University of Houston College of Pharmacy, Houston, TX, USA.
Microbiota-Host Interactions and Clostridia Research Group, Facultad de Ciencias de La Vida, Universidad Andrés Bello, Santiago, Chile; ANID - Millennium Science Initiative Program - Millennium Nucleus in the Biology of the Intestinal Microbiota, Santiago, Chile.
Anaerobe. 2021 Jun;69:102352. doi: 10.1016/j.anaerobe.2021.102352. Epub 2021 Feb 25.
Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies.
MATERIALS & METHODS: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis.
The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE.
This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.
非达霉素对艰难梭菌孢子的形成具有新颖的药理作用，包括抑制生长和持久的孢子附着。然而，非达霉素附着在孢子上的机制尚未经过严格的显微镜研究。
使用带有非达霉素-bodipy 复合物（绿色荧光）的共聚焦显微镜，可视化三种不同核糖体型的艰难梭菌孢子和外孢囊或孢子外壳蛋白编码基因失活的艰难梭菌突变体孢子中非达霉素的附着。测定非达霉素-bodipy 复合物的药理作用。共聚焦显微镜实验包括直接作用于艰难梭菌野生型和突变体孢子、外孢囊去除的作用以及直接附着于比较孢子形成生物枯草芽孢杆菌。
非达霉素-bodipy 复合物的 MIC 为 1 mg/L，而非标记的非达霉素为 0.06 mg/L，增加了 16 倍。使用共聚焦显微镜，观察到非达霉素进入有活力的艰难梭菌细胞的细胞内定位，与它的 RNA 聚合酶作用机制一致，并抑制孢子的生长。非达霉素-bodipy 复合物在艰难梭菌孢子的核心外被可视化，与膜染色染料 FM4-64 无共定位。外孢囊去除减少了非达霉素-bodipy 与艰难梭菌孢子的结合。在艰难梭菌突变体孢子中，观察到 CdeC 和 CotE 等孢子表面蛋白的非达霉素-bodipy 减少。
本研究可视化了非达霉素直接附着于艰难梭菌孢子，而外孢囊和孢子外壳蛋白的特定突变体减少了这种附着。这些数据提供了关于非达霉素抗孢子特性的深入了解。
