熱帶念珠菌基因NDT80調控菌絲生成的機制探討

Abstract

熱帶念珠菌(Candida tropicalis)是一種臨床上常見的伺機性病原真菌，具有非常多不同的細胞型態，例如酵母菌(yeast)、菌絲(hyphae)型態等，其中菌絲型態在以往的研究也被提及與念珠菌的致病能力具有關聯性，包括幫助念珠菌的細胞入侵組織、形成生物膜等，因此本實驗室參考以往對於白色念珠菌的研究，探討在調控白色念珠菌的菌絲與生物膜生成相關的 6 個重要同源基因是否也同樣在熱帶念珠菌中有類似或是不同的現象。其中我們發現轉錄因子 NDT80 在白色念珠菌中與熱帶念珠菌中在胺基酸序列、生物膜生成、以及致病能力方面都與白色念珠菌有相同現象，然而調控菌絲生成的能力表現出完全相反的功能。在白色念珠菌中 NDT80 為菌絲生成的正向調控者(activator)，然而在熱帶念珠菌中則是菌絲生成的負向調控者(repressor)。因此我希望藉由RNA-seq的方式，探討熱帶念珠菌NDT80突變株在調控菌絲生成的相關路徑以及機制是否與白色念珠菌有所不同。本研究分別將熱帶念珠菌野生株以及NDT80突變株在四種不同的固態培養基( YPD、Serum、RPMI1640、Spider )進行點盤以及液態培養基觀察菌絲生成能力，發現在四種培養基中相較於野生株，NDT80突變株都表現出大量生成菌絲的現象。另外我們也將白色念珠菌的NDT80基因送入熱帶念珠菌的NDT80突變株進行觀察，構築Ctndt80Δ::CaNDT80菌株，發現原先在白色念珠菌中扮演正向調控者的NDT80並無法在熱帶念珠菌中同樣去促進菌絲生成，代表在兩個物種間NDT80影響菌絲生成並非是完全相同的調控路徑。藉由基因表現分析，發現NDT80突變株中其他與菌絲生成相關的重要基因有顯著上調的現象。RNA-seq結果分析完成後，藉由Gene Ontology分析將熱帶念珠菌NDT80突變株中表現量改變的基因分群進行初步篩選並與前人研究比較後，發現有7個基因可能與NDT80影響到菌絲生成有關，這些基因可能牽涉到多個重要調控路徑，例如cAMP/PKA訊號途徑或是法尼醇(farnesol)等多個參與的菌絲生成路徑等，未來也需要進一步驗證找出NDT80可能具體參與到的路徑以及在路徑中的功能為何。

Candida tropicalis, a commonly encountered opportunistic pathogenic fungus in clinical settings, exhibits various cell morphologies including yeast and hyphal forms. The hyphal form has been associated with the pathogenic ability of Candida species, facilitating tissue invasion and biofilm formation. Therefore, inspired by previous studies on Candida albicans, our laboratory aimed to investigate whether six important homologous genes involved in regulating hyphal and biofilm formation in C. albicans show similar or distinct patterns in C. tropicalis. Among them, the transcription factor NDT80 in C. tropicalis exhibited similarities to C. albicans regarding amino acid sequence, biofilm formation, and pathogenicity. However, its role in regulating hyphal formation showed an opposite function. In C. albicans, NDT80 acts as a positive regulator (activator) of hyphal formation, whereas in C. tropicalis, it acts as a negative regulator (repressor). Hence, I aimed to investigate, through RNA-seq analysis, whether the pathways and mechanisms involved in regulating hyphal formation in the NDT80 mutant strain of C. tropicalis differ from those in C. albicans. In this study, the wild-type strain and NDT80 mutant strain of C. tropicalis were cultured on four different solid media (YPD, Serum, RPMI1640, Spider) and in liquid media to observe their hyphal formation ability. Compared to the wild-type strain, the NDT80 mutant strain exhibited enhanced hyphal formation in all four media. Additionally, we introduced the NDT80 gene from C. albicans into the NDT80 mutant strain of C. tropicalis, creating the Ctndt80Δ::CaNDT80 strain. Interestingly, the NDT80 gene, which functions as a positive regulator of hyphal formation in C. albicans, failed to promote hyphal formation in C. tropicalis. This indicates that the regulation of hyphal formation by NDT80 is not entirely conserved between these two species. Furthermore, gene expression analysis revealed significant upregulation of other important genes related to hyphal formation in the NDT80 mutant strain. After analyzing the RNA-seq results, a preliminary screening of differentially expressed genes in the NDT80 mutant strain of C. tropicalis was performed using Gene Ontology analysis. Comparison with previous studies identified seven genes that are potentially associated with NDT80-mediated regulation of hyphal formation. These genes may be involved in multiple critical regulatory pathways, such as the cAMP/PKA signaling pathway or the farnesol pathway, which are involved in hyphal formation. Further investigations are required to validate the specific pathways and functions in which NDT80 may be involved.