Georgieva Maria, Stojceski Filip, Wüthrich Fabian, Sosthène Carole, Blanco Pérez Laura, Grasso Gianvito, Jacquier Nicolas
Institute of Microbiology, University Hospital and University of Lausanne, Lausanne, Switzerland.
Dalle Molle Institute for Artificial Intelligence, IDSIA USI-SUPSI, Lugano, Switzerland.
J Biol Chem. 2025 Jan;301(1):108018. doi: 10.1016/j.jbc.2024.108018. Epub 2024 Nov 26.
Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics against antibiotic-resistant pathogens. TAT-RasGAP is an AMP with broad range antibacterial activity, but its mechanism of action is unknown. In this study, we analyzed a strain of Escherichia coli with extensive resistance to TAT-RasGAP but not to other AMPs that we obtained after twenty passages during an in vitro resistance selection experiment. This strain accumulated four mutations. One of these is a point mutation in bamA, which encodes an essential protein involved in the folding and proper insertion of outer membrane proteins. The mutation resulted in a change of charge in a surface-exposed negatively charged loop of the BamA protein. Using CRISPR-Cas9-based targeted mutagenesis, we showed that mutations lowering the negative charge of this loop decreased sensitivity of E. coli to TAT-RasGAP. In silico simulations unveiled the molecular driving forces responsible for the interaction between TAT-RasGAP and BamA. These results indicated that electrostatic interactions, particularly hydrogen bonds, are involved in the stability of the molecular complex, representing a predictive fingerprint of the TAT-RasGAP - BamA interaction strength. Interestingly, BamA activity was only partially affected by TAT-RasGAP, indicating that BamA may function as a specific receptor for this AMP. Our results indicate that binding and entry of TAT-RasGAP may involve different mechanisms compared to other AMPs, which is in line with limited cross-resistance observed between different AMPs. This limited cross-resistance is important for the clinical application of AMPs towards drug-resistant pathogens.
抗菌肽（AMPs）是对抗抗生素耐药病原体的经典抗生素的有前景的替代物。TAT-RasGAP是一种具有广泛抗菌活性的抗菌肽，但其作用机制尚不清楚。在本研究中，我们分析了一株在体外耐药性选择实验中经过二十代传代后获得的对TAT-RasGAP具有广泛耐药性但对其他抗菌肽不耐药的大肠杆菌菌株。该菌株积累了四个突变。其中一个是bamA中的点突变，bamA编码一种参与外膜蛋白折叠和正确插入的必需蛋白。该突变导致BamA蛋白表面暴露的带负电荷环中的电荷发生变化。使用基于CRISPR-Cas9的靶向诱变，我们表明降低该环负电荷的突变会降低大肠杆菌对TAT-RasGAP的敏感性。计算机模拟揭示了负责TAT-RasGAP与BamA之间相互作用的分子驱动力。这些结果表明，静电相互作用，特别是氢键，参与了分子复合物的稳定性，代表了TAT-RasGAP - BamA相互作用强度的预测指纹。有趣的是，BamA活性仅受到TAT-RasGAP的部分影响，表明BamA可能作为这种抗菌肽的特异性受体发挥作用。我们的结果表明，与其他抗菌肽相比，TAT-RasGAP的结合和进入可能涉及不同的机制，这与不同抗菌肽之间观察到的有限交叉耐药性一致。这种有限的交叉耐药性对抗菌肽在耐药病原体临床应用中很重要。
