天然素材Tryptanthrin對隱球菌的抗菌效果及細胞週期之影響

Abstract

本研究致力於尋找對人類病原真菌念珠菌及隱球菌具抗真菌效果之活性物質，希望能找出具抑制能力或和現有藥物有協同作用的化合物。透過快速大量篩選小分子化合物，包括天然素材與美國食品藥品管理局核准之藥品，找出有抗真菌能力的化合物。從篩選結果中發現，來自蜜蜂腸胃道共生之放線菌所分泌的次級代謝物色胺酮tryptanthrin對隱球菌屬有抑制能力，對新型隱球菌的最小抑制濃度為2 ug/mL，對格特隱球菌為4 ug/mL。進一步實驗結果發現，tryptanthrin與鈣調蛋白免疫抑制劑(FK506)有協同作用。Tryptanthrin的抗隱球菌能力為劑量依賴性的，並且為抑菌型藥物而非殺菌型。我們也發現tryptanthrin在37 ºC時抑菌能力比30 ºC或25 ºC更佳。同時，tryptanthrin對臨床抗藥性隱球菌菌株T1和89-610也具抑菌作用。為了找到tryptanthrin可能參與的藥物標靶或途徑，我們用藥物敏感性的測試篩選突變株庫。相較於野生株，49個突變株對藥物更敏感或有抗性，其中有一部分突變株參與在細胞週期的調控。螢光激發流式細胞分選和基因表現定量結果顯示隱球菌的細胞週期調控可能和tryptanthrin呈現的抑制作用相關。綜合以上研究，天然素材tryptanthrin具有抗真菌能力並和FK506有協同作用，有潛力作為隱球菌症的治療策略，尤其是抗藥性菌株。

This study aims to identify bioactive compounds that exert novel antifungal activity alone or exhibit synergistic effect with an existing antifungal agent against human fungal pathogens Cryptococcus and Candida. We screened several compound libraries including natural products, agricultural fungicides and FDA-approved drugs in order to identify compounds that exert antifungal activity. Among selected compounds, tryptanthrin secreted from Nocardiopsis alba, an actinobacterium existed in the intestine of honeybee, was chosen for further characterization because of its potent inhibition activity against Cryptococcus species with the minimal inhibition concentration (MIC) of 2 ug/mL for C. neoformans and 4 ug/mL for C. gattii. We further found that tryptanthrin showed synergistic effect with FK506, an immunosuppressant, based on broth dilution and checkerboard assays. Tryptanthrin inhibits the growth of Cryptococcus cells in a dose-dependent manner and shows fungistatic activity instead of fungicidal. We also found that tryptanthrin is more effective at 37 ºC compared with to 30 ºC or 25 ºC. Meanwhile, tryptanthrin demonstrated antifungal activity against two clinical azole-resistant C. neoformans isolates, T1 and 89-610. In order to identify potential targets or pathways that tryptanthrin involves in, we screened the Cryptococcus deletion mutant library by drug susceptibility test. Forty-nine deletion mutants were found to be more susceptible or resistant than that of the wild-type, and some of these mutants were involved in in cell cycle regulation. Fluorescence-activated cell sorting and gene expression quantification results support that cell cycle regulation in C. neoformans may be linked to tryptanthrin. In summary, the natural product tryptanthrin shows novel antifungal activity alone or in combination with FK506, leading possible therapeutic strategies for cryptococcosis caused by Cryptococcus species, especially the azole-resistant isolates.