Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins

The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity towards Gram-positive bacteria that include pathogens and resistant strains. For further development of these compounds, it was necessary to identify the mechanism of their action and characterize their interaction with eukaryotic cells/organisms in more detail. Here, we show that at their bactericidal concentrations LPPOs localize to the plasmatic membrane in bacteria but not in eukaryotes. In an in vitro system we demonstrate that LPPOs create pores in the membrane. This provides an explanation of their action in vivo where they cause serious damage of the cellular membrane, efflux of the cytosol, and cell disintegration. Further, we show that (i) LPPOs are not genotoxic as determined by the Ames test, (ii) do not cross a monolayer of Caco-2 cells, suggesting they are unable of transepithelial transport, (iii) are well tolerated by living mice when administered orally but not peritoneally, and (iv) are stable at low pH, indicating they could survive the acidic environment in the stomach. Finally, using one of the most potent LPPOs, we attempted and failed to select resistant strains against this compound while we were able to readily select resistant strains against a known antibiotic, rifampicin. In summary, LPPOs represent a new class of compounds with a potential for development as antibacterial agents for topical applications and perhaps also for treatment of gastrointestinal infections.

现有抗生素在治疗传染病方面的优势正受到威胁，这主要归因于耐药细菌菌株数量的增加。这一趋势促使我们寻求新的抗菌化合物。近期，我们发现了一系列被称为脂磷酰氧氮素（LPPOs）的化合物，这些化合物对包括病原体和耐药菌株在内的革兰氏阳性细菌表现出选择性细胞毒性。为进一步开发这些化合物，有必要阐明其作用机制并详细表征其与真核细胞/生物体的相互作用。在本研究中，我们发现LPPOs在杀菌浓度下能够定位于细菌的质膜中，而在真核生物中则不行。在体外实验系统中，我们证明了LPPOs能够在质膜上形成孔洞，这为其在体内的作用提供了合理解释，即它们能够造成细胞膜的严重损伤、细胞质外流以及细胞解体。此外，我们进一步证明：(i) LPPOs经艾姆斯试验证实不具有遗传毒性；(ii) 无法穿透Caco-2细胞单层，表明它们无法进行跨上皮转运；(iii) 当口服给药时，LPPOs在活鼠体内具有良好的耐受性，但腹腔给药则不然；(iv) 在低pH值下稳定，表明它们能够耐受胃内的酸性环境。最后，使用其中一种最有效的LPPOs，我们尝试并未能筛选出对该化合物具有耐药性的菌株，而能够轻易筛选出对已知抗生素利福平具有耐药性的菌株。总之，LPPOs代表一类新型化合物，具有作为局部应用的抗菌剂开发的潜力，或许也可用于治疗胃肠道感染。