| Author: | Zeng, Ping |
| Title: | De novo designed phenol-soluble-modulins-inspired peptides as effective antimicrobial agents |
| Advisors: | Wong, Kwok-yin (ABCT) Chan, Kin-fai (ABCT) Chen, Sheng (ABCT) |
| Degree: | Ph.D. |
| Year: | 2021 |
| Subject: | Staphylococcus aureus Antibiotics Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xxiv, 255 pages : color illustrations |
| Language: | English |
| Abstract: | Human health is currently facing a serious threat from bacterial pathogens. In particular, the aggravating clinical problems due to antimicrobial resistance call for alternative routes of drug development. Peptides are thought to be promising candidate compounds that can be used to combat this intractable problem. Among them, phenol-soluble-modulins (PSMs) are a large family of cytolytic peptide toxins that include seven members PSM α1-4, β1-2, and δ-toxin. They are produced and secreted by pathogenic Staphylococcus aureus (SA) to restrain the growth of other bacterial strains in close vicinity. However, individual members of PSMs exhibit only very weak antibacterial activity, implying that they may act synergistically in specific biological functions. By extracting potential useful structural motifs and applying rational design, we reasoned that PSMs could provide valuable templates for structural modification into promising antimicrobial peptides (AMP) of high potency and selectivity. Therefore, in this work, we have constructed a small library of PSM-inspired cationic AMPs via standard solid-phase peptide synthesis for a detailed evaluation of their bioactivity, antibacterial mechanism, and the potential in clinical and industrial applications. (1) Firstly, peptide zp3 (GIIAGIIIKIKK-NH2) was found to possess significantly improved physicochemical properties when compared to PSMs, as well as favourable antibacterial activity against Escherichia coli (EC), Bacillus subtilis (BS), and multiple clinically isolated multidrug-resistant bacteria. In addition, its cytotoxicity, hemolytic activity, and in vivo toxicity was relatively low. Peptide zp3 could penetrate the bacterial membrane, thereby causing leakage of the bacterial cytoplasm. Moreover, it inhibited biofilm formation in Acinetobacter baumannii (AB) at micromolar level, eliminating approximately 50% of preformed biofilm at 32 μM after 6 h treatment. Mechanistic studies revealed that the bactericidal mode of zp3 was attributed to the combined effect of affecting ions balance at low concentration, inducing permeability alteration, and causing formation of pores in the cell membrane at high concentration. Applications on medical textiles also showed that zp3 exhibit good antibacterial activity. (2) Secondly, seven linear cationic hexadecapeptides based on zp3 were designed, synthesized, and characterized for subsequent investigation. One of them, namely zp16, demonstrated significant synergy with glycopeptide antibiotics, such as vancomycin and teicoplanin, when tested on highly pathogenic Klebsiella pneumoniae (KP), with fractional inhibitory concentration (FIC) index as low as 0.03. Checkerboard assay indicated that, in the presence of 8 μM zp16, the minimum inhibitory concentration (MIC) of vancomycin towards carbapenem-resistant KP94 was reduced from >128 to 1 μM. In addition, the vancomycin-zp16 combination exhibited negligible toxicity in vitro and in vivo. Further efficacy studies confirmed that the survival rate of combination therapy at 100 mg/kg of zp16 and vancomycin was 30% higher than that of single-drug treatment. Peptide zp16 could strengthen the damaging effect of vancomycin on membrane permeability and membrane potential, leading to markedly reduced biofilm formation and rapid bacterial eradication. This new combination strategy not only broadened the antibacterial spectrum of glycopeptide antibiotics on pathogens ranging from SA to KP, but also offered an alternative direction to treat pathogenic KP infection. (3) On the other hand, peptide zp16 also exhibited potent bioactivity against the Gram-negative, plant-specific pathogen Pectobacterium carotovorum (PC). It inhibited the growth of planktonic bacterial cells at micromolar concentration and reduced the live bacterial count on cabbage leaf. Furthermore, zp16 treatment would significantly up-regulate intracellular reactive oxidative stress (ROS) in a dose-dependent manner, which was believed to cause structural damage to membranes and proteins. Proteomics analysis suggested that the expression level of some critical proteins related to arginine and peptidoglycan biosynthesis was significantly down-regulated after transitory zp16 treatment. In other words, peptide zp16 kills PC not only by physically damaging the cellular envelope but also by interfering with multiple metabolic processes. Given the safety consideration of pesticides as food preservatives, a peptide approach such as usage of zp16 may be a promising alternative to address problems of vegetable soft rot induced by PC. (4) Next, to discover AMPs with improved proteolytic stability and more potent bioactivity, thirteen linear peptides derived from zp3 were designed, synthesized, and characterized. One of them, zp65, whose sequence is GIOAOIIIOIOO-NH2, demonstrated bactericidal effect against common Gram-negative strains including enterohemorrhagic EC, Salmonella (SL), and Citrobacter freundii (CF). Encouragingly, the proteolytic stability of zp65 was much higher than its analog which consist of only natural amino acids. Isothermal titration calorimetry (ITC) showed that zp65 has a strong binding affinity to lipopolysaccharide (LPS) ,with Kd = 1.3 μM, suggesting that the drug target is on the bacterial envelope. This peptide also suppressed the membrane potential of Escherichia coli O157:H7 (O157) in a dose-dependent manner. Surprisingly, peptide zp65 did not induce disruption of membrane permeability even at a high concentration of 4-fold MIC. By employing confocal microscopy, it was found that zp65 molecules, which were labelled with a fluorescein isothiocyanate (FITC) at the N-terminal, mainly aggregated on the membrane. These results suggested that the bactericidal mode of zp65 may be mainly attributed to cell membrane depolarization. At last, the application of zp65 on real life food systems was evaluated. The minced lean beef experiment indicated that the maximum reduction of O157 reached 1.46 log colony-forming unit (CFU) per gram on day 1 after zp65 treatment at the dosage of 40 μg/g. Compared with the untreated cooked beef sample, the CFU of the zp65-treated group remained at a much lower level after 10-days storage. Subsequently, zp65 was proven to exhibit the ability to reduce O157 viable counts in fresh tomato juice. Ingestion of O157-contaminated juice containing zp65 treatment would rescue up to 40% larvae in animal infection experiments. In summary, based on the amino acid sequences of PSMs, a small library of novel engineering AMPs was designed. The lead peptides, such as zp3, zp16, and zp65, exhibited favourable antimicrobial spectrum and application potential, hence they deserve in-depth studies for further development as antimicrobial agents. |
| Rights: | All rights reserved |
| Access: | open access |
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