Müller Matthias, Mayrhofer Sigrid, Sudjarwo Wisnu Arfian A, Gibisch Martin, Tauer Christopher, Berger Eva, Brocard Cécile, Toca-Herrera José L, Striedner Gerald, Hahn Rainer, Cserjan-Puschmann Monika
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Institute of Bioprocess Science and Engineering, BOKU University, Vienna, Austria.
Institute of Molecular Biotechnology, BOKU University, Vienna, Austria.
J Bacteriol. 2025 May 22;207(5):e0045624. doi: 10.1128/jb.00456-24. Epub 2025 Apr 4.
Plectasin, an antimicrobial peptide, was initially isolated from the saprophytic fungus . This peptide, a member of the cysteine-stabilized α-helix and β-sheet family, has demonstrated potent antimicrobial activity against gram-positive pathogens, including strains resistant to conventional antibiotics. Our CASPON platform process enables the production of substantial quantities of plectasin, facilitating investigations on the activity and the mode of action of this recombinantly produced peptide. To this end, we developed an activity assay that reflects the growth inhibition of selected model bacteria, allowing for statistical analysis and evaluation of reproducibility. The mode of action was investigated using transmission electron microscopy and atomic force microscopy. The latter provided new insights into alterations in the cell surface of gram-positive bacteria treated with plectasin at the single-cell level. While the cell diameter remained unaltered, the roughness increased by up to twofold, and the cell stiffness decreased by approximately one-third in the four gram-positive bacterial strains tested. Statistical analysis of these morphological changes provides further insights into the effects and efficiency of antimicrobial peptides targeting pathogen cell walls.
The rise of antibiotic-resistant bacteria is a major threat to global health. Antimicrobial peptides (AMPs) offer a promising way to combat this. With the CASPON technology, we produced the AMP plectasin comprising three disulfide bonds using . The activity of purified plectasin with and without a CASPON fusion tag was determined for four gram-positive and four gram-negative bacteria. As anticipated, only gram-positive bacteria showed a growth inhibition response to un-tagged plectasin. Plectasin treatment on gram-positive bacteria was visualized via electron microscopy. Evaluation of atomic force microscopy indicated that plectasin treatment led to increased roughness but maintained thickness. Based on our study, we assume that the CASPON technology can be employed in the future for the production and characterization of medical-grade AMPs.
杀菌肽最初是从腐生真菌中分离出来的一种抗菌肽。这种肽属于半胱氨酸稳定的α螺旋和β折叠家族成员，已证明对革兰氏阳性病原体具有强大的抗菌活性，包括对传统抗生素耐药的菌株。我们的CASPON平台工艺能够大量生产杀菌肽，便于对这种重组生产的肽的活性和作用方式进行研究。为此，我们开发了一种活性测定方法，该方法反映了所选模型细菌的生长抑制情况，可进行统计分析和重现性评估。使用透射电子显微镜和原子力显微镜研究了其作用方式。后者在单细胞水平上为用杀菌肽处理的革兰氏阳性细菌的细胞表面变化提供了新的见解。在所测试的四种革兰氏阳性细菌菌株中，虽然细胞直径保持不变，但粗糙度增加了两倍，细胞硬度降低了约三分之一。对这些形态变化的统计分析为靶向病原体细胞壁的抗菌肽的效果和效率提供了进一步的见解。
抗生素耐药细菌的出现是对全球健康的重大威胁。抗菌肽为应对这一问题提供了一种有前景的方法。利用CASPON技术，我们生产了包含三个二硫键的抗菌肽杀菌肽。测定了有无CASPON融合标签的纯化杀菌肽对四种革兰氏阳性和四种革兰氏阴性细菌的活性。正如预期的那样，只有革兰氏阳性细菌对未标记的杀菌肽表现出生长抑制反应。通过电子显微镜观察了杀菌肽对革兰氏阳性细菌的处理情况。原子力显微镜评估表明，杀菌肽处理导致粗糙度增加但厚度保持不变。基于我们的研究，我们认为CASPON技术未来可用于医疗级抗菌肽的生产和表征。
