雙功能螢光探針用於選擇性追踪和標記宿主內的耐藥細菌

Abstract

多重抗藥性革蘭氏陰性細菌成為公共衛生領域的嚴重威脅。了解抗性細菌和寄主微生物群如何相互作用可以促進新抗生素的開發。但是，迄今為止，由於缺乏成像工具，它們的相互作用仍不清楚。在這裡，我們正在開發熒光探針來追蹤它們在宿主體內的空間和時間關係。 在我們的探針Ceph-couCF3-GU中，螢光基團香豆素衍生物以頭孢菌素保護，並與導向單元相連，其功能分別為偵測具有抗藥性細菌和增強細菌靶向選擇性。在去除β-內酰胺酶的反應後，可以開啟螢光團報告耐藥細菌內的β-內酰胺酶活性。通過特定激發波長藉此分辨水解產物，並通過螢光監測β-內酰胺酶活性。我們的前探針Ceph-couCF3-alkyne可以在細菌體內測定中水解並釋放出螢光，在沒有導向單元時，前探針無法利用穿膜蛋白穿越細胞膜，但在臨床菌株中，可以利用擴散的方式穿膜。此外，在IVIS（體內光學成像系統）中，我們的前探針顯示低螢光檢測極限（20 uM探針與20 nM β-內酰胺酶），證明了我們的探針在小鼠模型中體內即時檢測的潛力。接著，利用點擊化學將被用於在前探針上綴合引導單元，包括腸細菌素類似物（鐵響應單位），β-麥芽糖糊精（一般營養單位）或細胞穿透肽（CPPs，通用穿透單位）以產生最終的Ceph-couCF3-GU探針，而我們的最終探針Ceph-couCF3-GU，在不同的臨床菌中，可以利用擴散能力的不同而有不同的選擇性，進而達到選擇性偵測細菌的目的。

Multiple resistant Gram-negative bacteria became serious threats in public health. Understanding how resistant bacteria and host microbiota interact could facilitate the development of new antibiotics. However, up to date, their interaction remains unclear due to the lack of imaging tools. Here, we are developing fluorogenic probes to track their spatial and temporal relationship inside hosts. In our probe Ceph-couCF3-GU, coumarin is capped with substrates, such as cephalosporin, and linked to guiding units, whose functions are to report specific resistance and to enhance bacterial targeting selectivity, respectively. Upon decapping reaction of β-lactamase, the fluorophore can be turned on to report β-lactamase activity inside resistant bacteria. Through specifically exciting the hydrolyzed product, β-lactamase activity can be monitored by fluorescence. Our pre-probe, Ceph-couCF3-alkyne, can be turned on but cell-impermeable in the cell-based assay. And then, in the clinical resistance bacteria, our pre-probe can diffuse through outer membrane. Moreover, in IVIS (in vivo optical imaging system), our pre-probe showed low fluorescence detection limit (20 uM probe with 20 nM β-lactamase), demonstrating its potential for in vivo real-time detection in a mice model. Furthermore, we use the click chemistry to conjugate guiding units on the pre-probe, including enterobactin analogues (iron-responsive units), β-maltodextrins (general nutrient units) or cell-penetrating peptides (CPPs, universal penetrating units) to produce the final Ceph-couCF3-GU probes. Finally, the final Ceph-couCF3-GU probes can selectively detect different clinical resistance bacteria by different diffuse ability.