Search

Staphylococcus hyicus, considered as a leading pathogen of exudative epidermitis, is a serious threat to humans and animals. The emergency of bacterial resistance to antibiotics, especially in human and animal health fields, leads to an urgent need of exploration of new antimicrobial agents. In this study, NZX, a plectasin-derived peptide, was firstly expressed in Pichia pastoris X-33 and was purified by cation exchange chromatography, followed by detection of its antibacterial activity in vitro and in vivo. The results showed that the total secreted protein concentration in fermentation supernatant was up to 2820 mg/L (29 °C) after 120-h induction in a 5-L fermentor. The yield of NZX reached up to 965 mg/L with a purity of 92.6%. The recombinant expressed NZX had a strong antimicrobial activity, high stability, and low toxicity. The minimal inhibitory concentrations (MICs) of NZX and ceftriaxone (CRO) against Gram-positive bacteria were 0.46 to 0.91 μM and 6.04 to 12.09 μM, respectively. The time-killing curves showed that S. hyicus NCTC10350 was killed completely by 2× and 4 × MIC of NZX within 24 h. NZX also exhibited the intracellular activity against S. hyicus in Hacat cells. After treatment with NZX (10 mg/kg) and CRO (60 mg/kg), the survival rates of mice were 100% and 83.3%, respectively. NZX inhibited the bacterial translocation, downregulated pro-inflammatory cytokines (TNF-α/IL-1β/IL-6), upregulated the anti-inflammatory cytokine (IL-10), and ameliorated multiple-organ injuries (the liver, spleen, lung, and kidney). This study provides evidence that the expressed NZX has the potential to become a powerful candidate as novel antimicrobial agents against S. hyicus infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11-0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4 × MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6-45.6 and 1.7-3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI = 0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI = 0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI = 0.125-0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI = 8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]