以鐵載體為基礎的探針開發及其於弧菌感測上之應用

Abstract

弧菌(Vibrio)為一與人類生活高度相關之革蘭氏陰性菌之菌屬。鑑於這些微生物其所造成的疾病已在全球不斷增長，此促使我們想開發一個弧菌專屬的探針。為此，於本研究中，我們將目標放在一個在限鐵環境下會被諸多細菌所運用之運鐵機制，機制中細菌利用一與鐵具有高親和力的特定小分子稱鐵載體(siderophore)來螯取環境中鐵資源，並專一地將鐵給運送至特定菌種上。利用此途徑我們可藉此建構一個高效率且專一用以標記出弧菌的方法。在此我們先官能基化一個由副溶血弧菌(V. parahaemolyticus)所分泌的鐵載體 vibrioferrin，而得到一個延伸物，並於該物上銜接一個具有炔結構的化合物以便與一含有疊氮結構之螢光素(fluorescein)進行銅催化之疊氮與炔的環加成反應而得到最終產物 VSP1。利用流式細胞術，我們發現在限鐵條件下，在經由 VSP1 處置過後，除了副溶血弧菌外在其他弧菌如霍亂弧菌(V. cholerae)及創傷弧菌(V. vulnificus)上皆可觀察到明顯的螢光反應。而在其他物種如金黃色葡萄球菌(S. aureus)、枯草桿菌(B. subtilis)、大腸桿菌(E. coli)及腸道沙門氏菌(S. enterica)上則無顯著差異。至於在鐵充足的條件下，則所有的細菌當中皆無呈現螢光增強反應。另外在螢光顯微圖上，也可觀察到探針的物種選擇性。當中，在經由我們的探針處置過後，只有弧菌方能被標記於在螢光顯微圖上，而其他菌種如金黃色葡萄球菌及大腸桿菌則無法被顯示出來。這些實驗結果指出我們所開發出的探針，其在限鐵環境下能用於選擇偵測弧菌的潛力。此外，其於霍亂弧菌與創傷弧菌上所顯示的結果，也代表著 vibrioferrin 可能為一可供其他物種使用之鐵載體。

Vibrio is a genus of gram-negative bacteria that is highly relevant to our life. Given that illnesses caused by these microbes have escalating globally, it behooves us to develop a Vibrio-specific probe. To this end, we take our aim at iron acquisition mechanism mediated by siderophore, a small molecule used to specifically transport iron into bacteria under iron-limited condition. By hijacking the pathway, we may find us an efficient and specific way to label Vibrio spp. Herein we first functionalize vibrioferrin (VF), a siderophore secreted by V. parahaemolyticus. Then an alkyne-containing linker is harbored onto this siderophore analog to undergo copper catalyzed azide-alkyne cycloaddition with an azdio-containing fluorescein. On flow cytometry, upon treatment with our probe, VSP1, under iron-limited conditions, significant fluorescent increases can be found not only in its secretor, V. parahaemolyticus but also in other vibrio spp., V. cholerae and V. vulnificus, while no differences are observed in S. aureus, B. subtilis, E. coli and S. enterica. On the contract, when in an iron-rich media, no enhancements are observed in the bacteria tested. The strain dependency is also demonstrated on fluorescent images, where vibrios can be labelled, while S. aureus and E. coli give no response. The results suggest that our probe has the potential for specific labelling vibrios under ironlimited condition. In addition, the unexpected outcome given by V. cholerae and V. vulnificus, indicating VF may be an undiscovered xenosiderophore.