Gut microbes exacerbate systemic inflammation and behavior disorders in neurologic disease CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that carries mutations in NOTCH3. The clinical manifestations are influenced by genetic and environmental factors that may include gut microbiome. RESULTS: We investigated the fecal metagenome, fecal metabolome, serum metabolome, neurotransmitters, and cytokines in a cohort of 24 CADASIL patients with 28 healthy household controls. The integrated-omics study showed CADASIL patients harbored an altered microbiota composition and functions. The abundance of bacterial coenzyme A, thiamin, and flavin-synthesizing pathways was depleted in patients. Neurotransmitter balance, represented by the glutamate/GABA (4-aminobutanoate) ratio, was disrupted in patients, which was consistent with the increased abundance of two major GABA-consuming bacteria, Megasphaera elsdenii and Eubacterium siraeum. Essential inflammatory cytokines were significantly elevated in patients, accompanied by an increased abundance of bacterial virulence gene homologs. The abundance of patient-enriched Fusobacterium varium positively correlated with the levels of IL-1ß and IL-6. Random forest classification based on gut microbial species, serum cytokines, and neurotransmitters showed high predictivity for CADASIL with AUC = 0.89. Targeted culturomics and mechanisms study further showed that patient-derived F. varium infection caused systemic inflammation and behavior disorder in Notch3R170C/+ mice potentially via induction of caspase-8-dependent noncanonical inflammasome activation in macrophages. CONCLUSION: These findings suggested the potential linkage among the brain-gut-microbe axis in CADASIL. Video Abstract.

Synthetic peptides that form nanostructured micelles have potent antibiotic and antibiofilm activity against polymicrobial infections.

The emergence of multidrug-resistant bacterial pathogens is a growing threat to global public health. Here, we report the development and characterization of a panel of nine-amino acid residue synthetic peptides that display potent antibacterial activity and the ability to disrupt preestablished microbial biofilms. The lead peptide (Peptide K6) showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus in culture and in monocultures and mixed biofilms in vitro. Biophysical analysis revealed that Peptide K6 self-assembled into nanostructured micelles that correlated with its strong antibiofilm activity. When surface displayed on the outer membrane protein LamB, two copies of the Peptide K6 were highly bactericidal to Escherichia coli. Peptide K6 rapidly increased the permeability of bacterial cells, and resistance to this toxic peptide occurred less quickly than that to the potent antibiotic gentamicin. Furthermore, we found that Peptide K6 was safe and effective in clearing mixed P. aeruginosa-S. aureus biofilms in a mouse model of persistent infection. Taken together, the properties of Peptide K6 suggest that it is a promising antibiotic candidate and that design of additional short peptides that form micelles represents a worthwhile approach for the development of antimicrobial agents.

The design of cell-selective tryptophan and arginine-rich antimicrobial peptides by introducing hydrophilic uncharged residues.

Antimicrobial peptides (AMPs) are considered to be powerful weapons in the fight against traditional antibiotic resistance due to their unique membrane-disruptive mechanism. The combination of traditional and classical hydrophobic tryptophan (W) residues and hydrophilic charged arginine (R) residues is considered as the first choice for the minimalist design of AMPs due to its potent performance in antibacterial activity. However, some W- and R-rich AMPs that are not rationally designed and contain excessive repeats of W and R residues may cause severe cytotoxicity and hemolysis. To address this issue, we designed the (WRX)n (where X = hydrophilic uncharged amino residues; n = number of repeat units) series engineered peptides with high cell selectivity by introducing hydrophilic uncharged threonine (T), serine (S), glutamine (Q) or asparagine (N) residues into the minimalist design of W- and R-rich AMPs. The results showed that the introduction of these hydrophilic uncharged amino residues, especially T residues, significantly improved the cell selectivity of the W- and R-rich engineered peptides. Among (WRX)n series engineered peptides, T6 presents a mixture structure of ß-turn and α-helix. It has broad spectrum and potent antibacterial activity (no activity against probiotics), good biocompatibility, high selectivity index, strong tolerance (physiological salts, serum acid, alkali, and heat conditions), rapid and efficient time-kill kinetics, and no tendency of resistance. Studies on antibacterial mechanism show that T6 exert antibacterial activity mainly by disrupting bacterial cell membrane and inducing the accumulation of reactive oxygen species in bacterial cells. Furthermore, T6 exhibited potent antibacterial and antiinflammatory capabilities in vivo in a mouse peritonitis-sepsis model infected with Escherichia coli. In conclusion, our study confirms an effective strategy for the minimalist design of highly cell selective W- and R-rich AMPs by introducing hydrophilic uncharged T residues, which may trigger widespread attention to hydrophilic uncharged amino acid residues, including T residues, and provide new insights into the design of peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: We have introduced hydrophilic uncharged T, S, Q or N residues into the minimalist design of W- and R-rich engineered peptides and found that the introduction of these hydrophilic uncharged amino residues, especially the T residues, can significantly improve the cell selectivity of W- and R-rich engineered peptides. The target compound T6 showed potent antibacterial activity, high cell selectivity, strong tolerance, good in vivo efficacy and killed bacteria through multiple mechanisms mainly membrane-disruptive. These findings may spark widespread interest in hydrophilic uncharged amino acid residues, and provide new insights into the design of peptide-based antimicrobial biomaterials.

Targeted Antimicrobial Agents as Potential Tools for Modulating the Gut Microbiome.

The gut microbiome plays a pivotal role in maintaining the health of the hosts; however, there is accumulating evidence that certain bacteria in the host, termed pathobionts, play roles in the progression of diseases. Although antibiotics can be used to eradicate unwanted bacteria, the side effects of antibiotic treatment lead to a great need for more targeted antimicrobial agents as tools to modulate the microbiome more precisely. Herein, we reviewed narrow-spectrum antibiotics naturally made by plants and microorganisms, followed by more targeted antibiotic agents including synthetic peptides, phage, and targeted drug delivery systems, from the perspective of using them as potential tools for modulating the gut microbiome for favorable effects on the health of the host. Given the emerging discoveries on pathobionts and the increasing knowledge on targeted antimicrobial agents reviewed in this article, we anticipate targeted antimicrobial agents will emerge as a new generation of a drug to treat microbiome-involved diseases.

pH-Responsive Antimicrobial Peptide with Selective Killing Activity for Bacterial Abscess Therapy.

The unusual acidic pH of the abscess milieu is an adverse factor that decreases the therapeutic efficacy of traditional antibiotics. Moreover, avoiding both the undesired killing of commensal bacteria and the development of drug resistance remains difficult during abscess therapy. Hence, we synthesized a series of pH-responsive antimicrobial peptides equipped with efficient bacterial killing activity at pH 6.5 and inactivity at pH 7.4. Among the peptides, F5 exhibited outstanding pH-responsive antimicrobial activity and low toxicity. Fluorescence spectroscopy and electron microscopy illustrated that F5 killed bacteria via a membrane-disruptive mechanism at acidic pH values. Mouse cutaneous abscesses revealed that F5 was equipped with excellent therapeutic ability to reduce the bacterial load and cytokines without causing skin toxicity. In summary, this study reveals a strategy for selectively killing bacteria under the pathologic conditions of abscess sites while avoiding the elimination of commensal bacteria under normal physiological pH levels.

The Trp-rich Antimicrobial Amphiphiles With Intramolecular Aromatic Interactions for the Treatment of Bacterial Infection.

Antibiotic resistance is emerging as a hot issue with the abuse and overuse of antibiotics, and the shortage of effective antimicrobial agents against multidrug resistant bacteria creates a huge problem to treat the threatening nosocomial skin and soft tissue infection. Antimicrobial peptides (AMPs) exhibite enormous potential as one of the most promising candidates of antibiotic to fight against pathogenic infections because of its unique membrane penetration mechanism to kill pathogens, whereas the clinical application of AMPs still faces the challenges of production cost, stability, safety, and design strategy. Herein, a series of Trp-rich peptides was designed following the principle of paired Trp plated at the ith and ith+4 position on the backbone of peptides, based on the template (VKKX)4, where X represents W, A, or L, to study the effect of intramolecular aromatic interactions on the bioactivity of AMPs. Through comparing the antimicrobial performance, hemolysis, cytotoxicity, and stability, VW5 which is equipped with the characters of direct antimicrobial efficacy (GM=1.68µM) and physical destruction of bacterial membrane (SEM and electron microscopy) stood out from the engineering peptides. VW5 also performed well in mice models, which could significantly decrease the bacterial colony (VW5 vs infection group, 12.72±2.26 vs 5.52±2.01×109CFU/abscess), the area of dermo-necrosis (VW5 vs infection group, 0.74±0.29 vs 1.86±0.98mm2) and the inflammation cytokine levels at the abscess site without causing toxicity to the skin. Overall, this study provides a strategy and template to diminish the randomness in the exploration and design of novel peptides.

Selective Antifungal Activity and Fungal Biofilm Inhibition of Tryptophan Center Symmetrical Short Peptide.

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.

Hybrid Antimicrobial Peptide Targeting Staphylococcus aureus and Displaying Anti-infective Activity in a Murine Model.

Broad-spectrum antimicrobial peptides (AMPs) kill bacteria indiscriminately, increasing the possibility of an ecological imbalance in the microbiota. To solve this problem, new types of AMPs, which kill pathogenic bacteria without breaking the micro-ecological balance of the body, were proposed. Here, we successfully designed a targeting AMP, S2, which is a fusion peptide composed of a species-specific targeting domain and broad-spectrum AMP domain. In the current study, S2 showed specific killing activity against Staphylococcus aureus, and almost no resistance induced compared to penicillin. Mechanism studies indicated that S2 killed S. aureus by destroying the bacterial membrane. Meanwhile, S2 possessed excellent salt-tolerance properties and biocompatibility. Importantly, S2 exhibited perfect treatment efficacy against an S. aureus subcutaneous infection model and remained nontoxic. In conclusion, this study provides a promising strategy for designing specific AMPs against growing bacterial infections.

Targeted and Intracellular Antibacterial Activity against S. agalactiae of the Chimeric Peptides Based on Pheromone and Cell-Penetrating Peptides.

The significance of the complex bacterial ecosystem in the human body and the impediment of the mammalian membrane against many antibiotics together emphasize the necessity to develop antimicrobial agents with precise antimicrobial and cell-penetrating activities. A simple and feasible method for generating dual-function antimicrobial peptides inspired by highly hydrophobic peptide pheromone and cationic cell-penetrating peptides is presented. Furthermore, the extension of the peptide candidate library is achieved by modifying the charged domain. The bacteria-selective peptides L1, L2, L10, and L11 kill Streptococcus agalactiae by disrupting the membrane structure, and the targeted mechanism is suggested where the peptides offset the entrapment of S. agalactiae rather than of other bacteria. Moreover, L2 and L10 possess intracellular antibacterial activity and carrier property, which is mainly dependent on endocytosis. Given their suitable biocompatibility, high tolerance, no drug resistance, and effective antimicrobial capacity in a mouse mastitis model, L2 and L10 can be powerful weapons against S. agalactiae pathogen infection.

Peptides With Triplet-Tryptophan-Pivot Promoted Pathogenic Bacteria Membrane Defects.

Development of probiotic-ineffective antimicrobial peptides (AMPs)-based coatings that can kill pathogenic bacteria at low concentrations but are essentially harmless (even high concentrations) to probiotic organisms is a relatively new trend for therapy against GI tract infections. In this study, a series of triplet-tryptophan-pivot peptides with various hydrophilic amino acids was constructed. One AMP in particular, S7, showed bactericidal activity against Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli and antibiotic-resistant Staphylococcus aureus, yet was shown to be harmless to Lactobacillus rhamnosus, a key GI tract commensal. Furthermore, antibacterial mechanism assays, drug resistance assays, and mouse model tests suggested that S7 was useful in a clinical setting as it proved to significantly reduce bacterial load and cytokines (TNF-α, IL-6; P < 0.05) with a low probability of resistance via bacterial membrane physical destruction and formation of intracellular ROS. Combined, the results show that a triplet-tryptophan-pivot peptide containing a pair of serine residues was an excellent pathogen-selective candidate for medical devices and was potentially useful in food preservation, crop protection, and human health.

Conversion of Broad-Spectrum Antimicrobial Peptides into Species-Specific Antimicrobials Capable of Precisely Targeting Pathogenic Bacteria.

Currently, the majority of antibiotics in clinical use have broad activity spectra, killing pathogenic and beneficial microorganisms indiscriminately. The disruption of the ecological balance of normal flora often results in secondary infections or other antibiotic-associated complications. Therefore, targeted antimicrobial therapies capable of specifically eliminating pathogenic bacteria while retaining the protective benefits of a normal microflora would be advantageous. In this study, we successfully constructed a series of Enterococcus faecalis-targeted antimicrobial peptides from wide-spectrum antimicrobial peptide precursors. These peptides are designed based on fusion of the species-specific peptide pheromone cCF10 and modification of the active region of the antimicrobial peptide. The results showed that cCF10-C4 possessed specific antimicrobial activity against E. faecalis and was not active against other types of bacteria tested. The specificity of this hybrid peptide was shown by the absence of antimicrobial effects in the pheromone-substituted derivative. Further studies indicated that cCF10-C4 and its parent peptide C4 exert their activities by damaging cytoplasmic membrane integrity. The present study reveals the application potential of these molecules as "probiotic" antimicrobials for the control of specific bacterial infections, and it also helps to elucidate the design and construction of species-specific antimicrobials with precise targeting specificity.

Short, symmetric-helical peptides have narrow-spectrum activity with low resistance potential and high selectivity.

Broad-spectrum antibiotics have, until now, been the mainstay of antibiotic therapy. However, the increasing threat of drug-resistant bacteria and the ecological imbalance of normal microbial communities have forced a reconsideration of the best strategies to treat such pathogens. Therefore, antibacterial agents with specific abilities of eliminating pathogens may provide long-term protection. Antimicrobial peptides (AMPs), which can be optimized by modifying their primary sequences, are regarded as potentially valuable in development of pathogen-specific agents. To obtain efficient narrow-spectrum AMPs, database-filtering technology, which filters the most probable amino acid composition, positive charge, sequence length and hydrophobic content of peptides against Gram-negative bacteria, was taken as the first step. Then, the filtered parameters were distributed and modified into an α-helical symmetrical structure by considering the structure-function relationship of synthesized antimicrobial peptides. Finally, short, safe and stable peptides against Escherichia coli, Salmonella pullorum and Pseudomonas aeruginosa were successfully identified. The potential peptides F1 and F4 showed low cell toxicity, low resistance potential and low salt sensitivity. CD spectroscopy of the peptides illustrated that F1 and F4 exhibited a tendency towards an α-helical structure in a membrane-mimetic environment. Indeed, fluorescence spectroscopy and electron microscopy analyses indicated that the shorter potential sequence F4 killed the bacteria by causing physical destruction of the bacterial membrane and cytosol leakage. In the mouse model test, F4 reduced the bacterial load in major organs and the cytokine (TNF-α, IL-6, and IL-1ß) levels in serum significantly (P < 0.05). Collectively, this symmetric-helical distribution, dependent on database-filtering parameters, is a promising strategy for designing effective smart AMPs with high cell selectivity, and it also provides new insights into the design and optimization of pathogen-specific biomaterials.

Short Symmetric-End Antimicrobial Peptides Centered on ß-Turn Amino Acids Unit Improve Selectivity and Stability.

Antimicrobial peptides (AMPs) are excellent candidates to combat the increasing number of multi- or pan-resistant pathogens worldwide based on their mechanism of action, which is different from that of antibiotics. In this study, we designed short peptides by fusing an α-helix and ß-turn sequence-motif in a symmetric-end template to promote the higher cell selectivity, antibacterial activity and salt-resistance of these structures. The results showed that the designed peptides PQ and PP tended to form an α-helical structure upon interacting with a membrane-mimicking environment. They displayed high cell selectivity toward bacterial cells over eukaryotic cells. Their activities were mostly maintained in the presence of different conditions (salts, serum, heat, and pH), which indicated their stability in vivo. Fluorescence spectroscopy and electron microscopy analyses indicated that PP and PQ killed bacterial cells through membrane pore formation, thereby damaging membrane integrity. This study revealed the potential application of these designed peptides as new candidate antimicrobial agents.

Combating Drug-Resistant Fungi with Novel Imperfectly Amphipathic Palindromic Peptides.

Antimicrobial peptides are an important weapon against invading pathogens and are potential candidates as novel antibacterial agents, but their antifungal activities are not fully developed. In this study, a set of imperfectly amphipathic peptides was developed based on the imperfectly amphipathic palindromic structure R n(XRXXXRX)R n ( n = 1, 2; X represents L, I, F, or W), and the engineered peptides exhibited high antimicrobial activities against all fungi and bacteria tested (including fluconazole-resistant Candida albicans), with geometric mean (GM) MICs ranging from 2.2 to 6.62 µM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi = 1.64 µM) while showing low toxicity and high salt and serum tolerance. It also had dramatic LPS-neutralizing propensity and a potent membrane-disruptive mechanism against microbial cells. In summary, these findings were useful for short AMPs design to combat the growing threat of drug-resistant fungal and bacterial infections.

Central ß-turn increases the cell selectivity of imperfectly amphipathic α-helical peptides.

Although membrane lytic antimicrobial peptides (AMPs) show enormous potential for addressing mounting global antibiotic resistance, therapeutic applications are hindered by their weak antimicrobial activity, high toxicity, salt sensitivity and poor understanding of structure-activity relationships. To investigate the effects of different parameters on the biological activities of AMPs, a rational approach was adopted to design a series of short cationic α-helical peptides comprising the Ac-WxKyWxzzyKxWyK-NH2 sequence, where x: cationic residues (Arg or Lys), y: hydrophobic residues (Ala, Val, Ile or Leu), and zz: ß-turn (rigid D-Pro-Gly turn or flexible Gly-Gly turn). The peptides showed a more helical structure as the concentration of membrane-mimetic solution increased. The peptide RL with a central D-Pro-Gly turn (x: Arg, y: Lys, zz = D-Pro-Gly) exhibited broad-spectrum antimicrobial activities (2-8 µM) against ten types of clinically relevant microorganisms and even maintained its activity in the presence of physiological salts and showed excellent selectivity toward bacterial cells over human red blood cells and mammalian cells. However, the toxicity was increased after the removal of D-Pro-Gly turn. Additionally, the bactericidal activity was reduced when the D-Pro-Gly turn was replaced by a Gly-Gly turn. Fluorescence spectroscopy and electron microscopy analyses indicated that RL and its derivatives killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. In conclusion, these findings clearly generalized a potential method for designing or optimizing AMPs, and the peptide RL is a promising therapeutic candidate to combat antibiotic resistance. STATEMENT OF SIGNIFICANCE: We proposed a rational approach to design imperfectly amphiphilic peptides and identified RL (Ac-WRKLWRpGLKRWLK-NH2) in particular that shows strong antibacterial properties, low toxicity and high salt resistance. The ß-turn unit inserted into the central position of cationic α-helical peptides, especially the D-Pro-Gly turn, significantly increase the cell selectivity of the synthetic amphiphiles. The findings demonstrate a potential method for designing and/or optimizing AMPs, which would facilitate the development of strategies to design peptide-based antimicrobial biomaterials in a variety of biotechnological and clinical applications.

Antibacterial activities and molecular mechanism of amino-terminal fragments from pig nematode antimicrobial peptide CP-1.

High manufacturing costs and weak cell selectivity have limited the clinical application of naturally occurring peptides when faced with an outbreak of drug resistance. To overcome these limitations, a set of antimicrobial peptides was synthesized with the general sequence of (WL)n, where n = 1, 2, 3, and WL was truncated from the N-terminus of Cecropin P1 without initial serine residues. The antimicrobial peptide WL3 exhibited stronger antimicrobial activity against both Gram-negative and Gram-positive microbes than the parental peptide CP-1. WL3 showed no hemolysis even at the highest test concentrations compared to the parental peptide CP-1. The condition sensitivity assays (salts, serum, and trypsin) demonstrated that WL3 had high stability in vitro. Fluorescence spectroscopy and electron microscopy indicated that WL3 killed microbes by means of penetrating the membrane and causing cell lysis. In a mouse model, WL3 was able to significantly reduce the bacteria load in major organs and cytokines (TNF-α, IL-6, and IL-1ß) levels in serum. In summary, these findings suggest that WL3, which was modified from a natural antimicrobial peptide, has enormous potential for application as a novel antibacterial agent.

Short, multiple-stranded ß-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

The ß-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple ß-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded ß-hairpin peptides with the same ß-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded ß-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8µM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple ß-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. STATEMENT OF SIGNIFICANCE: We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded ß-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide-based antimicrobial biomaterials. Further exploration of these AMPs will allow for diverse biotechnological and clinical applications such as biomedical coating, food storaging, and animal feeding.

High specific selectivity and Membrane-Active Mechanism of the synthetic centrosymmetric α-helical peptides with Gly-Gly pairs.

We used a template-assisted approach to develop synthetic antimicrobial peptides, which differ from naturally occurring antimicrobial peptides that can compromise host natural defenses. Previous researches have demonstrated that symmetrical distribution patterns of amino acids contribute to the antimicrobial activity of natural peptides. However, there is little research describing such design ideas for synthetic α-helical peptides. Therefore, here, we established a centrosymmetric α-helical sequence template (y + hhh + y)n (h, hydrophobic amino acid; +, cationic amino acid; y, Gly or hydrophobic amino acid), which contributed to amphipathicity, and a series of centrosymmetric peptides was designed with pairs of small amino acids (Ala and Gly), which were utilized to modulate the biological activity. The centrosymmetric peptides with 3 repeat units exhibited strong antimicrobial activity; in particular, the Gly-rich centrosymmetric peptide GG3 showed stronger selectivity for gram-negative bacteria without hemolysis. Furthermore, beyond our expectation, fluorescence spectroscopy and electron microscopy analyses indicated that the GG3, which possessed poor α-helix conformation, dramatically exhibited marked membrane destruction via inducing bacterial membrane permeabilization, pore formation and disruption, even bound DNA to further exert antimicrobial activity. Collectively, the Gly-rich centrosymmetric peptide GG3 was an ideal candidate for commercialization as a clinical therapeutic to treat gram-negative bacterial infections.

Antimicrobial activity and membrane-active mechanism of tryptophan zipper-like ß-hairpin antimicrobial peptides.

Antimicrobial peptides (AMPs) with amphipathic ß-hairpin structures have been demonstrated to possess potent antimicrobial activities and great cell selectivities. However, our understanding of ß-hairpin antimicrobial peptides lags behind that of α-helices, mainly because it is difficult for short peptides to form robust ß-hairpin structures. Tryptophan zipper (trpzip) peptides are among the most stable ß-hairpin peptides known to fold spontaneously without requiring covalent disulfide constraint or metal binding. To develop model ß-hairpin AMPs with small size and remarkable stability, a series of amphiphilic linear peptides were designed based on the trpzip motif. The sequence of designed peptides is (WK) n (D) PG(KW) n -NH2 (n = 1, 2, 3, 4, 5), and the antimicrobial activity and membrane interaction mechanism of the peptides were evaluated. The results showed that these peptides readily fold into ß-hairpin structures in aqueous and membrane-mimicking environments and exhibit broad-spectrum antimicrobial activities against both gram-positive and gram-negative bacteria. The antibacterial potency of the peptides initially increased and then decreased with increasing chain length. WK3, a 14-residue peptide, displayed excellent antimicrobial activity with minimal hemolytic activity and cytotoxicity, suggesting that it possesses great cell selectivity. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence spectroscopy, and flow cytometry indicated that representative peptides WK3 and WK4 exert their activities by permeabilizing the microbial membrane and damaging cell membrane integrity. This study reveals the application potential of the designed peptides as promising antimicrobial agents for the control of infectious diseases, and it also provides new insights into the design and optimization of highly stable ß-hairpin AMPs with great antimicrobial activities and cell selectivities.