艱難梭菌（C. difficile）是一種革蘭氏陽性、厭氧、 會產生孢子的 細菌。在艱難梭菌感染 (CDI)時，兩種主要的分泌性毒素，毒素 A (TcdA)和毒素 B (TcdB)是 造成 病理學的主要原因，例如會造成腹瀉等臨床症狀。而CDI依賴休眠孢子而發生。因此萌發被認為是攝入孢子後造成發病的第一步。艱難梭菌孢子萌發的機制是對牛磺膽酸鹽 (TA) 和甘氨酸的組合作出反應。另一方面，一些化合物如鵝脫氧膽酸鹽已被認為是孢子萌發抑制劑。然而，這類化合物的角色尚未得到充分研究探討。在這本篇研究中，我們的目標是尋找新的孢子萌發抑制劑。一開始，我們使用表型微陣列 (PMs) 來篩選潛在抑制劑。在我們的篩選結果中，與其他化合物相比，發現其中一種化合物菊粉可以有效抑制孢子萌發。接下來，為了能夠找到抑制孢子萌發的最佳濃度。我們透過OD動力學測定、菌落形成和DPA釋放測定皆表明7% 菊粉能有效抑制孢子萌發。此外，我們想探討菊粉如何對孢子萌發產生抑制作用。我們的首要探討目標是鈣誘導萌發途徑，而從結果發現菊粉並不會抑制鈣的效果 ，因為趨勢與沒有額外添加菊粉組的趨勢相同。其次，我們發現不同的孢子 密度會影響孢子萌發效率。因此，在這部分，我們使用了更高濃度的 TA和更高密度的孢子來測試菊粉效果。結果表明，高濃度的TA以及高密度孢子皆降低菊粉抑制效果 。由於較高的TA和較高密度的孢子密度這兩者都與TA受體有很大的關係，我們假設 TA 受體，CspC 在這當中有相當的重要作用，並可能影響菊粉效 果。為了進一步研究，我們建構 兩個過表達菌株 R20291-cspBAC和 -cspBA。結果表明，在 7% 菊粉狀況下，R20291-cspBAC與對照組 R20291-pML兩者相比沒有任何差異，但 R20291-cspBAC的萌發在早期有延遲的現象。為了進一步確認 CspC已成功過表達，我們使用了有nisin啟動子的穿梭載體 pMTL84151-pcpr帶有cspC-His tag來進行證明。結果當中發現在 7% 菊粉狀況下， R20291-cspC His在萌發早期也有被延遲的現象。綜合上述，我們發現一種潛在的抑制孢子萌發的化合物菊粉。並且也發現在較高濃度的TA和較高密度的孢子條件下，菊粉的作用會被減弱。在表達 CspC 的菌株中菊粉的作用可以延遲孢子萌發的時間 。這些發現表明菊粉可以成為艱難梭菌孢子萌發的潛在抑製劑 。

Clostridioides difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacterium. During C. difficile infection (CDI), two major secretory toxins, toxin A (TcdA) and toxin B (TcdB) are the major cause of pathologies, such as clinical symptoms like diarrhea. CDI relies on dormant spores. Germination is identified as the first step in pathogenesis following ingestion of spores. The mechanism of spore germination in C. difficile is in response to the combination of taurocholate (TA) and glycine. Besides, some compounds such as chenodeoxycholate have been described as spore germination inhibitors. However, these kinds of compounds have not been fully discussed. In this study, we aim to look for novel inhibitors for spore germination. In the beginning, we used phenotype microarrays (PMs) to screen potential inhibitors. In our screening results, one of the nutritional sources, inulin, can efficiently inhibit spore germination compared to other compounds. Next, we tried to determine the best concentration of inulin to inhibit spore germination. Through OD kinetic assay, colony formation, and DPA release assay, the results showed that 7% inulin can effectively inhibit spore germination. Moreover, we also tried to investigate the inhibition mechanism of inulin on spore germination. First, we focused on the calcium-induced germination pathway, and the results showed that inulin didn’t inhibit calcium effect. Second, we found different densities of spores could influence germination efficiency. Therefore, we used the higher concentration of TA and higher density spores to test the inulin effect. The results showed the higher TA and higher spore density could decrease the inulin effect. Because higher TA and higher density spores have a strong relationship with the TA receptor, we assumed the TA receptor, CspC plays an important role and can influence the inulin effect. To further study, we used two overexpression strains, R20291-cspBAC and -cspBA. The result showed that R20291-cspBAC compared to the control group R20291-pML didn't have any difference under 7% inulin treatment, but the germination of R20291-cspBAC was delayed in the early stage. To further confirm the CspC was successfully overexpressed, we used shuttle vector, pMTL84151-pcpr, with cspC His tag to create R20291-cspC His. R20291-cspC His was also delayed in the early stage. Together, we found a potential inhibitor, inulin, for spore germination. The following results revealed that the effect of inulin was decreased under the condition of higher TA and higher density spores. In the cspC-expressing strain, the effect of inulin could delay the early germination process. These findings demonstrated that inulin can be a potential inhibitor for C. difficile spore germination.

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