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High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus

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Abstract

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.

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Acknowledgments

The authors wish to acknowledge Prof. Shi Xianming, Ph.D., in the Bor Luh Food Safety Center, Shanghai Jiaotong University, for donating all the MRSA clinical isolates and Prof. Yang Fuquan, Ph.D., in the Proteomics Platform Laboratory at the Institute of Biophysics, Chinese Academy of Sciences, for his coordination of the MALDI-TOF MS analysis. This research was supported by the National Natural Science Foundation of China (nos. 30972125 and 31001026) and the Project of National Support Program for Science and Technology in China (nos. 2013BAD10B02 and 2011BAD26B02).

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  1. Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, China

    Yong Zhang, Da Teng, Ruoyu Mao, Xiumin Wang, Di Xi, Xiaoyuan Hu & Jianhua Wang

  2. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St, Haidian District, Beijing, 100081, People’s Republic of China

    Yong Zhang, Da Teng, Ruoyu Mao, Xiumin Wang, Di Xi, Xiaoyuan Hu & Jianhua Wang

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  1. Yong Zhang
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Correspondence to Jianhua Wang.

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Yong Zhang and Ruoyu Mao contributed equally to this paper.

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Zhang, Y., Teng, D., Mao, R. et al. High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus . Appl Microbiol Biotechnol 98, 681–694 (2014). https://doi.org/10.1007/s00253-013-4881-2

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