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There is an urgent need to discover new active drugs to combat methicillin-resistant Staphylococcus aureus, which is a serious threat to humans and animals and incompletely eliminated by antibiotics due to its intracellular accumulation in host cells, production of biofilms, and persisters. Fungal defensin-like peptides (DLPs) are emerging as a potential source of new antibacterial drugs due to their potent antibacterial activity. In this study, nine novel fungal DLPs were firstly identified by querying against UniProt databases and expressed in Pichia pastoris, and their antibacterial and anti-biofilm ability were tested against multidrug-resistant (MDR) S. aureus. Results showed that among them, P2, the highest activity and expression level, showed low toxicity, no resistance, and high stability. Minimal inhibitory concentrations (MICs) of P2 against Gram-positive bacteria were < 2 μg/mL. P2 exhibited the potent activity against intracellular MDR S. aureus (bacterial reduction in 80-97%) in RAW264.7 macrophages. P2 bound to/disrupted bacterial DNA, wrinkled outer membranes and permeabilized cytoplasmic membranes, but maintained the integrity of bacterial cells. P2 inhibited/eradicated the biofilm and killed 99% persister bacteria, which were resistant to 100× MIC vancomycin. P2 upregulated the anti-inflammatory cytokine (IL-10) and downregulated pro-inflammatory cytokines (TNF-α/IL-1β) and chemokine (MCP-1) levels in RAW 264.7 macrophages and in mice, respectively. Five milligram per kilogram P2 enhanced the survival of S. aureus-infected mice (100%), superior to vancomycin (30 mg/kg), inhibited the bacterial translocation, and alleviated multiple-organ injuries (liver, spleen, kidney, and lung). These data suggest that P2 may be a candidate for novel antimicrobial agents against MDR staphylococcal infections.

Bovine mastitis is mainly caused by Staphylococcus aureus, which is difficult to eliminate, prone to escape from antibacterial agents, and may cause recurring infections due to the intracellular nature of its infection and multidrug resistance. In this study, the intracellular activities of the NZ2114 derivative peptide H18R (H2) against methicillin-resistant S. aureus (MRSA) and multidrug-resistant bovine S. aureus strains were investigated in bovine mammary epithelial MAC-T cells and mouse mammary glands. The minimum inhibitory concentrations of H2 against S. aureus were 0.5‒1 μg/ml; H2 displayed a lower cytotoxicity than its parental peptide NZ2114 (survival rates of MAC-T cells: 100% [H2 treatment] vs 60.7% [NZ2114 (256 μg/ml) treatment]). H2 was internalized into MAC-T cells mainly via clathrin-mediated endocytosis, and distributed in the cytoplasm. The intracellular inhibition rates against MRSA ATCC43300, the mastitis isolates S. aureus CVCC 3051 and E48 were above 99%, 99%, and 94%, respectively; these were higher than those in case of vancomycin (23-47%). In the mouse model of S. aureus E48-induced mastitis, after treatment with 100 μg of H2 and vancomycin, bacterial numbers in each mammary gland were reduced by 3.96- and 1.59-log CFU, respectively. Additionally, similar to NZ2114 and vancomycin, H2 alleviated the histopathological damage of the mammary tissue and polymorphonuclear neutrophil infiltration in the alveoli. These results suggest that H2 can be used as a safe and effective candidate for treating S. aureus-induced mastitis.

Currently, more antimicrobial drug candidates are urgently needed to combat the rise in drug-resistance among pathogenic microbes. A new antimicrobial peptide, MP1102, a variant of NZ2114, was designed, evaluated, and overexpressed in Pichia pastoris. The total secreted protein in cultures reached 695 mg/l, and the concentration of the recombinant MP1102 (rMP1102) was 292 mg/l. rMP1102 was purified from the fermentation supernatant by one-step cation exchange chromatography to obtain a yield of 197.1 mg/l with 96.4 % purity. rMP1102 exhibited potent activity against Gram-positive bacteria, and its minimum inhibitory concentrations (MICs) for four Staphyloccocus aureus (S. aureus) strains ranged from 0.028 to 0.11 μM, and it had stronger activity (MIC = 0.04 to 0.23 μM) to 20 clinical isolates of MRSA (cMRSA) than rNZ2114 (MIC = 0.11 to 0.90 μM). rMP1102 was shown to kill over 99.9 % of tested S. aureus cells within 6 h when treated at one, two, and four times its MIC and over 90 % of S. aureus cells within 12 h at concentrations of 5, 10, and 20 mg/kg in a mouse thigh infection model. The higher sensitivity of MRSA to MP1102 than to its parental peptide, NZ2114, indicated by this initial pharmacodynamic analysis suggests a possible difference in the killing mechanism of these two molecules. rMP1102 caused less than 0.05 % hemolytic activity at 128 μg/ml and exhibited good thermostability from 20 to 80 °C, with its highest activity being observed at pH 8.0. These results suggest that this yeast expression system is feasible for large-scale production, and rMP1102 exerted stronger activity against S. aureus than NZ2114 via a different mechanism and exhibited potential as a new antimicrobial agent for S. aureus, especially MRSA infections.

A novel antimicrobial peptide MP1106 was designed based on the parental peptide plectasin with four mutational sites and a high level of expression in Pichia pastoris X-33 via the pPICZαA plasmid was achieved. The concentration of total secreted protein in the fermented supernatant was 2.134 g/l (29 °C), and the concentration of recombinant MP1106 (rMP1106) reached 1,808 mg/l after a 120-h induction in a 5-l fermentor. The rMP1106 was purified using a cation-exchange column, and the yield was 831 mg/l with 94.68 % purity. The sample exhibited a narrow spectrum against some Gram-positive bacteria and strong antimicrobial activity against Staphylococcus aureus at low minimal inhibitory concentrations (MICs) of 0.014, 1.8, 0.45, and 0.91 μM to S. aureus strains ATCC 25923, 29213, 6538, and 43300, respectively. Meanwhile, rMP1106 showed potent activity (0.03-1.8 μM) against 20 clinical isolates of methicillin-resistant S. aureus (MRSA). In addition, rMP1106 exhibited a broad range of thermostability from 20 to 100 °C. The higher antimicrobial activity of rMP1106 was maintained in neutral and alkaline environments (pH 6, 8, and 10), and its activity was slightly reduced in acidic environments (pH 2 and 4). The rMP1106 was resistant to the digestion of pepsin, snailase, and proteinase K and was sensitive to trypsin. It exhibited hemolytic activity of only 1.16 % at a concentration of 512 μg/ml and remained stable in human serum at 37 °C for 24 h. Furthermore, the activity of rMP1106 was minorly affected by 10 mM dithiothreitol and 20 % dimethylsulfoxide. Our results indicate that MP1106 can be produced on a large scale and has potential as a therapeutic drug against S. aureus.

A novel specifically targeted antimicrobial peptide (STAMP) that was especially effective against methicillin-resistant Staphylococcus aureus (MRSA) was designed by fusing the AgrD1 pheromone to the N-terminal end of plectasin. This STAMP was named Agplectasin, and its gene was synthesized and expressed in Pichia pastoris X-33 via pPICZαA. The highest amount of total secreted protein reached 1,285.5 mg/l at 108 h during the 120-h induction. The recombinant Agplectasin (rAgP) was purified by cation exchange chromatography and hydrophobic exchange chromatography; its yield reached 150 mg/l with 94 % purity. The rAgP exhibited strong bactericidal activity against S. aureus but not Staphylococcus epidermidis or other types of tested bacteria. A bactericidal kinetics assay showed that the rAgP killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in both Mueller-Hinton medium and human blood within 10 h when treated with 4× minimal inhibitory concentration. The rAgP caused only approximately 1 % hemolysis of human blood cells, even when the concentration reached 512 μg/ml, making it potentially feasible as a clinical injection agent. In addition, it maintained a high activity over a wide range of pH values (2.0-10.0) and demonstrated a high thermal stability at 100 °C for 1 h. These results suggested that this STAMP has the potential to eliminate MRSA strains without disrupting the normal flora.

NZ2114 is a promising candidate for therapeutic application owing to its potent activity to Staphylococcus aureus. Our objective was to identify NZ2114 derivatives with improved activity through substitution of His16 and His18 with residues Arginine and Lysine. Eight mutants were designed and expressed in Pichia pastoris X-33 via pPICZαA. Five of them exhibited strong antimicrobial activity against S. aureus at low minimal inhibitory concentrations (MICs) of 0.057-0.454 μM. Among them, H1, H2, and H3 showed ideal pharmacodynamic effects on methicillin-resistant S. aureus ATCC43300. The total protein level of H1, H2, and H3 reached 1.70, 1.77 and 1.54 g/l at 120 h of induction in the 5-l fermenter, respectively. They killed over 99.9% of pathogens within 1.5 h at 2× and 4× MIC. The post antibiotic effect of H1, H2 and H3 to S. aureus ATCC43300 was 2.94, 1.75 and 1.55 h at 2× MIC, which was similar with their original peptide NZ2114 (1.43 h) and vancomycin (1.72 h). The fractional inhibitory concentration index (FICI) indicated indifferent effects between H1, H2, H3 and vancomycin, ampicillin, rifampicin. Additionally, they had low hemolysis and high stability in different environments (temperature, pH, proteases, and saline ions). All results indicate that H1, H2, and H3 can be produced in large-scale and have potential as therapeutic drugs against MRSA. [ABSTRACT FROM AUTHOR]