example, aminoglycoside is selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, plazomicin, streptomycin and tobramycin. In another example, macrolide is selected from the group consisting of azithromycin, clarithromycin, erythromycin and fidaxomicin. In another example, fluoroquinolone is selected from the group consisting of ciprofloxacin, delafloxacin, gemifloxacin,levofloxacin, moxifloxacin, norfloxacin, and ofloxacin. In another example, P-lactam is selected from the group consisting of carbapenem, cephalosporin, cephamycin, clavam, monobactam and penicillin. In another example, glycopeptide is selected from the group consisting of dalbavancin, oritavancin, teicoplanin, telavancin and vancomycin. In another example, oxazolidinone is selected from the group consisting of linezolid and tedizolid. In another example, ansamycin is selected from the group consisting of streptovaricin, rifamycin, naphthomycin, geldanamycin and ansamitocin. In some examples, the antibiotic is selected from the group consisting of colistin, ceftazidime, ciprofloxacin, streptomycin, clarithromycin, vancomycin, linezolid, rifampicin, and rifabutin. In a particular example, the antibiotic is colisitin. In one example, synthetic P- hairpin AMP as disclosed herein may be administered simultaneously with an antibiotic as disclosed herein.

[0102] To achieve the first function and second function of the multi-functional nanonets formed by the synthetic P-hairpin AMP as disclosed herein, i.e. for treating a condition associated with a bacterial infection, or an immune -related disease, the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg. In one example, the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 2.5-4 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 3-5 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 4-6 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 5-7 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 6-8 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 7-9 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 8-10 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 2.5 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 5 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 10 mg / kg.

[0103] The minimum inhibitory concentration (MIC) of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is 4-32 pM. In one example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is 4-10 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is 10-20 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosedherein towards a bacteria is 20-30 pM. In another example, the MIC of the synthetic P- hairpin AMP as disclosed herein towards a bacteria is about 4 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 6 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 8 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 10 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 12 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 14 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 16 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 18 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 20 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 22 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 24 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 26 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 28 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 30 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria is about 32 pM.

[0104] To achieve the third function of the multi-functional nanonets formed by the synthetic P-hairpin AMP as disclosed herein, i.e. for delaying the development of antibiotic resistance, the synthetic P-hairpin AMP is administered in a sub-inhibitory concentration of 1-4 pM. In one example, the sub-inhibitory concentration is 1 pM. In another example, the sub-inhibitory concentration is 2 pM. In another example, the sub- inhibitory concentration is 3 pM. In another example, the sub-inhibitory concentration is 4 pM. When used together with an antibiotics, to delay the development of antibiotic resistance, the synthetic P-hairpin AMP as disclosed herein is not administered as the main active therapeutic, but as an adjuvant in combination to support the function of the main antibiotic. Hence, it is given at sub-inhibitory concentration.

[0105] The synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered via a route selected from the group consisting ofintravenous, intraperitoneal, intratracheal, intramuscular, intradermal, subcutaneous, oral, topical, and intranasal administration.

[0106] The synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered at a frequency depending on the specific disease to be treated and / or the administration frequency of an antibiotic. In some examples, the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered at a frequency selected from the group consisting of one, two, three, four, or six times a day.

[0107] The synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered for a duration depending on the specific disease to be treated and / or the administration duration of an antibiotic. In some examples, the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered for a duration selected from the group consisting of about 1 day, about 2 days, about 1 week, about 10 days, about 15 days, about 1 month, about 3 months, about 6 months, about 1 year, or greater than 1 year. The treatment may be continual for days, weeks, months, or even years.

[0108] In another aspect, the present disclosure refers to a kit comprising the pharmaceutical or adjuvant composition of as disclosed herein, and a dispenser and / or applicator. The dispenser and / or applicator facilitates dispensing and applying the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein, and may be designed for convenient application and long-term storage of the AMP and compositions. The packaging or dispenser may include a bottle, tube, spray bottle, a syringe or other dispenser. In certain examples, the composition is packaged in a concentrated form, and diluted to a desired concentration upon use by the end user. In some examples, the composition is formulated and packaged in a manner suitable for long-term storage to maintain efficacy of the composition.

[0109] The kit may further include additional components to facilitate application of the composition to the affected area, such as, for example, a brush, sponge, cotton swab, syringe, needle, nebulizer, or the like.

[0110] Other components of the kit may include, but are not limited to, one or more antibiotics selected from the group consisting of colistin, ceftazidime, ciprofloxacin, streptomycin, clarithromycin, vancomycin, linezolid, rifampicin, and rifabutin.

[0111] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0112] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0113] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0114] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0115] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0116] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0117] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0118] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0119] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0120] Example 1 - Experimental Section 1 - "Trap and Kill" Activity

[0121] 1.1 Materials

[0122] Glutaraldehyde (grade I, for use as electron microscope fixative), cation- adjusted Mueller Hinton broth (MHB), Mueller Hinton agar (MHA), LPS from E. coli O111:B4, LTA from S. aureus, cyclophosphamide (Pharmacopoeia reference standard), and PI were obtained from Sigma (St. Louis, MO). Osmium tetroxide (OsO4) was obtained from Ted Pella (Redding, CA). Poly-l-lysine was obtained from Electron Microscopy Sciences (Hatfield, PA). DAPI was obtained from Invitrogen (Carlsbad, CA). FM4-64 was obtained from Molecular Probes (Eugene, OR). KI 14 was obtained from Santa Cruz (Dallas, TX). Trypsin-EDTA was obtained from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was obtained from ThermoFisher Scientific (Waltham, MA)

[0123] Peptides, including fluorescently labelled peptides, were synthesized by GL Biochem (Shanghai, China). Using reversed-phase high-performance liquid chromatography (RP-HPLC), their purity was determined to be at least 95% by the manufacturer.

[0124] 1.2 Bacterial Strains

[0125] E. coli ATCC 25922 and S. aureus ATCC 29737 were obtained from American Type Culture Collection (Manassas, VA). E. coli and K. pneumoniae clinical isolatescarrying mcr-1 were kind gifts from Dr. Jeanette Teo (National University Hospital, Singapore).

[0126] 1.3 MIC determination

[0127] The MICs of the peptides were determined using broth microdilution method.

[0052] Stock solutions of peptides were diluted into MHB in a 96-well plate to yield twice the highest tested concentration. Two-fold serial dilution of the peptides was performed. Bacteria culture in mid-log phase was diluted to obtain an initial inoculum of 106colony-forming units (CFU) mL1. A volume of bacteria inoculum equal to peptide sample volume was added to each well. Following 18-20 h of incubation (37°C, 120 rpm), ODeoo values were measured using Tecan Infinite M200 (Mannedorf, Switzerland), or Hidex Sense 425-301 (Turku, Finland). MIC value for each peptide was determined as the lowest peptide concentration achieving at least 90% reduction in ODeoo compared to that of drug-free control.

[0128] 1.4 Field-emission SEM

[0129] SEM sample processing was performed following the below procedure: Bacteria culture in mid-log phase was diluted to yield an initial ODeoo value of 0.1. Peptides (20 pM) were added to the diluted culture (1.5 mL). After being incubated for 30 min at 37°C with shaking, the incubation mixtures were washed thrice in phosphate - buffered saline (PBS). The samples were fixed overnight in 2.5% glutaraldehyde (in PBS) at 4°C. After fixation, the samples were washed twice in PBS, then re-suspended in 500 pL PBS. Sample droplets were added to cover the surface of cover slips, which had been previously coated with poly-l-lysine. After 30 min, unbound cells were washed away with PBS. Post-fixation with 1% OsO4 was performed for 30 min. After removal of OsCL, the cover slips were washed twice with distilled water. The samples were dehydrated using increasing concentrations of ethanol (50%, 75%, 95%, 3x100%), followed by critical point drying using Leica EM CPD300 (Wetzlar, Germany), and sputter gold coating using Leica EM ACE200 (Wetzlar, Germany). The samples were viewed using JEOL JSM- 6701F (Tokyo, Japan). For each sample slide, at least five independent fields were imaged to ensure representativeness. Images were processed using ImageJ to add scale bar (National Institutes of Health, Bethesda, MD).

[0130] 1.5 Confocal microscopy

[0131] Bacteria culture in mid-log phase was diluted to yield an initial ODeoo of 0.1. FITC-tagged BTT peptide was incubated with either bacteria sample (in MHB) or LPS(16 pg ml / 1in 1 mM Tris-HCl buffer) for 1 h. After washing thrice with 1 mM Tris-HCl, bacteria cells were stained with FM4-64 (1 pg ml / 1) (Molecular Probes, T3166) for 1 h. Cell-free samples were not subjected to this step. The sample was mounted onto glass slides, and air-dried. With mounting media added, the sample was viewed with FV 1000 TIRF inverted laser scanning confocal microscope (Olympus, Tokyo, Japan), using a lOOx oil immersion lens. Filter sets (Lx = 561 nm, ZEM = 603 nm) and (XEX= 488 nm, XEM = 520 nm) were used to visualize the FM4-64-stained cells and FITC-tagged peptides respectively.

[0132] Live / dead staining experiment followed a similar confocal sample preparation protocol. Instead of FM4-64, bacteria cells were stained with a mixture of DAPI (15 pg mL1) and PI (20 pg mL-1) in PBS buffer for 1 h. Filter sets (XEX = 405 nm, ZEM = 461 nm), (XEX = 561 nm, XEM = 603 nm), and (XEX = 488 nm, XEM = 520 nm) were used to visualize D API-stained cells, Pl-stained cells, and FITC-tagged peptides respectively.

[0133] 1.6 CD spectroscopy

[0134] Using CD spectroscopy, secondary structures of peptide (50 pM) was determined in IxPBS buffer. The peptide was measured alone, or in the presence of LPS or LTA (0.2 mg mL1). Using a quartz cell (1 mm path length), CD spectra in the 190-260 nm range were measured using Jasco CD Spectrometer (JASCO, Easton, MD) at a scanning speed of 60 nm min1. Results were averaged from 3 runs per sample. The recorded spectra of observed ellipticity were converted to mean residue ellipticity usingthe following equation: where 6Lis the mean residue ellipticity (deg cm2dmol1), 0obs is the observed ellipticity corrected for the blank at a given wavelength (mdeg), MRW is residue molecular weight (i.e. AfWnumber of amino acids), c is peptide concentration (mg mL1), and I is the path length (cm).

[0135] 1.7 K114 fluorescence assayThe amount of peptide amyloids was quantified using the KI 14 fluorescence dye. Stock solution of KI 14 (10 mM) was prepared in DMSO. Peptide (20 pM) was incubated with KI 14 (20 pM) in the presence of LPS or LTA (5 pg mL1), or PBS buffer alone. Using Hidex Sense 425-301, the fluorescence intensities (ZEX = 360 nm, XEM = 550 nm) were measured after 30 min of incubation at 37°C with shaking. PAPf39 was tested as a positivecontrol, with amyloid fibrillation induced by overnight agitation (37°C, 1400 rpm, 2 mg mL1in in PBS buffer).

[0136] To examine the proteolytic stability of the peptide amyloid using KI 14 assay, trypsin (15 nM) was added to the incubation mixtures containing peptide (20 pM), LPS (5 pg mL1), and KI 14 (20 pM) at different time points. Further details of the experimental scheme can be found in Figure 16a.

[0137] 1.8 Agglutination assay

[0138] Bacteria culture was diluted into PBS buffer to achieve a starting ODeoo of 0.075-0.078, as measured in a 96-well plate using the Tecan Infinite M200. Stock peptide solution (1 mM) was diluted into bacteria culture (1 mL) to achieve desired concentrations. After vortex mixing, the sample was transferred to a disposable plastic cuvette (12x12x4.5 mm). The ODeoo of the sample was measured at each time point over 8 h at room temperature, using the Tecan Infinite M200.

[0139] 1.9 DLS measurement

[0140] Incubation mixtures consisting of peptide (20 pM) and LPS or LTA (5 pg mL ’) were prepared in PBS, with peptide added last. Sample (40 pL) was immediately transferred to a low-volume quartz cuvette (12.5x12.5x45mm) for DLS measurements using the Litesizer™ 500 (Anton Paar, Graz, Austria). Series measurement mode was employed to monitor the change in hydrodynamic radius over time at 25°C. The average hydrodynamic size was analyzed using Kalliope™ software (Anton Paar).

[0141] 1.10 Serial passage

[0142] Overnight culture of E. coli M2 was diluted with MHB to a starting inoculum of 106CFU / mL. Peptide at different concentrations (0.25x to 2xMIC) was added to the diluted culture. After incubated for 24 h (37°C, 120 rpm), ODeoo values of samples were measured using Tecan Infinite M200 to identify the new MIC. Culture at highest peptide concentration with visual turbidity was diluted with MHB to yield an inoculum of 106CFU / mL, into which fresh peptide was added. The range of peptide concentrations was calculated based on the new MIC value. These steps were repeated daily for 14 days.

[0143] 1.11 In vivo efficacy in peritonitis model

[0144] All animal procedures were conducted in accordance with protocol R21-0162 approved by the Institutional Animal Care and Use Committee (IACUC) at National University of Singapore (Singapore). Female C57BL / 6NTac mice aged 6-8 weeks were acquired from InVivos Pte Ltd (Singapore, Singapore). Solutions for injections wereprepared fresh in pharmaceutical grade saline and sterilized by filtering through a 0.22 pm membrane.Peritonitis model was conducted by following an published protocol. Briefly, healthy mice were rendered neutropenic by giving single i.p. injection (0.5 mL) of cyclophosphamide on day -4 (150 mg kg1) and day -1 (100 mg kg1). On day 0, mice were inoculated with E. coli M2 (109CFU mL-1) by giving i.p. injection (0.1 mL). At 30 min post-inoculation, mice were given i.p. injection (0.5 mL) of a single dose (2.25 mg kg-1) of BTT1-3A, BTT2-4A, colistin, or saline control (n = 5 mice per group, n = 4 for control group). At 2 h post-treatment, mice were euthanized by carbon dioxide asphyxiation, followed by cervical dislocation. Peritoneal lavage was performed by injecting sterile PBS (3 mL) into the peritoneal cavity, followed by abdominal massage and collection of peritoneal fluid (1-2 mL). Blood (0.3-0.5 mL) was collected through cardiac puncture. Liver, spleen, and kidney were surgically removed and immersed in 0.1% Triton X-100 (in PBS). Tissue homogenization was performed using gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) following a pre-set protocol. Cell aggregates were removed using a 30 pm mesh MACS SmartStrainer (Miltenyi Biotec). Blood, peritoneal fluid, and tissue homogenate were plated on LB agar and incubated overnight for colony counting. For multiple-dose experiment (n = 5 mice per group), At 30 min after bacterial inoculation, mice were given three i.p. injections every 1 h (0.5 mL each, 2.25 mg kg-1) of BTT1-3A, BTT2-4A, colistin, or saline control. 5 h after the first treatment injection, mice were euthanized for collection of blood and peritoneal fluid, which were plated on LB agar for colony counting.

[0145] For ex vivo microscopic imaging of nanonets formation, peritoneal fluid collected from non-infected mice (n = 2) were used. E. coli M2 culture in mid-log phase was diluted to an initial ODeoo of 0.1. After centrifugation at 6,000 rpm for 5 min, cell pellet was resuspended in the peritoneal fluid. BTT1-3A or BTT2-4A (20 pM) were added to the sample (0.5 mL). After being incubated for 30 min at 37°C with shaking, the incubation mixtures were washed thrice in PBS, and processed for SEM viewing as described above.

[0146] 1.12 In vivo toxicity of peptide administration via intraperitoneal route

[0147] All animal studies were performed in accordance with the NACLAR guidelines style husbandry as per AAALAC International Complaint. The guidelines of the Animal Ethics Committee of the SingHealth Singapore Association for Assessment and Accreditation of Laboratory Animal Care were also satisfied. Toxicity studies wereperformed by following the protocol 2019 / SHS / 1491 approved by the IACUC at the Singapore Eye Research Institute (Singapore). On the study termination day, efforts were made to minimize discomfort, pain and suffering. The animals were euthanized humanely using CO2 followed by harvesting of vital organs for laboratory analysis.

[0148] Male C57BL / 6J aged 7-8 weeks old were purchased from In Vivos (Singapore). Mice (5 per cage) were kept in ventilated cages (Tecniplast, USA) at a room temperature of 20 to 25°C, humidity of 50 to 60%, 10-15 air exchanges per hour with 100% fresh air and a 12 / 12-hour light / dark cycle. Mice were fed with standardized feed (LabDiet for rodents) and water (Hydropac® Alternative Watering System) from an animal’s ad libitum (free feeding). All materials, including IVCs, lids, feeders, bottles, bedding, and water, were autoclaved before use. All animal manipulations were performed in a class II laminar flow biological safety cabinet. Mice (n = 5 per treatment group) were given a single i.p. injection of BTT1-3A or BTT2-4A at three different doses (2.5, 5 and 10 mg / kg) then monitored for 24 h. Mice were observed for mortality twice (a.m. and p.m.). Body weight and animal behavior were recorded and scored: piloerection, subdued normal behaviour patterns, interaction with peers, hunched transiently, transient vocalization, oculonasal discharge, normal respiration, tremor, convulsion, prostration, and self-mutilation. Prior to euthanasia, blood and urine was collected for blood chemistry, urinalysis and cell count evaluation. Kidneys were harvested for histology analysis via the standard H & E staining. Since the urine initially collected was insufficient for urine pathological analysis, a new batch of mice (n = 3 per treatment group) were subjected to identical procedures for urine collection.

[0149] Blood samples were submitted to the Department of Pathology at the Singapore General Hospital (Singapore) to determine the cell count and biochemical markers of kidney toxicity: creatinine, glucose, urea, total protein, bilirubin, alkaline phosphatase and alkaline transaminase. The following parameters were determined from the whole blood cell analysis: white blood cell (WBC), red blood cell (RBC), lymphocyte, monocyte, neutrophils, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, and mean corpuscular hemoglobin concentration. More than 1 mL of urine was pooled from each treatment group of mice for determining urine RBC, urine WBC, epithelial cell, urine casts, and urine crystals. For H & E staining, kidneys were fixed with 10% formalin and embedded in paraffin, followed by vertical bisection. Embedded tissues were sliced into 4 pm thick sections, mounted on glass slides,and incubated at 75 °C for 30 min. After deparaffinization using xylene for 10 min, the specimens were rehydrated using a graded ethanol series (95%, 85%, and 70% ethanol). The specimens were washed and treated with hematoxylin for 2 min, washed in running tap water for 1 min, and then incubated with acid alcohol for Is. The specimens were incubated with ammonia water solution for 1 s and washed in running tap water for 10 min. After counterstaining in eosin solution for 90 s, the specimens were dehydrated using an ethanol gradient (70%, 80%, 90%, and 100%). Finally, the specimens were mounted with mounting medium, and the kidney was observed under microscopy.

[0150] 1.13 Statistical Methods

[0151] Statistical significance of inter- groups differences was determined by performing one-way analysis of variance (ANOVA) and Bonferroni post-hoc test, or Student’s t test (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). GraphPad Prism 7.03 was used for statistical tests, graph drawing, and curve fitting.

[0152] Example 2- Results section 1 - "Trap and Kill" Activity

[0153] 2.1 Peptide sequence and mechanism

[0154] A series of synthetic P-hairpin antimicrobial peptides (AMPs) were synthesized, which are capable of self-assembling into nanonets in response to the presence of bacteria, hence conferring specificity. The sequences of the peptides are rationally designed with two functional modules: (i) a hairpin turn as the ‘recognition module’ for interacting with bacterial membrane components to initiate peptide conformational change and amyloid nucleation, and (ii) side strands as the ‘structural module’ for P-sheet formation and molecular stacking during the fibrillation process (Figure la). The peptides were shown to undergo amyloid fibrillation in response to both Gram-positive and Gram-negative bacteria, with lipoteichoic acid (LTA) and lipopolysaccharide (LPS) respectively identified as the prominent amyloid nucleators in the bacterial membranes. As the peptide molecules in solution encounter LPS or LTA on the bacterial surface, amyloid nucleation is initiated. Amyloid fibrils undergo elongation as hairpin peptides stack at the tail-end of the fibril. Once the nanofibrils grow sufficiently long, they start to form physical cross-links. Eventually, a mature interlocking network of fibrils entraps other bacteria in the surroundings (Figure lb). As the peptides displayed both trapping and killing functionalities, they present a multifunctional upgrade from the trap-only nanonets in nature, which are dependent on other immune components for biocidal effect. By manipulating only amino acids at the turn sequence, it wasdemonstrated that critical parameters of the nanonets, including antimicrobial activity, fibrillating capacity, and proteolytic stability, can be fine-tuned. Of interest, peptide BTT2-4A displayed potent antimicrobial and agglutinating activity even against colistinresistant clinical isolates. Detailed in vivo evaluations demonstrated significant antimicrobial efficacy and lack of systemic toxicity by nanonet-forming peptides.

[0155] 2.2 Gram-negative bacteria induce nanonets formation by BTT2

[0156] A series of de novo designed synthetic P-hairpin AMPs with identical side strands of amphipathic Leu-Lys repeat units but transplanted turn sequences (Table 1) was previously reported in N. D. T. Tram et.al, 2021. They were capable of selective antimicrobial activity against Gram-negative bacteria, among which BTT2 was a potent candidate. Using scanning electron microscopy (SEM), an expansive network of nanofibrils entangling Escherichia coli (E. coll) cells was observed, after these Gramnegative bacteria were incubated with BTT2 for 30 min (Figure 2a). Such net-like nanostructures were absent from untreated bacteria culture (Figure 2b), or after treatment with other active BTT peptides. With diameter distributed around 30-39 nm (Figure 3b), the gross morphology of BTT2 nanofibrils closely resembled that of human defensin 6 (HD6) and human P-defensin 1 nanonets self-assembled in the human colon.

[0157] Table 1. Sequence, molecular weight (MW), and hydropathy value of the peptides.Peptide name Peptide sequencea)MWb)BTT1 EKEKEKETGKEKEKE-NH2 1736.3 2.1BTT2 EKEKEKVpPTKEKEKE-NH21859.5 -0.3BTT3 EKEKEKpGKEKEKE-NH21619.2 -2.6BTT4 EKEKEKINGKKEKEKE-NH21877.5 -3.9BTT6 EKEKEKpNKEKEKE-NH21676.2 -5.7BTT7 EKEKEKRDGKEKEKE-NH21793.3 -9.0BTT2-As AAAAAAVpPTAAAAAA-NH21264.4 21.9BTT2-At EKEKEKAAAAKEKEKE-NH21749.3 6.6BTT1-1A EKEKEKATGKEKEKE-NH21694.2 0.1BTT1-2A EKEKEKEAGKEKEKE-NH21706.3 4.6BTT1-3A EKEKEKETAKEKEKE-NH2(SEQ ID NO:1)1 / 5U’4 4’JBTT2-1A EKEKEKApPTKEKEKE-NH21831.4 -2.7BTT2-2A EKEKEKVAPTKEKEKE-NH21833.4 3.1BTT2-3A EKEKEKVpATKEKEKE-NH21833.4 3.1a)‘p’: d-Proline; ‘-NH2’: amidation at C-terminal; putative turn residues are highlighted in grey.b)MW was calculated using a web-based tool (https: / / pepcalc.com / )c)Hydropathy value was computed by adding the hydropathic index of individual residues. A higher hydropathy value indicates a greater degree of hydrophobicity.

[0158] To demonstrate bacterial cell entanglement with the nanofibrils, E. coli cells were stained with the bacterial membrane dye A-(3-Triethylammoniumpropyl)-4-(6-(4- (Diethylamino) Phenyl) Hexa-trienyl) Pyridinium dibromide (FM4-64) and co-incubated with fluorescein isothiocyanate (FITC)-labelled BTT2 peptides. In buffer alone, negligible green fluorescence was observed (Figure 4a). Only in the presence of bacteria, the confocal images showed large aggregates with intense green fluorescence (Figure 2c), suggesting the bacteria-responsiveness of BTT2 nanonet self-assembly. Most of the bacteria (labeled red) were found to co-localize with the peptide aggregates (labeled green), demonstrating the ability of nanonets to entrap bacteria. The entrapped bacteria exhibited a range of fluorescence color from orange to yellow, indicating that the nanonets adopted a three-dimensional architecture and immobilized bacteria at varying depths. In contrast, untreated E. coli were scattered across the microscopic field in the absence of any aggregate (Figure 4b). It was observed that BTT2 was the only active BTT peptide to generate bacteria-responsive nanonets, with FITC-labelled BTT3, BTT4, and BTT6 not forming E. co / z-trapping green clusters under identical treatment conditions (Figure 4c).

[0159] 2.3 EPS from the Gram-negative membrane induces amyloid formation by BTT2

[0160] To explore the specificity of nanonet formation, FITC-BTT2 was incubated with cell-free LPS extracted from E. coli and visualized using confocal microscopy. Green meshes similar in appearance to E. co / z-induced nanonets could be observed (Figure 2d). In contrast, BTT2 did not form any aggregate in aqueous buffer alone (Figure 4a), hence supporting the specificity of the phenomenon. Using SEM, it was confirmed that BTT2 formed nanonets in the presence of LPS (Figure 5c), but not MHB alone (Figure 5b). The morphology of LPS-induced nanonets highly resembled those observed in the presence of E. coli cells (Figure 2a). This is of significance since LPS occurs in abundance in the outer membrane of Gram-negative bacteria, and comprises the first point of contact for xenobiotics.

[0161] To gain structural insights into peptide arrangement within the nanofibrils, a fluorescence assay was conducted using the amyloidophilic dye (trans, trans)- 1-bromo- 2,5-bis-(4-hydroxy)styrylbenzene (KI 14) (Figure le). The fluorescence emission of KI 14 undergoes red-shift and an increase in intensity when KI 14 binds specifically to cross-P structures typical of amyloid-like fibrils. In aqueous buffer alone, all peptides displayed a baseline fluorescence level of 0.1-0.2 a.u., with BTT2 being the only exception with an intensity of 0.57 a.u.. In the presence of LPS, however, the amyloid content of BTT2 was significantly increased as shown by the 2-fold increase in KI 14 fluorescence. This increase was not observed with other peptides. Though BTT1 also exhibited a notable increase in amyloid structures with the addition of LPS, its KI 14 fluorescence remained 2.3-fold lower than that of BTT2. Overall, the results suggested that interactions with LPS caused BTT2 molecules to self-assemble into nanofibrils with amyloid-like structures. The unique aggregative behavior of BTT2 among active BTT peptides agrees with the confocal observations.

[0162] The secondary structure of BTT2 was next evaluated using CD spectroscopy. In aqueous buffer, CD spectrum of BTT2 displayed a minimum around 198 nm and nearzero signals from 210 nm, which indicated random-coiled structures (Figure 6b). When incubated with LPS, BTT2 exhibited CD spectra characteristic of P-sheet structures, as evidenced from a maximum at 196 nm and a minimum at 218 nm (Figure 7a). The spectra were then analyzed using the P-Structure Selection (BeStSel) method to estimate the secondary structure composition. As the concentration of either LPS or BTT2 increased, the parallel P-sheet content and hairpin turn content of the peptide increased (Figure 7b- 7c). This indicates that LPS -peptide interactions effected conformational transition to hairpin turns, which were in turn crucial for the early nucleation phase of amyloid formation.

[0163] Of note, extensive BTT2 nanonet formation was previously observed to occur within 30 min of exposure to E. coli, which is much faster than amyloidogenic proteins and peptides in nature. For instance, Ap requires at least two days for mature nanofibrils to form in aqueous solution, while protegrin-1 a few hours. Amyloidogenesis typically follows a nucleation-dependent growth kinetics with an initial lag phase, which can be shortened with the addition of preformed nuclei to the mixture. Using KI 14 fluorescence assay, the amyloid extent of BTT2 was observed when incubated with LPS over 3 h. The resulting kinetic curve exhibited virtually no lag phase, with KI 14 fluorescence rapidlyincreasing in the first hour (Figure 8). Taken together, the membrane LPS layer serves as a nucleation site for the peptide molecules to gather and assemble, thereby expediting the fibrillation kinetics (Figure lb).

[0164] Ionic strength of the solvent was shown in literature to impact the extent and kinetics of the fibrillation process. Electrolytes can provide screening to mitigate electrostatic repulsion between the cationic peptides and promote self-assembly. To prevent variation in ionic strength from affecting the results, biophysical and structural studies of peptide amyloids were performed in the same solvent system (PBS buffer) throughout this work.

[0165] 2.4 Gram-positive bacteria and LT A can induce nanonet formation by BTT2

[0166] To examine the bacteria spectrum of BTT2 nanonet formation, the activation of peptide fibrillation by Gram-positive strains was examined next. SEM images revealed the formation of bacteria-trapping nanonets in Staphylococcus aureus (S. aureus) culture treated with BTT2 for 30 min (Figure 2f), but not in untreated bacteria (Figure 2g). Using confocal microscopy for visualizing FfTC-labelled BTT2 after being incubated with S. aureus for 1 h, bacteria-trapping aggregates were observed, similar to those observed with E. coli (Figure 2h), which were absent in untreated bacteria (Figure 9a). In contrast, FITC- labelled BTT1 (Figure 9b) and BTT3 (Figure 9c) formed very small aggregates at the cellular surfaces and did not appear to promote bacterial agglutination.

[0167] SEM micrographs also revealed inter-strain differences in gross morphology of the peptide nanonets. Whereas S. aureus triggered the formation of cluttered nanofibrils with rough nodules along their length (Figure 2f), E. coli induced the selfassembly of thinner and more loosely packed nanofibrils (Figure 2a). However, such differences were not reflected in their secondary structure compositions (Table 2 and 3), indicating that peptide molecules with similar conformations could be arranged in a different manner within the scaffold of amyloid fibrils to yield a range of fibril morphology.

[0168] Table 2. The percentage of secondary structures adopted by 50 pM of peptides in the presence of 100 pg mL1LPS was estimated using the web-based tool BeStSel (http: / / bestsel.elte.hu / index.php) to analyze the corresponding CD spectra in Figures 8A- B.

[0169] Table 3. The percentage of secondary structures adopted by 50 pM of fibrillating peptides in the presence of 100 pg mL1LTA was estimated using the webbased tool BeStSel (http: / / bestsel.elte.hu / index.php) to analyze the corresponding CD spectra in Figure S8C.

[0170] LTA is a cell wall component characteristic of Gram-positive bacteria, and shares many structural features with LPS (e.g. negatively charged phosphate groups, sugar units, fatty acid chains). Cell-free LTA isolated from S. aureus was shown to be able to induce BTT2 to undergo amyloido genesis, with BTT2 exhibiting a 7-fold increase in KI 14 fluorescence compared to buffer alone (Figure 2e). Overall, the KI 14 results showed a similar trend to those obtained in the presence of LPS. The ability of cell-free LTA to induce nanonets formation by BTT2 was confirmed using SEM imaging (Figure 5d). These nanonets were similar in appearance to those triggered by S. aureus cells (Figure 2f).

[0171] 2.5 Both side strand and turn residues are critical to BTT2 amyloidogenesis

[0172] To explore the modular significance of the BTT2 sequence, residues at either the side strands or the reverse turn were substituted with Ala to obtain BTT2-As andBTT2-At respectively (Table 1). As a chemically inert and structurally minimal amino acid, Ala substitution enables the study of the essentiality of the replaced amino acids to the overall fibrillation capacity of BTT2. In the presence of either LPS or LTA, BTT2- As and BTT2-At formed relatively minimal amyloid structures, with KI 14 fluorescence at least 13-fold and 2-fold lower that that of BTT2 respectively. This indicated that both the side strand and turn modules played critical roles in mediating BTT2 self-assembly.

[0173] To verify whether these analogs formed nanonets in the presence of LPS, dynamic light scattering (DLS) was employed to monitor the size of peptide aggregates over time. By gathering information on the scattering of incident laser light, this technique can determine the hydrodynamic radius of a hypothetical spherical particle with a similar diffusion rate to the particle of interest. Whereas the hydrodynamic radius of BTT2 rapidly increased over time to reach tens of microns over 3 h (Figure lOd), that of BTT2- At remained at 200-300 nm even after 1 h of being exposed to LPS (Figure 10c). These results indicated that BTT2-At formed small amorphous aggregates instead of expansive nanonets in the presence of LPS. Overall, Ala substitutions performed on either the turn or side strand residues completely abolished the fibrillating capacity of the resulting analogs.

[0174] Using CD spectroscopy, these functional modules were again observed to play important roles in facilitating structural changes by BTT2 molecules. Unlike the parent peptide, neither BTT2-As nor BTT2-At could form P-sheet structures in the presence of LPS. Instead, their CD spectra were characteristic of random-coiled structures (Figure 10b).

[0175] 2.4 Fibrillating capacity of P-hairpin peptides can be modulated by turn sequence modification

[0176] In the original BTT series, variations in the turn sequence alone gave rise to a significant difference in fibrillating capacity between BTT2 and the remaining peptides. BTT1 was found to be most potent broad spectrum but did not possess fibrillating capacity. The next question was if BTT1 could be imparted with similar amyloidogenic behavior through sequence modification. Thus, Ala scanning was performed at the turn sequence of BTT1, where individual residues were sequentially replaced by Ala to yield BTT1-1A to BTT1-3A (Table 1).

[0177] BTT1-3A was found to be the only analogue capable of forming bacteria- responsive nanonets. It exhibited a significant increase in amyloid structures upon theintroduction of LPS or LTA, with a KI 14 fluorescence comparable to that of BTT2 (Figure I la). In contrast, the fluorescence intensity of BTT1-1 A and BTT1-2A remained low similar to the parent BTT1 peptide. Using SEM, the ability of BTT1-3A to generate extensive nanonets was observed when incubated with either E. coli (Figure 1 lb) or S. aureus (Figure 11c). Although the E. co / z-induced BTT1-3A nanofibrils appeared more cluttered than BTT2 counterpart, their diameter distribution closely resembled each other with an average of ~35 nm (Figure 3a).

[0178] Using CD spectroscopy, the extent of structured conformations formed by the peptides in the presence of UPS or LTA was found to correlate with their fibrillating capacity. In aqueous buffer, the BTT1 analogs were determined to be predominantly random coils. In the presence of LPS, their CD spectra were representative of P-sheet structures, with BTT1-3A displaying more prominent signals at the characteristic peaks (i.e. maximum at 196 nm and minimum at 218 nm) than BTT1-1A and BTT1-2A (Figure 6a). This finding supported the finding that peptide folding events triggered by LPS binding facilitated amyloid formation. Similar to what was observed with BTT2, there were no obvious changes in the structural composition of BTT1-3A when the amyloid inducer was switched from LPS to LTA (Table 2 and 3).

[0179] Preliminary structure-activity analysis revealed that greater hydrophobicity of BTT1-3A versus BTT1, as reflected by a higher hydropathy value (4.3 vs 2.1, Table 1), appeared to favor its fibrillating capacity, indicating that hydrophobic interactions among peptide monomers were in part important to the amyloid formation process. However, this is not the case for BTT1-2A, which did not display a change in aggregative behavior despite having a higher hydropathy value of 4.6. As outlined in N. D. T. Tram et.al, 2021, the turn sequence of BTT1 followed a sequence motif computationally and experimentally demonstrated to strongly recognize LPS. With the polar Thr in BTT1 replaced by the non-polar Ala in BTT1-2A, homology to the LPS-binding motif was compromised in the latter peptide, potentially accounting for its inability to favorably interact with LPS and undergo fibrillation.

[0180] To study the in vitro bacteria-trapping efficiency of BTT1-3A nanonets, the agglutination assay was employed. Against both E. coli and S. aureus, BTT1-3A caused a significant decrease in normalized ODeoo as compared to the untreated controls (Figure l ld-e). Bacterial agglutination by BTT1-3A resulted in the rapid sinking of the aggregates, effectively reducing the bulk turbidity of the sample. At 80 pM, BTT1-3Acaused >50% decrease in normalized ODeoo after 2 h of incubation. At 20 |aM, the same effect was achieved after 5-6 h of incubation. It was observed that BTT1-3A displayed clearly greater agglutinating activity than the parent peptide BTT1 at equimolar concentrations, indicating that the bacteria-agglutinating efficacy correlated with the extent of amyloid fibrillation. The turbidity of both E. coli and S. aureus cultures treated with 20 pM BTT1 remained high after 8 h, indicating the failure of the parent peptide to agglutinate the bacteria (Figure 38a-b).

[0181] To evaluate if the antimicrobial activity of BTT1 has been altered after having conferred the capacity to fibrillate in BTT1-3A, broth microdilution method was employed to determine its minimum inhibitory concentration (MIC90). Against both Gram-positive and Gram-negative ATCC strains, BTT1-3A displayed antibacterial potency comparable to that of BTT1 (Figure Ilf). Overall, converted the kill-only BTT1 was successfully converted to the trap-and-kill BTT1-3A by replacing a single residue at the hairpin turn region.

[0182] 2.5 Antimicrobial activity against drug-resistant bacteria can be conferred on fibrillating peptides through turn sequence tuning

[0183] Next, the possibility of modulating antimicrobial potency of the fibrillating peptides against Gram-negative strains carrying the mcr-1 resistance gene was evaluated, which causes resistance to the last-line antibiotic colistin. Although BTT2 was capable of nanonets formation and bacterial entrapment, it was previously determined to be relatively inactive against mcr-1 -positive clinical isolates. It was sought to improve the activity of BTT2 against antibiotic -resistant Gram-negative strains by modifying its turn sequence via Ala scanning, yielding BTT2-1A to BTT2-4A (Table 1).

[0184] Of the four analogs, only BTT2-2A exhibited a substantial decrease in LPS- and LTA-induced KI 14 fluorescence, indicating a reduction in amyloid formation (Figure 12a). On the contrary, KI 14 fluorescence of the other three analogs remained comparable to that of the parent peptide BTT2. Structure- activity analysis revealed the importance of conformational stability to the process, which is in agreement with reports that turn folding was essential to the early nucleation and fibril maturation phases of amyloido genesis. The turn sequence of BTT2-2A was designed by replacing a D-Pro in BTT2 with Ala. D-Pro is known to be a turn-promoting residue, so its replacement could have caused a decrease in hairpin folding stability. Using CD spectroscopy, it was shown that BTT2-2A was the only BTT2 analog to adopt random-coiled structures in thepresence of LPS, whereas the spectra of other peptides were characteristic of P- sheet structures (Figure 6b). BeStSel analysis of the spectra of BTT2-1A,-3A,-4A revealed a high percentage of parallel P-sheets (>30%, Table 2-3). In addition, structural plasticity seemed critical to peptide fibrillation capacity. Despite a high content of hairpin turn which likely impairs its structural flexibility (Table 2), BTT1 was not observed to form nanonets. The finding was in line with report that excessive rigidity as a result of additional turn-stabilizing disulfide bridge impeded amyloidogenesis by Ap mutants. Structural flexibility was proposed to enable the transition between hairpin and extended conformations during fibril elongation.

[0185] Then the amyloidogenic BTT2 analogs were screened for antimicrobial activity against a panel of mcr- 1 -positive clinical isolates (Figure 12b). As compared to BTT2, only BTT2-4A was consistently more potent than the other peptides, with MICs at 16 pM against most strains (vs 32 pM for parent BTT2). Given the more potent activity of BTT1 against mcr-1 strains, its fibrillating analog BTT1-3A was also tested for antimicrobial activity against the same panel. Notably, BTT1-3A displayed comparatively poor antimicrobial activity with MICs of 32 pM and above against most strains (Figure 13). Among BTT peptides, there appeared to be minimal correlation between the antimicrobial potency and fibrillating capacity. While BTT1 displayed more potent activity than BTT2, the former was unable to generate amyloid nanofibrils (Figure 2e and 9b). The ability to tune BTT1 to yield nanonets in response to mcr-1 -positive clinical isolates in the form of BTT 1-3 A, however, did not improve its antimicrobial activity. Using membrane- sensitive fluorescence dyes, previously it was shown that the active BTT peptides, including the fibrillating BTT2, displayed a conventional antimicrobial mechanism of membrane disruption. It was postulated that, after coming into contact with the bacterial membrane surfaces, the fibrillating BTT peptides can undergo two different pathways: (1) amyloidogenesis to generate nanonets for trapping activity, and (2) membrane insertion as monomers or small oligomers to exert killing activity. As peptide molecules participate in amyloidogenesis, they become less available in solution to effect membrane disruption. This presents a trade-off between the two functionalities which we need to balance when designing multifunctional trap-and-kill AMPs.

[0186] The formation of nanonets when BTT2-4A was incubated with E. coli ATCC 25922 was observed using both SEM (Figure 12c), and confocal microscopy (Figure 14a).FITC-labelled BTT2-4A was also observed to form large green clusters in the presence of LPS (Figure 15B). In contrast, visible aggregates were absent in untreated bacteria control (Figure 5) or when the peptide was incubated in aqueous buffer alone (Figure 15a). The resistance gene mcr-1 encodes for a phosphoethanolamine transferase which catalyzes modifications of lipid A, resulting in a decrease in net negative charge of LPS. However, it was observed that this mutation did not interfere with the efficacy of LPS as an amyloid nucleator. Using SEM and confocal microscopy, it was observed that BTT2- 4A successfully formed bacteria-trapping nanonets when incubated with the mcr-1- positive strain E. coli M2 (Figure 12d and 14b).

[0187] Using the agglutination assay, slightly weaker agglutinating activity was observed by BTT2-4A against E. coli ATCC 25922 than BTT1-3A. At 20 pM, BTT2-4A failed to lower the normalized ODeooto below 50% the starting value (Figure 12e). At 40 and 80 pM, however, there was minimal difference between the agglutination curves of the two peptides. In contrast, the non-amyloidogenic BTT2-2A exhibited negligible agglutinating activity against both E. coli and S. aureus, even at 80 pM (Figure 38a-b). Against E. coli M2, BTT2-4A caused an even faster decrease in sample turbidity than the ATCC strain, lowering the normalized ODeoo to 48.7% after just 3 h of incubation (Figure 12e). Similarly, BTT1-3A appeared to be more effective at agglutinating E. coli M2 than E. coli ATCC 25922 (Figure 38c). The higher susceptibility of this mcr-1 -positive strain to nanonets entrapment could be explained by higher intrinsic clumping tendency of M2 cells, with normalized ODeoo of untreated bacteria significantly lowered after 8 h of incubation (Figure 12e). Taken together, the results suggest that the mechanism of bacteria-trapping activity of the nanonets involves: (1) electrostatic interactions between the positively charged Lys residues and anionic components of bacteria membranes, and (2) physical entrapment by the cross-linked nanofibrils. It was postulated that the latter mode plays a more central role, since the non-fibrillating BTT2-2A exerted clearly poorer agglutinating activity than the fibrillating analogues despite similar net positive charge.

[0188] Overall, by replacing a single amino acid at the turn segment of BTT2, BTT2- 4A was obtained with improved antimicrobial potency against clinically relevant antibiotic -resistant strains, while preserving the ability to form bacteria-trapping nanonets. Antimicrobial activity of our trap-and-kill nanonets is clinically valuable. Trap- only nanonets such as HD6 are reliant on host antimicrobial components for bacterial killing, which could be evaded by certain pathogenic strains. In comparison, the trap-and-kill peptide nanonets of the present invention offer more therapeutic values, since it is possible to adjust their antimicrobial profile to meet the clinical needs through rational designs of the peptide sequence. Besides, entrapped microbes can eventually escape from the mesh as luminal fluids flow past the nanonets -bacteria complex, further highlighting the importance of potent antimicrobial activity with rapid bactericidal kinetics.

[0189] 2.6 Proteolytic stability of peptide nanonets varies with turn sequence modification

[0190] Hydrolysis by peptidases is a mechanism employed by the human body to clear pathogenic amyloid fibrils and prevent them from reaching harmful levels. However, this physiological process can potentially interfere with the in vivo performance of the peptide nanonets of the present disclosure by accelerating their degradation. To evaluate the stability against proteolytic degradation, the KI 14 fluorescence of fibrillating BTT peptides was monitored in the presence of trypsin, a serine protease that preferentially cleave peptide sequences at Lys and Arg. A higher KI 14 signal was indicative of a higher amount of amyloid structures remaining after trypsin exposure, hence greater trypsin stability. In brief, trypsin was added to the peptide incubation mixture in different setups: (1) before (Trypsin pre-incub ation), (2) at the same time (Co-incubation), or (3) after the addition of LPS (Pre-formed fibrils), or (4) added in the absence of LPS (Non-specific aggregation). For each set-up, exact timepoints when individual components were added to the incubation mixtures are described in Figure 16a.

[0191] Pre-formed BTT2 fibrils maintained consistent normalized KI 14 fluorescence in the range of 90-100% over 3 h of trypsin exposure (Figure 16b), indicating that mature BTT2 fibrils were very stable against proteolytic degradation. Whereas pre-formed fibrils of BTT1-3A retained fluorescence intensity comparable to BTT2 over time, that of BTT2- 4A gradually decreased to 65% after 3 h of trypsin incubation (Figure 16c). When BTT2 was pre- or co-incubated with trypsin, a significant decrease in fluorescence intensity (<55%) was observed after 2 h of exposure as compared to the non-trypsinized control fluorescence of -100%. This finding suggested that intermediate aggregative species were more susceptible to hydrolysis by trypsin. Then, whether the remaining fibrillating peptides in the series performed any better than BTT2 was explored. With regards to coincubated stability, the normalized KI 14 fluorescence of both BTT1-3A (95%) and BTT2-3A (75%) was significantly higher than that of BTT2 (34%) after 3 h of trypsin incubation (Figure 16d). While the normalized fluorescence of BTT2-4A was also higherthan BTT2, it was comparatively low at 65%. On the other hand, all the tested peptides were determined to be mostly unstable when pre-exposed to trypsin for 30 min before LPS was added to the incubation mixture. The normalized KI 14 fluorescence of all peptides decreased to <60% after 3 h of trypsin exposure (Figure 16e). BTT1-3A was the only peptide whose KI 14 fluorescence was >70%.

[0192] To examine the effect of trypsin exposure on the trapping functionality, the bacteria-agglutinating activity of BTT1-3A and BTT2-4A in the presence of trypsin was tested. Whereas the agglutination curve of BTT1-3A was minimally affected by trypsin co-incubation up to 8 h, the BTT2-4A curve shifted upwards by 10-20% over most of the incubation duration (Figure 17). This difference in behavior between the two peptides mirrored their relative changes in amyloid content (Figure 16d). Overall, trypsin exposure did not appear to be overly detrimental to the in vitro bacteria-trapping efficacy of the peptide nanonets.

[0193] In summary, BTT peptide analogs with modifications of up to four residues at the turn region could significantly differ in their proteolytic stability. BTT 1-3 A was determined to be the most robust peptide under all tested incubation schemes. Whereas the impact of trypsin degradation on amyloid structures was the most significant when peptides were pre-exposed to trypsin, its inhibitory effect on pre-formed nanofibrils was clearly less prominent.

[0194] After characterizing the in vitro proteolytic stability of the nanonets in buffer, their stability in simulated serum condition was examined. Using SEM, extensive nanonets formation by BTT2-4A in MHB in the presence of FBS at both 10% and 50% v / v were observed (Figure 18). The morphology of the nanofibril strands did not appear to be affected under serum condition. Both fibrillating peptides were demonstrated to be able to self-assemble into nanonets in biological matrices after incubation with E. coli inoculated into ex vivo murine peritoneal fluid (Figure 19a-b). In comparison, no fibrillar structures in untreated E. coli incubated in the same biological matrix was observed (Figure 19c). Notably, the agglutinating activity of the disclosed peptides was observed to selectively impact bacteria cells, without causing clumping or cellular deformation of murine red blood cells in the proximity (Figure 19d).

[0195] 2.7 Trapping functionality contributes to the overall anti-infective efficacy

[0196] Previously, BTT2 at IxMIC was observed to cause a 100-fold decrease in the colony count of E. coli ATCC 25922 cultures after just 1 h of treatment. At 2xMIC, a1000-fold decrease in colony count was achieved within the same timescale. With such a rapid killing kinetics, it was possible that most bacteria cells in the surroundings had been eliminated before expansive nanonets could form. Under such circumstances, contributions of the trapping functionality to the overall anti-infective efficacy of the nanonets would be minimal.

[0197] To evaluate the viability of the E. coli cells trapped by peptide nanonets, a live / dead staining approach using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide (PI) for confocal viewing was employed. Both fluorescence dyes specifically bind to nucleic acids in the nuclei of bacteria cells. While DAPI stains both live and dead bacteria in a non-selective manner, PI can only pass through permeabilized membranes to selectively stain dead cells. After 1 h of incubating bacteria with FITC- labelled peptides, a mix of live (blue) and dead (red) cells among those co-localized with the green nanonet clusters was observed (Figure 20). To quantify the proportion of live cells, the number of live and dead cells was counted. On average, 50.4%, 51.7%, and 23.6% of the E. coli cells entrapped by BTT2, BTT2-4A, and BTT1-3A nanonets respectively remained viable after 1 h of treatment (Table 4). This supported the clinical utility and relevance of the trapping functionality in gathering bacteria for other antimicrobial components in solution to exert their biocidal effect more easily.

[0198] Table S4. Live / dead count and percentage dead entrapped E. coli ATCC 25922 cells after 1 h of incubation with FITC-labelled fibrillating peptides (Figure 20). 10 independent microscopic fields from two sample replicates were considered for each peptide treatment. Only fields with at least 30 total cells were taken into consideration. Only cells co-localized with green aggregates were counted.

[0199] To assess the rate of resistance development against the disclosed nanonets- forming peptides, serial passage assay against E. coli M2 was performed (Figure 21), with resistance defined as >4-fold increase in MIC. The antimicrobial potency of BTT1-3A, BTT2, and BTT2-4A remained unchanged over 14 days. MICs of BTT1-3A and BTT2 varied between 0.5x and 2x from the respective starting MIC, indicating a lack of resistance evolution. While reaching 4-fold increase on day 4 and 10-11, MIC of BTT2-4A promptly returned to 2x fold-change, which suggests the mounted resistance was likely unstable. In contrast, colistin became significantly less effective, with MIC displaying 8-fold increase from day 6. This provides further evidence of the clinical utility of our peptides as a therapeutic option against drug-resistant pathogens.

[0200] 2.8 Nanonet-forming peptides displays in vivo efficacy against drug-resistant pathogens and lack of systemic in vivo toxicity up to 24 h post-administration

[0201] After establishing the in vitro antimicrobial potency of the peptides, their in vivo efficacy was explored using a peritonitis model in neutropenic mice. C57BL / 6NTac mice infected with mcr- 1 -positive E. coli M2 were given a single dose of intraperitoneal (i.p.) treatment injection at 30 min post-inoculation. The mice were euthanized at 2 h post-treatment, then body fluid and organs were collected for quantifying bacteria load via colony counting on agar. Treatment with of BTT1-3A and BTT2-4A (2.25 mg kg1) achieved a >3 -log and >2-log reduction in CFU / mL in blood compared to saline control respectively (p < 0.002) (Figure 22a). While colistin treatment at equivalent dose also caused a significant decrease in bacteria load, no statistically significant difference to peptide treatment was observed, indicating comparable efficacy. Although peptide effect on the bacteria load in peritoneal fluid was less prominent (~l-log reduction in CFU / mL versus saline control), it was again comparable to colistin treatment (p > 0.5). The effect of a single i.p. dose of both peptide and colistin on bacteria burden in liver and spleen was minimal (Figure 23), suggesting that the peptides were not sufficiently robust to reach the murine organs in adequate quantity and chemical integrity. Overall, the findings of immediate peptide effect at lowering the bacteria burden in blood and peritoneal fluid of the mice are promising for clinical application, but further improvement to the formulation is required to achieve greater antimicrobial efficacy at distant organs.

[0202] Then the efficacy of a multiple-injection regimen was evaluated. At 30 min post-inoculation with E. coli M2, the mice were given three i.p. treatment injections (2.25 mg kg1) 1 h apart, then euthanized at 5 h post-treatment for quantification of bacteria load. Compared to saline control, mice treated with BTT1-3A displayed a 4-log and 3- log reduction in CFU / mL in peritoneal fluid and blood respectively (p < 0.05) (Figure 22b). BTT2-4A injections achieved a similar effect in peritoneal fluid, but less pronounced effect in blood. Similar to single-injection studies, the peptides of the present disclosure displayed statistically comparable antimicrobial efficacy to colistin.

[0203] Systemic in vivo toxicity of BTT1-3A and BTT2-4A was evaluated in mice at 24 h after a single i.p. administration at three different doses (2.5, 5, and 10 mg / kg). Normal murine behavior was observed throughout the treatment duration, without any signs of discomfort or distress. At 24 h post-treatment, mice treated with peptides at all tested doses displayed negligible changes in whole blood cell profile (Table 5), and body weight (Table 7). Their urine test results displayed an absence of blood and epithelial cells, casts, and crystals (Table 6), indicating healthy renal functions. Blood biochemical analysis revealed an absence of statistically significant differences between peptide - treated mice and saline-administered mice in concentrations of urea and creatinine and liver function biomarker (ALT) (Figure 22c-g), which suggest that peptide administration did not cause acute toxicity to vital murine organs. Examination of the kidney histology images revealed no apparent change between saline- and peptide-treated groups (Figure 22h), thus further supporting a lack of in vivo toxicity. The blood cell counts were comparable for all the groups, in agreement with the lack of toxicity by the peptides (Table 5).

[0204] Table 5. Whole blood cell analysis of mice (n = 5). Data are presented as mean + SD.

[0205] Table 6. Urine test results of mice (n = 3).

[0206] Table 7. Body weight of mice pre- and post-treatment (n = 5). Data are presented as mean ± SD.

[0207] Example 3 - Conclusion 1 - "Trap and Kill" Activity

[0208] In nature, the initiation of self-assembly by functional peptides is typically controlled at the cellular level via signaling pathways in response to host detection of pathogens. For the development of synthetic biomimetics, there is a need to replace such intricate regulatory machineries with simpler stimuli-induced designs using biochemical cues unique to the pathophysiology of infection sites. Until now, the majority of synthetic bacteria-agglutinating nanofibrils did not implement such a self-assembly switch, instead allowing for non-specific fibrillation in aqueous solution in the absence of bacteria. In this work, peptides was successfully designed that self-assembled into expansive nanonets selectively in the presence of bacteria. LT A and LPS were identified to provide nucleation sites for initiating peptide amyloidogenesis on the cellular envelope of Grampositive and Gram-negative bacteria respectively. With these amyloid inducers being ubiquitous among bacteria, it was believed that the fibrillating capacity of the disclosed peptides has a wide scope of application. A recent report of bacteria-responsive nanofibrils employed gelatinase as the bacteria- specific stimulus. However, this enzyme is not expressed by many clinically relevant strains in need for novel therapies such as E. coli or E. faecalis. It can also be overexpressed by the tumor milieu, potentially giving rise to off-target fibrillation in comorbid patients. These observations further highlight the superior specificity of BTT peptide nanonets in comparison to state-of-the-artsdesigns in the field. In addition, it was demonstrated that clinically significant parameters such as antimicrobial activity, fibrillation capacity, and proteolytic stability can be finetuned through rational design of the peptide sequence. Representative nanonet-forming peptides displayed excellent in vivo efficacy and a lack of in vivo toxicity up to 24 h posttreatment. Overall, the present disclosed antibacterial trap-and-kill peptide nanonets exhibited promising clinical potentials as an alternative anti-adhesion strategy to circumvent antibiotic resistance.

[0209] Example 4 - Experimental Section 1 - Anti-inflammatory properties

[0210] 4.1 Materials

[0211] Cation-adjusted Mueller Hinton broth (MHB), Mueller Hinton agar (MHA), Dulbecco's Modified Eagle's Medium (DMEM), dexamethasone, LTA from S. aureus, lipopolysaccharide (LPS) from E. coli O111:B4, fluorescein isothiocyanate (FITC)- labelled LPS, and propidium iodide (PI) were obtained from Sigma (St. Louis, MO). 4', 6- diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific (Waltham, MA). CellTiter-Glo® luminescent cell viability assay kit was obtained from Promega (Madison, WI). (trans, trans)-l-Bromo-2,5-bis-(4-hydroxy)styrylbenzene (KI 14) was obtained from Santa Cruz (Dallas, TX). Peptides were procured from GL Biochem (Shanghai, China), with their purity confirmed to be at least 95% by the manufacturer using reversed-phase high-performance liquid chromatography (RP- HPLC). Mouse Mono / Mono OptEIA™ ELISA Sets for quantifying cytokines were obtained from BD Biosciences (Franklin Lakes, NJ).

[0212] 4.2 Bacteria strains and mammalian cell cultures

[0213] E. coli ATCC 25922 and P. aeruginosa ATCC 9027 were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Clinical isolate of mcr-1- positive E. coli M2 was a kind gift from Dr. Jeanette Teo (National University Hospital, Singapore). RAW 264.7 cell cultures were maintained in high-glucose DMEM supplemented with 10% heat-inactivated FBS, and cultured under a humidified atmosphere at 37°C and 5% CO2.

[0214] 4.3 FITC-LPS trapping assay

[0215] Incubation mixture of FITC-LPS (2 pg / mL) and peptide at different concentrations was prepared in phosphate buffered saline (PBS). A peptide-free control was also prepared. Sample was incubated for 1 h (37°C, 120 rpm), followed bycentrifugation (16,000 rpm, 10 min). 100 pL of the supernatant was gently collected. Fluorescence intensity of FITC-LPS (ki.x = 492 nm, XEM = 525 nm) in the supernatant was measured using TECAN Infinite M200 microplate reader (TECAN, Mannedorf, Switzerland). FITC-LPS trapping index was calculated according to the followingwhere Fsampie, Fbiank and Fcontroi respectively refer to fluorescence intensity of the test sample, blank containing only PBS buffer, and the peptide-free control which was subjected to identical processing procedures.

[0216] For the pre-incubation experiment, peptide at different concentrations were pre-incubated with LPS (2 pg / mL) for 1 h (37°C, 120 rpm). FITC-LPS (2 pg / mL) was then added followed by incubation for 1 h (37°C, 120 rpm) then centrifugation (16,000 rpm, 10 min). 100 pL of the supernatant was gently collected. Fluorescence intensity of FITC-LPS (XEX= 492 nm, ZEM = 525 nm) in the supernatant was measured using TECAN Infinite M200. FITC-LPS trapping index was calculated using equation (1).

[0217] 4.4 KI 14 fluorescence assay

[0218] Incubation mixture of LPS (2 pg / mL), KI 14 (20 pM), and peptide at different concentrations were prepared in PBS buffer. A positive control containing LPS (5 pg / mL), KI 14 (20 pM) and BTT2 (20 pM) was also prepared, which we previously established to form extensive amyloid nanofibrils. Sample was incubated for 1 h (37°C, 120 rpm), then fluorescence intensity of KI 14 (ki.x = 360 nm, XEM = 550 nm) was measured using TECAN Infinite M200 and normalized against the positive control.

[0219] 4.5 Confocal microscopy

[0220] Peptide (64 pM) was incubated with FITC-LPS (32 pg / mL) in PBS buffer for 1 h. A peptide-free control was also prepared. Samples were mounted onto glass slide and air-dried, then imaged under a lOOx oil immersion lens using FV 1000 TIF inverted laser scanning confocal microscope (Olympus, Tokyo, Japan). A filter set of ki.x= 488 nm and XEM = 520 nm was used to visualize FITC-LPS.

[0221] 4.6 Cytokine trapping assay

[0222] Incubation mixture of LPS or LTA (5 pg / mL), cytokine (2 ng / mL), and peptide at different concentrations was prepared in PBS buffer. A peptide-free control was also prepared. After 1 h of incubation (37°C, 120 rpm), sample was centrifuged (16,000 rpm,10 min). 100 pL of the supernatant was gently collected and quantified for cytokine using commercial ELISA kits (BD Biosciences), and normalized against the control.

[0223] 4.7 Minimum inhibitory concentration (MIC) assay

[0224] MIC assays were conducted using broth microdilution method. LPS at different concentrations was added to a two-fold dilution series of peptide or colistin in MHB. To investigate the effect of peptide co-treatment on colistin MIC, LPS (100 pg / mL) and peptide (10, 20, or 40 pg / mL) were added to a series of colistin concentrations. Bacterial culture in mid-log phase were adjusted to an optical density at 600 nm (ODeoo) of 0.07, followed by lOOx dilution to give an initial inoculum of 106colony-forming units (CFU) / mL. An equal volume of bacteria culture was added to the mixture, followed by incubation for 18-20 h (37°C, 120 rpm). ODeoo was measured using TECAN Infinite M200, and MIC was recorded as the lowest peptide concentration to cause >90% decrease in the ODeoo relative to drug-free control.

[0225] 4.8 Quantification of LPS-induced cytokine production by macrophages

[0226] RAW 264.7 cells (200,000 cells / mL) were incubated for 24 h. For pretreatment experiment, cells were pre-treated with peptide at different concentrations for 1 h, followed by the addition of LPS (0.1 or 1 pg / mL) an incubation for 24 h at 37 °C. Blank wells received only the culture medium without LPS. Dexamethasone (1 pM) was tested as a positive control. After incubation, the plate was centrifuged (l,000xg, 5 min, 4°C) and the supernatant was collected and quantified for TNF-a and IL-6 using commercially available ELISA kits according to the manufacturer’s instructions. Results were presented as % cytokine release relative to the peptide-free control.

[0227] 4.9 cell viability assay

[0228] RAW 264.7 cells (200,000 cells / mL) were seeded into 96-well tissue culture plates and incubated for 24 h. Cells were treated with fresh culture media containing peptide at different concentrations and incubated for another 24 h. Blank control wells received only the medium but no cells. Quantity of viable cells were determined using CellTiter-Glo® luminescent cell viability assay kit. Results were normalized against drug-free control.

[0229] 4.10 Murine model of LPS-induced acute lung injury

[0230] Female BALB / c mice of 6-8 weeks old (InVivos, Singapore) were maintained in a 12-h light / dark cycle with food and water available ad libitum. The animals were divided into 5 groups (n = 5-6 mice per group): saline control, 5 pg LPS, LPS + 0.03mg / kg BTT1-3A, LPS + 0.1 mg / kg BTT1-3A, and LPS + 0.02 mg / kg dexamethasone (positive control). Dexamethasone dose is equivalent to clinical inhaler dose. After the mice were lightly anesthetized with isoflurane (Piranal, Mumbai, India), 5 pg LPS in 40 pL saline was administered intratracheally to induce acute lung infection. 2 h later, BTT1- 3A or dexamethasone was administered intratracheally. At 4 h post-treatment, mice were euthanized to collect bronchoalveolar lavage fluid (BALF) for biochemical analyses.

[0231] 4.11 BALF analysis

[0232] Mice were anesthetized with ketamine / medetomidine (110 and 1.5 mg / kg respectively). Tracheotomy was performed and a cannula was inserted into the mouse trachea. Ice-cold PBS (3x0.5 mL) was instilled into the lungs, and BALF was collected and kept at -80°C until analysis. Total cell counts were performed blinded. Differential cell count was determined by using the BD Fortessa flow cytometer and analyzed with FlowJo software (BD Bioscience, Franklin Lakes, NJ). Leukocytes were identified as CD45+ cells, neutrophils as CDl lb+ / Gr-l+ cells, macrophage as CDl lc+ / Siglec-F+ cells. Cytokine levels in BALF were analyzed using mouse TNF-a and IL-6 Mono / Mono OptEIA™ ELISA kits.

[0233] 4.12 Statistical analysis

[0234] Statistical significance of differences between data groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). GraphPad Prism 7.03 was employed for all statistical tests, graph plotting, and curve fitting.

[0235] Example 5- Results section 2 - Anti-inflammatory properties

[0236] Inspired by the trap-and-kill immune defense strategy ubiquitous in nature, synthetic P-hairpin antimicrobial peptides (AMPs) have been designed, with the ability to self-assemble into amyloid-like nanonets specifically in response to bacterial endotoxins. These peptide nanonets, which comprise extensively cross-linked nanofibrils, displayed unique trap-and-kill dual-functionalities, which present a direct upgrade from the trap-only nanonets in nature which are reliant on other host immune components for antimicrobial activity. Upon coming into contact with LPS, the peptide can either: (1) undergo fibrillation to form cross-linked nanonets for physical trapping of nearby bacteria cells, or (2) fold into hairpin conformations for membranolytic antimicrobial activity. The side strands of the disclosed peptides carry three Leu-Lys repeat units, and the resulting polycationicity proved integral to facilitating interactions between the peptides andendotoxins. As such, it is hypothesized that the fibrillating peptides could display antiinflammatory functionality by capturing free endotoxins and pro-inflammatory cytokines in solution via electrostatic attractions. Selective trapping of pro-inflammatory cytokines is highly coveted for sepsis management, given the complex concoction of pro- and antiinflammatory cytokines typically faced in a cytokine storm. Endotoxin trapping can impede its engagement with host immune and endothelial cells, including activation of Toll-like receptor 4 (TLR4) and induction of nitric oxide production. By alleviating the damage of endotoxemia, the multifunctional nanonets potentially offer a holistic anti- infective therapy that mitigates both upstream and downstream ramifications of infectious pathogenesis. As the first report of multifunctional peptide nanonets, this work will help accentuate the clinical potential of this class of biomaterials. In comparison to recently explored anti-septic strategies for capturing endotoxins and cytokines (e.g. bioengineered extracellular vesicles, and telodendrimer nanotrap), the synthesis of the disclosed short linear peptides is far more streamlined with well-established solid-phase peptide synthesis (SPPS) approach. In addition, the current system offers superior flexibility as specific functionalities of the nanonets can be readily fine-tuned through simple manipulation of the peptide sequence, as illustrated herein.

[0237] 5.1 Peptide nanonets bind and entrap bacterial endotoxins

[0238] From the library of [3-hairpin AMPs disclosed herein, two representative fibrillating peptides previously demonstrated to display potent trap-and-kill activity against pathogenic bacteria, BTT1-3A and BTT2-4A, were selected for investigating their anti-inflammatory activity, as compared to the non-fibrillating peptide BTT2-2A. Their sequence and properties are listed in Table 8. As shown in transmission electron microscopy (TEM) images (Figure 24), BTT2-4A formed expansive nanonets selectively in the presence of bacterial LPS, but not buffer alone. To study the in vitro LPS-trapping ability of the nanonets, a fluorescence assay using fluorescein isothiocyanate (FITC)- labelled LPS was employed. After incubating the peptides with FITC-LPS for 1 h, nanonets self-assembled through stacking of the peptide monomers with LPS molecules acting as amyloid-nucleating sites, thus capturing the LPS in solution. The sample was then centrifuged to pellet the nanonet-bound LPS, and the amount of free LPS in the supernatant was measured via FITC fluorescence for calculating the FITC-LPS trapping index. A higher index reflects greater LPS-trapping efficacy. As expected, the fibrillating BTT2-4A and BTT1-3A displayed higher FITC-LPS trapping indices than the non-fibrillating BTT2-2A, in a concentration-dependent manner (Figure 25). LPS-trapping efficacy of BTT2-4A (0.52 at 80 pM) was moderately higher than that of BTT1-3A (0.44 at 80 pM) despite identical peptide net charge of +7 (Table 8). This agrees with the previous analysis that variations in amphipathicity pattern of the turn sequence can considerably impact LPS-binding affinity. Vortexing of the centrifuged samples resulted in the release of most of the FITC-LPS back to the supernatant (Figure 26), thus ruling out photobleaching during processing as a cause for the observed decrease in fluorescence signals. Confocal microscopy was used to visualize nanocomplexation between FITC- LPS and the nanonets. After 1 h of incubation with the fibrillating peptides BTT2-4A and BTT1-3A, FITC-LPS was observed to form large aggregates with intense green fluorescence (Figure 25b), whose size was comparable to FITC-labelled peptide aggregates previously observed. In contrast, incubating FITC-LPS with the non- fibrillating BTT2-2A or in the absence of peptide did not yield visible aggregates. Taken together, the findings suggest that free LPS in solution could be readily captured by peptide nanonets.[a]p: D-Pro; -NFL: amidation at C-terminal; turn residues are highlighted in gray.[b]Net charge at neutral pH was calculated using an online web-based tool (https : / / pepcalc .com / )[c]Hydropathy value, which quantifies hydrophobicity, was calculated by adding up hydropathic index of every amino acid in the sequence.

[0239] To gain mechanistic insights into the endotoxin trapping process, peptide nanonets were pre-formed during 1 h pre-incubation with non-labelled LPS, followed by exposure to FITC-LPS for quantification of trapping efficacy like above. This set-up simulates situations where administered peptides first self-assemble to form nanonets in response to bacteria cells before encountering the circulated endotoxins. Overall, trapping capacity of pre-formed nanonets increased with peptide concentration up to ~50 pM, above which a plateau was observed (Figure 25c). From the concentration-responsecurve, the LPS-trapping ability of BTT2-4A nanonets (EC50 = 12.2 pM) was slightly more potent than BTT1-3A (EC50 = 15.1 pM). KI 14 fluorescence assay was conducted to quantify amyloid formation during the pre-incubation period. The fluorescence dye KI 14 exhibits a red-shift and higher fluorescence when it binds to amyloid fibrils. BTT1-3A induced a higher KI 14 fluorescence than BTT2-4A at all tested concentrations (Figure 27), indicating a greater amount of pre-formed nanonets during pre-incubation with nonlabelled LPS. The negative correlation between the quantity of pre-formed nanonets (BTT2-4A < BTT1-3A) and subsequent extent of FITC-LPS trapping (BTT2-4A > BTT1-3A) can be explained by a dual endotoxin trapping mechanism (Figure 25d): (1) scaffold integration - EPS served as amyloid-nucleating foci and was integrated within the elongating nanofibrils, and (2) post-binding - LPS was electrostatically bound to the pre-formed nanofibrils. Compared to BTT1-3A, a lesser extent of pre-formed BTT2-4A nanonets implies that more BTT2-4A molecules remained in solution for additional nanonets formation with FITC-LPS, followed by greater FITC-LPS trapping via mechanism (1). Meanwhile, more limited binding pockets on pre-formed BTT2-4A nanonets would allow for less FITC-LPS trapping via mechanism (2), assuming that the binding sites for KI 14 and LPS highly correlate. These sites comprise both external surfaces and internal structures of the pre-formed fibrils. Altogether, mechanism (1) appears to play a more prominent role.

[0240] Previously it was observed that LTA, a biomolecule characteristic of Grampositive membranes, induced the formation of nanonets with different morphology and behaviors than LPS. To investigate whether nanonets induced by Gram-positive bacteria can also capture free LPS, pre-formed nanonets was triggered using LTA in place of LPS during the pre-incubation step, then the extent of FITC-LPS trapping was measured. Overall, LTA-induced nanonets displayed an endotoxin-trapping profile very similar to LPS-induced counterparts (Figure 28), thus suggesting that the nanonets as disclosed herein can retain therapeutic utility against polymicrobial infections of both Grampositive and Gram-negative pathogens.

[0241] 5.2 LPS-trapping activity of nanonets enables restoration of colistin activity

[0242] Endotoxin-trapping activity of the nanonets can be utilized for various biomedical applications. Colistin is a last-resort antibiotic that disrupts bacteria membranes by interacting with membrane-bound LPS, and its activity can be impaired by competitive binding from extracellular LPS whose abundance markedly elevatesduring sepsis. By capturing free LPS, it was hypothesized that the disclosed nanonets could safeguard colistin from their interference, thus enabling membrane interactions and antimicrobial activity by the antibiotic (Figure 29a). To this end, broth microdilution method was employed to determine the activity of colistin and peptides under different conditions, where a lower minimum inhibitory concentration (MIC) signifies more potent antimicrobial activity. Against E. coli ATCC 25922, the individual MICs of colistin and BTT2-4A in the absence of LPS were 2 and 10 pM, which increased 8-fold and 4-fold in the presence of LPS (100 pg / mL) (Figure 29b). In combination, the addition of BTT2-4A at a sub-MIC concentration (20 pM) was observed to lower the LPS-impaired MIC of colistin 8-fold from 16 pM to 2 pM (Figure 29c), thus restoring colistin activity. For comparison, gold nanosheets recently reported by Liao et al only achieved a 2-fold reduction in colistin MIC, but requires a very high gold concentration of 500 pg / mL. BTT2-4A also exhibited similar antibiotic-potentiating effect against other Gramnegative strains. When tested with the colistin-resistant E. coli M2 clinical isolate and P. aeruginosa ATCC 9027, BTT2-4A at sub-MIC concentrations yielded respectively 4- fold and 8-fold decrease in LPS-impaired MIC of colistin to 4 pM (Figure 29c). The significant improvement in antibacterial potency of colistin might be explained by a threshold peptide concentration above which nanonets formation drastically increased, which in turn elevated the endotoxin-trapping capacity of the peptide.

[0243] 5.3 Nanonets selectively bind pro -inflammatory cytokines through electrostatic interactions

[0244] Next, the interactions between the disclosed peptides and inflammation mediators was explored (Figure 30a). After incubating the peptides with LPS (5 pg / mL) and recombinant murine cytokine (2 ng / mL) for 1 h, the sample was centrifuged and the amount of free cytokine remaining in supernatant was quantified using standard enzyme- linked immunosorbent assay (ELISA). Whereas the non-fibrillating BTT2-2A failed to precipitate cytokines in the pellet, the fibrillating BTT2-4A exhibited substantial cytokine-trapping ability with a clear preference for tumor necrosis factor-a (TNF-a) and interleukin (IL)-6, which are secreted by macrophages during systemic inflammation. At 80 pM, BTT2-4A nanonets entrapped >70% of these two cytokines, but did not lower the quantity of free IL-4 and IL- 10 in the supernatant (Figure 30b). Pro-inflammatory cytokines (e.g. TNF-a and IL-6) mostly carry negative charges, whereas many antiinflammatory cytokines (e.g. IL-4 and IL- 10) are positively charged. The apparenttrapping selectivity of the disclosed nanonets towards the pro-inflammatory cytokines is indicative of electrostatic interactions mediated by the cationic Lys residues on the peptides. Of interest, BTT1-3A at 80 pM captured -50% of TNF-a but had negligible effect on IL-6. The higher hydropathy value of BTT2-4A signifies that it is more hydrophobic than BTT1-3A (Table 8). The corresponding greater binding affinity of BTT2-4A towards the near-neutral IL-6 suggests that hydrophobic interactions also contribute to cytokine-trapping activity of the nanonets. LTA-nucleated BTT2-4A and BTT1-3A nanonets also displayed selective trapping activity towards pro-inflammatory cytokines while not affecting anti-inflammatory cytokines (Figure 28), which supports the clinical potential of the nanonets against Gram-positive bacteria.

[0245] 5.4 In vitro and in vivo anti-inflammatory activity of the peptides

[0246] Then, the peptide effect on LPS -activated cytokine production was evaluated by the murine macrophage RAW 264.7 cells. When LPS is recognized by the membranebound TLR4 and CD 14 co-receptors on macrophages, signaling pathways are activated which promote the release of cytokines. When the cells were post-treated with BTT2-4A or BTT1-3A after LPS exposure, their cytokine release measured after 24 h was significantly lower than untreated control, with a more pronounced impact on IL-6 than TNF-a (Figure 31). In contrast, treatment with the non-fibrillating BTT2-2A had minimal effect on cytokine release despite an identical net charge among the peptides (Table 8). This establishes nanonet formation as a pre-requisite for mitigating LPS -induced cytokine release by macrophages. Peptide pre-treatment 1 h before LPS was added to the media did not yield noticeably greater anti-inflammatory effect than post-treatment (Figure 32). When LPS concentration was increased from 0.1 to 1 pg / mL, the percentage of cytokine release by peptide-treated macrophages remained similar. By showing that the peptides were not cytotoxic at all tested concentrations (Figure 33), it was possible to rule out lysis of the macrophages as a cause for the observed decrease in cytokine release.

[0247] Although BTT1-3A displayed considerably weaker LPS -trapping efficacy than BTT2-4A (Figure 25) and negligible trapping effect against IL-6 (Figure 30b), it was found to strongly inhibit LPS-induced IL-6 production by macrophages (Figure 31a). These findings suggest that the fibrillating peptides also have direct suppressive effect on cellular pathways governing cytokine release. A multi-pronged anti-inflammatory mechanism was thus proposed, which impacts the inflammation process at various stages: (1) Nanonets entrap LPS which prevents macrophage binding and activation, (2)Nanonets entrap released pro-inflammatory cytokines which mitigates their ability to trigger downstream inflammatory events, and (3) Monomeric peptide molecules unassociated in nanonets penetrate immune cells and inhibit intracellular cytokine release pathways.

[0248] With moderately greater in vitro anti-inflammatory activity in RAW 264.7 cells (Figure 31a and 28), BTT1-3A was selected for exploring in vivo efficacy. Animal experiments were performed in accordance to protocol no. R20-01283 approved by the Institutional Guidelines of the Animal Care and Use Committee (IACUC) at National University of Singapore. An endotoxin-induced acute lung injury model in mice was employed, whereby the mice were euthanized at 4 h post-treatment and bronchoalveolar lavage fluid (BALF) was collected post-mortem for analysis. The effect of LPS on local immune response in the mice was apparent when compared to saline control, as saline vehicle control induced minimal immune cell count and cytokine levels in BALF (Figure 31c). In mice treated with BTT1-3A via intratracheal administration, the level of pro- inflammatory cytokines TNF-a and IL-6 in BALF was significantly lowered in a concentration-dependent manner. An equimolar dose of BTT1-3A (0.1 mg / kg) achieved comparable efficacy to the positive control dexamethasone (0.02 mg / kg) at lowering TNF-a, but greater effect against IL-6 (p = 0.02). Neither BTT1-3A nor dexamethasone caused any change in the BALF immune cell count was observed, which indicates a lack of effect on immune cell chemotaxis. Overall, the disclosed fibrillating peptide exhibited promising in vivo activity at reducing endotoxin-activated immune response.

[0249] Example 6 - Conclusion 2 - Anti-inflammatory properties

[0250] In summary, the disclosed peptide nanonets displayed promising antiinflammatory activity both in vitro and in vivo, thus expanding their repertoire of functionalities beyond that of antibacterial trap-and-kill. The nanonets are postulated to specifically bind and entrap endotoxin and pro-inflammatory cytokines by exploiting the abundance of negative charges on these inflammation mediators. This could potentially overcome the limitation in activity spectrum associated with target- specific anti-septic strategies and minimize undesirable inactivation of the beneficial anti-inflammatory mediators. In addition, it was proposed that the LPS -trapping ability of the nanonets can be mobilized as adjuvant action to nullify antagonistic interactions from extracellular LPS and restore the activity of clinically relevant antibiotics. By expanding their arsenal offunctionalities, it is possible to promote further development of peptide -based nanonets as multi-purpose anti-infective biomaterials.

[0251] Example 7 - Results Section 3 - delayed resistance

[0252] Antibiotic resistance is major global healthcare issue, which significantly shorten the lifespan of any novel antibiotics. This disclosure introduces peptides as adjuvants to directly tackle this issue, thus extending the drug lifespan, and in turn boosting both their commercial and clinical values. The mechanism of action is potentially applicable in potentiating various classes of antibiotics.

[0253] Bacteria with restricted motility have been reported to exhibit extensive changes in their transcriptome and proteome and subsequent virulence and fitness. The increase in bacteria cluster size can impair their immune evasiveness and promote complement deposition and neutrophil phagocytosis. It was asked if immobilization by nanonet trapping could affect the bacteria’s response to subsequent antibiotic selection. In this work, BTT1-3A and BTT2-4A was tested as candidate nanonet-forming (i.e. fibrillating) peptides, alongside BTT1 and BTT4 as active but non-fibrillating peptides for comparison.

[0254] Checkerboard assays were initially performed using paired combinations of fibrillating peptides and mechanistically diverse antibiotics as an indirect measure of bacteria fitness change. These antibiotics from diverse classes typically display narrow spectrum antibiotics active against Gram-positive bacteria but inactive against Gramnegative P. aeruginosa and E. coli M2 strains. However, Figure 34A shows that when used in combination with another “trap-and-kill” fibrillating peptide BTT1-3A, synergy (as indicated by FICI <0.5) was observed with a limited number of otherwise inactive antibiotics. Another non-fibrillating BTT4 peptide used for comparison here displayed broader synergy than the fibrillating BTT1-3A. However, BTT1-3A far outperformed BTT1 and BTT4 at delaying resistance emergence against rifampicin, when tested at sub- MIC concentrations in combination treatments. While BTT1 and BTT4 moderately delayed the onset of resistance by around 2 and 4 days respectively, co-administration of BTT1-3A maintained the starting potency of rifampicin up to 18 days (Figure 34B). BTT2-4A was similarly effective at delaying rifampicin resistance, with only minor increase in MIC after 17 days. E. coli cultures after 18 days of serial passage were examined for their motility, using live-image fluorescence imaging to track cellular movement (Figure 34C) and soft agar assay (Figure 34D). Compared to rifampicin alone,the addition of non-fibrillating peptides caused negligible change in bacteria motility. The impact of combining BTT1-3A or BTT2-4A with rifampicin was clearly greater. These findings suggest that fibrillating peptides exerted a sustaining effect on bacteria that escaped the antibiotic selection and affected its fitness for growth.

[0255] To evaluate phenotypic changes due to prolonged exposure to sub-inhibitory concentration of BTT peptides, E. coli clones that were selected after 18 days of serial passage with peptides was subjected to repeated synergy testing and biofilm formation. As shown in Figure 35A, synergy was preserved only in clones treated with fibrillating peptides, P18_R1-3A and P18_R2-4A, as indicated by fold-change in FICI < 1 (Figure 35B). In contrast, clones treated with rifampicin alone or in combination with non- fibrillating peptides (P18_R, P18-R1, and P18-R4) rapidly acquired resistance, thus resulting in the loss of synergies. Overall, continual exposure to nanonet-forming peptides not only delay resistance of bacteria against antibiotic alone, but also maintain their susceptibility to synergistic interactions between antibiotic and AMPs. Additionally, the Pl 8 clones were examined using crystal violet staining for their capacity to form biofilms. Of interest, clones selected with sub-MIC of BTT peptides displayed impaired ability to form biofilm, but no significant difference was observed between fibrillating and non- fibrillating peptides (Figure 35C). This supports the utility of BTT peptides as adjuvants of antibiotics, for reduced motility on biofilm formation.

[0256] After establishing peptide interactions with Gram- negative-inactive drugs, antibiotics already active against Gram-negative pathogens (i.e. colistin, ceftazidime, ciprofloxacin) were next tested. Synergy was not detected in most of the pairwise combinations (FICI > 0.5). However, the fibrillating peptides again demonstrated resistance-delaying effects superior to non-fibrillating peptides when combined with ceftazidime (Figure 36A). While the MIC of ceftazidime alone drastically increased 32- fold after 7 days, the addition of BTT 1-3 A or BTT2-4A at sub-inhibitory concentrations maintained a consistently low ceftazidime MIC throughout 18 days. In comparison, resistance to ceftazidime emerged after 3 and 8 days with BTT1 and BTT4 respectively. The correlation with motility reduction was also established using the soft agar assay (Figure 36B). These findings illustrated the potential application of nanonet peptides as a resistance-delaying adjuvant even in combination with non-synergistic antibiotics.

[0257] Taken together, the data supports that peptide nanonets display multifunctionality that are not typically seen in classical antibiotic adjuvants in clinical use.

[0258] Example 8 - Discussion- delayed resistance

[0259] As shown in Figure 34A, lack of synergy was observed in a number of combinations between BTT peptides and Gram- negative-inactive antibiotics. It was also shown that the fibrillating peptides do not exhibit any synergy with Gram-negative-active antibiotics from different classes.

[0260] While there have been studies on the effect of motility reduction on resistance of antimicrobial agents like silver nanoparticles, it has never been explored as strategy to actively delay resistance to traditional small-molecular antibiotics. Serial passage is a resource- and time-consuming procedure, thus generally not suitable for large-scale screening.

[0261] Not every antibiotic -peptide combination displayed clear resistance delaying effect. For instance, resistance developed against streptomycin was observed within 6 days even when a fibrillating peptide BTT 1-3 A or BTT2-4A was added as an adjuvant (Figure 37). The MIC of the antibiotic increased > 16-fold after 10 days of serial passage, in combination with the fibrillating peptides. This further supports non-obviousness of the invention.

[0262] Industrial Applicability

[0263] This disclosure is to be applied in the field of anti-infective therapies for treating infections caused by antibiotic -resistant bacteria, as well as infection- or sepsis- associated inflammatory responses.

[0264] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.Reference• Tram, N. D. T., Selvarajan, V., Boags, A., Mukherjee, D., Marzinek, J. K., Cheng, B., Jiang, Z. C., Goh, P., Koh, J. J., Teo, J. W. P., Bond, P. J., & Ee, P. L. R. Acta biomaterialia, 2021; 735:214-224. • F.-H. Liao, T.-H. Wu, C.-N. Yao, S.-C. Kuo, C.-J. Su, U.-S. Jeng, S.-Y. Lin, Angew.Chem. Int. Ed. Engl. 2020, 59, 1430-1434.• Zhang G, Meredith TC, Kahne D. Curr. Opin. Microbiol. 2013 Dec 1; 16(6):779-85.• Percy, M.G. and Griindling, A., 2014. Anna. Rev. Microbiol., 68, pp.81-100.

Claims

Claims1. A synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules:(a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component to initiate amyloid nucleation; and(b) a structural module comprising a first side strand and a second side strand for P- sheet formation and molecular stacking during fibrillation; wherein the synthetic P-hairpin AMP comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of the bacterial membrane component or a bacterium.

2. The synthetic P-hairpin AMP of claim 1, wherein the hairpin turn comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, the first side strand comprises the sequence of SEQ ID NO: 5, and the second side strand comprises the sequence of SEQ ID NO: 6.

3. The synthetic P-hairpin AMP of claim 1, wherein the bacterial membrane component is selected from the group consisting of lipoteichoic acid (LTA) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gram-negative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

4. The synthetic P-hairpin AMP of claim 3, wherein the Gram-positive bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Eisteria monocytogenes.

5. The synthetic P-hairpin AMP of claim 3, wherein the Gram-negative bacterium is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera.

6. A pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP of any one of claims 1-5, and a pharmaceutically acceptable carrier or diluent.

7. The pharmaceutical or adjuvant composition of claim 6, wherein the composition is in a formulation selected from the group consisting of a parenteral formulation, a nasal spray, an eye drop, a mouth wash and oral gel.

8. A method for treating a condition associated with a bacterial infection, or an immune- related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, comprising administering the synthetic P-hairpin AMP of any one of claims 1-5, or the pharmaceutical or adjuvant composition of claim 6 or 7 to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to(a) trap and kill the bacterium; and / or(b) trap an endotoxin and a pro-inflammatory cytokine; and / or(c) trap and reduce motility of the bacterium.

9. Use of the synthetic P-hairpin AMP of any one of claims 1-5, or the pharmaceutical or adjuvant composition of claim 6 or 7, in the manufacture of a medicament for treating a condition associated with a bacterial infection, or an immune-related disease, or for delaying the development of antibiotic resistance in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets in the presence of a bacterial membrane component or a bacterium to(a) trap and kill the bacterium; and / or(b) trap an endotoxin and a pro-inflammatory cytokine; and / or(c) trap and reduce motility of the bacterium.

10. The method of claim 8 or the use of claim 9, wherein the bacterial infection is an infection of a Gram-positive bacterium or a Gram-negative bacterium selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, Eisteriamonocytogenes, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera.

11. The method of claim 8 or the use of claim 9, wherein the bacterial infection is an infection of an antibiotic -resistant bacteria.

12. The method or the use of claim 11, wherein the antibiotic-resistant bacteria is selected from the group consisting of colistin-and / or carbapenem- resistant Escherichia coli, colistin- and / or carbapenem- resistant Klebsiella aerogenes, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, colistin- and / or carbapenem- resistant Acinetobacter baumannii, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

13. The method of claim 8 or the use of claim 9, wherein the condition associated with a bacterial infection is selected form the group consisting of sepsis, septic arthritis, listeriosis, skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, abscesses, toxic shock syndrome (TSST-1), urinary tract infection (UTI), bloodstream infections, otitis media, sinusitis, meningitis, bacterial endocarditis, pharyngitis, cellulitis, impetigo, erysipelas, scarlet fever, necrotizing fasciitis, glomerulonephritis, rheumatic fever, myocarditis, arrhythmias, gastroenteritis, peritonitis, brucellosis, catscratch disease, cholera, Legionnaires' disease, pertussis, salmonella infections, shigellosis, tularemia, typhoid fever, acne, athlete's feet, onychomycosis, caries, periodontal disease, cold sore, rhinitis, and pink eye.

14. The method of claim 8 or the use of claim 9, wherein the immune-related disease is a bacterial infection-associated inflammation disease.

15. The method or use of claim 14, wherein the bacterial infection-associated inflammation disease is endotoxin-activated inflammation disease.

16. The method or use of claim 15, wherein the bacterial infection-associated inflammation disease is sepsis.

17. The method of any one of claims 8-16, or the use of any one of claims 9-16, wherein the nanonets trap a pro -inflammatory cytokine selected from the group consisting of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin- la (IL- la),interleukin- ip (IL-ip), interleukin- 12a (IL- 12a), interleukin- 12P (IL-12P), interleukin- 18 (IL- 18), and high mobility group box 1 (HMGB1).

18. The method of any one of claims 8-17, or the use of any one of claims 9-17, wherein the nanonets do not trap an anti-inflammatory cytokine selected from the group consisting of interleukin-4 (IL-4), interleukin- 10 (IL- 10), interleukin- 11 (IL- 11), interleukin- 13 (IL- 13), transforming growth factor beta 1 (TGF-pi), and interleukin- 18BP (IL-18BP).

19. The method of any one of claims 8-18, or the use of any one of claims 9-18, wherein the synthetic P-hairpin AMP or the composition is administered with an antibiotic.

20. The method or use of claim 19, wherein the antibiotic is selected from the group consisting of aminoglycoside, macrolide, fluoroquinolone, P-lactam, glycopeptide, oxazolidinone, and ansamycin.

21. The method of any one of claims 8-20, or the use of any one of claims 9-20, wherein the subject is a human.

22. The method of any one of claims 8-21, or the use of any one of claims 9-21, wherein the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg.

23. The method or use of claim 19 or 20, wherein the synthetic P-hairpin AMP is administered at a sub-inhibitory concentration of 1-4 pM.

24. The method of any one of claims 8-23, or the use of any one of claims 9-23, wherein the synthetic P-hairpin AMP is administered via a route selected from the group consisting of intravenous, intraperitoneal, intratracheal, intramuscular, intradermal, subcutaneous, oral, topical, and intranasal administration.

25. The method of any one of claims 8-24, or the use of any one of claims 9-24, wherein the synthetic P-hairpin AMP is administered at a frequency selected from the group consisting of one, two, three, four, and six times a day.

26. The method of any one of claims 8-25, or the use of any one of claims 9-25, wherein the synthetic P-hairpin AMP is administered for a duration selected from the group consisting of about 1 day, about 2 days, about 1 week, about 10 days, about 15 days, about 1 month, about 3 months, about 6 months, about 1 year, or greater than 1 year.

27. A kit comprising the pharmaceutical or adjuvant composition of claim 6 or 7, and a dispenser and / or applicator.

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