探討臨床熱帶念珠菌的菌絲生長與致病力

Abstract

熱帶念珠菌是一種臺灣常見的致病真菌，感染案例僅次於同為念珠菌屬的白色念珠菌，不過它卻具有更高的藥物抗性而且致病機制尚未釐清。熱帶念珠菌具有多型性的特徵，其中菌絲型(hyphal form)與致病能力有非常大的關係，歸因於菌絲能夠提升菌體的細胞入侵能力等等，因此希望能透過這次實驗探討熱帶念珠菌的型態與致病力以及基因表現的關聯性，藉此找到治療方法的突破點。在本研究中，266株臨床菌株在不同培養基培養並篩選出具有偏好特定型態的特異性菌株，YPD與Spider作為一般培養基；小牛血清、RPMI作為菌絲誘導培養基，其中兩株分離株(No. 77以及No. 205)在四種培養液中皆比標準菌株(MYA3404)生長更多菌絲，為菌絲型特異性菌株；另外發現三株分離株(No. 107、No. 220-2以及No. 236)則幾乎不生長菌絲，為酵母菌型特異性菌株。上述五株菌株挑選出來進行其他實驗來分析傳播能力，結果發現菌絲生成能夠有效的附著在瓊脂表面，增加菌體的附著能力；另外生物膜不會只因為較多的菌絲而有較高的生成量。接著透過動物實驗來分析致病力，在大蠟蛾致病實驗中菌絲型特異性菌株具有更高的致死率；但在小鼠致病實驗中，任何型態特異性菌株的致死率卻都比較低。在體內菌數檢測中，菌絲型特異性菌株在器官的菌數較少，這表示菌絲形態的菌體遷移能力較差，而菌體較小的酵母菌型更有機會透過循環系統散播，這些結果表明菌體在動物體內需要適當的型態轉換方能造成較嚴重的感染。最後進行基因表現分析來探討表徵與基因的關聯，不過結果與預期的略有不同，基因表現並未因為型態的特異性而出現特定趨勢。綜合實驗結果，菌絲與致病能力有很大的相關性，不過在生成生物膜與感染宿主的過程中，酵母菌型與菌絲型菌體之間的轉換與協力是最為重要的，型態轉換的抑制也許能成為治療念珠菌症的方法。

Candida tropicalis, a common pathogenic fungus in Taiwan, has an infection capability second only to Candida albicans within the Candida genus. However, it exhibits higher drug resistance, and its pathogenic mechanism has not been clarified. C. tropicalis displays polymorphic characteristics, and the hyphal form is closely associated with its virulence due to the enhanced cellular invasion capability. Therefore, this study aims to explore the correlation between the morphology, virulence, and gene expression of C. tropicalis, hoping to identify breakthroughs in treatment strategies. In this research, 266 clinical isolates were cultured on different media, including YPD, Spider, bovine serum, and RPMI-1640. Among these isolates, two isolates (No. 77 and No. 205) exhibited significantly more hyphal growth than the reference standard strain (MYA3404) across all four culture conditions, indicating their specific tendency towards the hyphal form. Furthermore, three isolates (No. 107, No. 220-2, and No. 236) showed minimal hyphal growth, suggesting their specific tendency towards the yeast form. The selected five strains were further subjected to additional experiments to analyze their dissemination capabilities. The results revealed that hyphal form Candida exhibited enhanced attachment to agar surfaces, thereby increasing their attachment ability. However, it was observed that higher hyphal growth did not necessarily correlate with higher biofilm production. Subsequently, animal experiments were conducted to assess the virulence in vivo. In Galleria mellonella, hyphal form isolates demonstrated higher mortality rates. However, in mice, all isolates with specific morphological tendencies exhibited lower mortality rates. In assessing fungal quantities within the host, hyphal form isolates showed lower fungal burden in the hemolymph and the organs, indicating their diminished ability to migrate within the host. Conversely, the yeast form isolates, with smaller fungal bodies, had a greater opportunity for dissemination through the circulatory system. These findings indicated that an appropriate morphological transition of the fungal cells is necessary for causing more severe infections within the host. Finally, gene expression analysis was conducted to explore the association between phenotypes and gene expression. However, the results differed from the expected patterns, as specific trends in gene expression did not correspond to the morphological tendencies. Based on the findings of this study, there is a significant correlation between hyphal form and virulence. Nevertheless, during biofilm formation and host infection processes, the transition and collaboration between yeast and hyphal forms of the fungal cells are crucial. Inhibiting the morphological transition may serve as a therapeutic approach for treating candidiasis.