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A recombinant fungal defensin-like peptide-P2 combats Streptococcus dysgalactiae and biofilms

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Abstract

Streptococcus dysgalactiae, considered one of the main pathogens that causes bovine mastitis, is a serious threat to humans and animals. However, the excessive use of antibiotics and the characteristic of S. dysgalactiae forming biofilms in mastitic teat canal have serious clinical implications. In this study, in vivo and in vitro multiple mechanisms of action of P2, a mutant of fungal defensin plectasin, against S. dysgalactiae were systematically and comprehensively investigated for the first time. P2 showed potent antibacterial activity against S. dysgalactiae (minimum inhibitory concentration, MIC = 0.23–0.46 μM) and rapid bactericidal action by 3.0 lg units reduction in 2–4 h. No resistant mutants appeared after 30-d serial passage of S. dysgalactiae in the presence of P2. The results of electron microscopy and flow cytometer showed that P2 induced membrane damage of S. dysgalactiae, causing the leakage of cellular content and eventually cell death. Besides, P2 effectively inhibited early biofilm formation, eradicated mature biofilms, and killed 99.9% persisters which were resistant to 100 × MIC vancomycin; and confocal laser scanning microscopy (CLSM) also revealed the potent antibacterial and antibiofilm activity of P2 (the thickness of biofilm reduced from 18.82 to 7.94 μm). The in vivo therapeutic effect of P2 in mouse mastitis model showed that it decreased the number of mammary bacteria and alleviated breast inflammation by regulating cytokines and inhibiting bacterial proliferation, which were superior to vancomycin. These data indicated that P2 maybe a potential candidate peptide for mastitis treatment of S. dysgalactiae infections.

Key points

•P2 showed potential in vitro antibacterial characteristics towards S. dysgalactiae.

•P2 eradicated biofilms, killed persisters, and induced cell death of S. dysgalactiae.

•P2 could effectively protect mice from S. dysgalactiae infection in gland.

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All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgments

We acknowledge Chunli Li and Tong Zhao from the Core Facility at the Institute of Microbiology at the Chinese Academy of Sciences (CAS) for their technical support with SEM, TEM, and flow cytometer analysis, and Dan Zhang from the Core Facility at the Center of Biomedical Analysis at Tsinghua University for her CLSM analysis.

Funding

This study was funded by the National Natural Science Fundation of China (31672456), the AMP & ATA direction of National Innovation Program of the Agricultural Science and Technology in CAAS (CAAS-ASTIP-2013-FRI-02) and Its Key Project of Alternatives to Antibiotics for Feed Usages (CAAS-ZDXT-2018008), and Tianjin Science and Technology Planning Project (18YFZCNC01130).

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Author notes

  1. Qingjuan Zhang and Na Yang contributed equally to this work.

Authors and Affiliations

  1. Team of AMP & Alternatives to Antibiotics, Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Qingjuan Zhang, Na Yang, Ruoyu Mao, Ya Hao, Xuanxuan Ma, Da Teng & Jianhua Wang

  2. Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Beijing, 100081, China

    Qingjuan Zhang, Na Yang, Ruoyu Mao, Ya Hao, Xuanxuan Ma, Da Teng & Jianhua Wang

  3. Tianjin Animal Science and Veterinary Research Institute, Tianjin, 300381, China

    Qingjuan Zhang & Huan Fan

  4. College of Life Sciences, Tianjin Normal University, Tianjin, 300387, China

    Qingjuan Zhang

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  1. Qingjuan Zhang
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  2. Na Yang
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  3. Ruoyu Mao
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  4. Ya Hao
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Contributions

QZ, NY, RM, DT, HF, and JW conceived and designed experiments. QZ carried out all the experiments. QZ, NY, DT, and JW contributed in writing. JW and HF contributed in funding acquisition. YH and XM contributed to materials and reagents. RM contributed in modifying figure.

Corresponding authors

Correspondence to Da Teng, Huan Fan or Jianhua Wang.

Ethics declarations

The animal protocol for this study was approved by the Animal Care and Use Committee of the Feed Research Institute, Chinese Academy of Agricultural Sciences (Beijing, China), and all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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All the authors declare no conflicts of interest (financial or non-financial).

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Zhang, Q., Yang, N., Mao, R. et al. A recombinant fungal defensin-like peptide-P2 combats Streptococcus dysgalactiae and biofilms. Appl Microbiol Biotechnol 105, 1489–1504 (2021). https://doi.org/10.1007/s00253-021-11135-y

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