白色念珠球菌a-費洛蒙運輸蛋白質Hst6對於費洛蒙誘導細胞附著、交配能力及White-Opaque型態轉換之探討

Abstract

白色念珠球菌 (Candida albicans) 為人類主要的伺機型致病真菌之一，對於免疫缺失的患者易造成致命的系統性感染。為了適應宿主體內多樣的微環境，白色念珠球菌發展出一種獨特的型態轉換，稱之為 white-opaque switching。而 white 細胞及 opaque 細胞之間最大的行為差異，在於其能否行有性生殖。opaque 細胞具有交配能力，藉由分泌與細胞性狀一致的費洛蒙 (a or α pheromone)，來活化相反性狀之細胞進行交配反應。雖然 white 細胞不具有交配能力，但是當接收到opaque 細胞所分泌的費洛蒙時，能誘導 white 細胞產生細胞附著及生成生物膜。過去實驗室研究發現，以 α pheromone 刺激 MTLa/a white 細胞後，由 cDNA 微陣列分析結果顯示 MFA1 (產生a pheromone前導蛋白質) 及 HST6 (產生a pheromone運輸蛋白質) 皆出現高表現的情形。因此，本研究假設 MFA1 及 HST6 可能會參與在 α pheromone 誘導的 white 細胞之細胞附著中，並進一步探討其所扮演的角色。於編號 P37005 之 MTLa/a 菌株剔除 HST6 後，會使得費洛蒙誘導之細胞附著量 (約5.2 x 107個細胞) 與野生株 (約13.4 x 107個細胞) 相比出現顯著下降；然而 MFA1 基因的剔除，卻不會影響費洛蒙誘導之細胞附著。先前研究指出，Cph1 是調控費洛蒙誘導之細胞附著形成的重要轉錄因子。在此，透過定量即時聚合酶鏈鎖反應分析顯示，HST6 基因的表現會受到 Cph1、Cek1 (mitogen-activated protein kinase，MAPK) 及 Ste2 (α pheromone受體蛋白質) 所調控。除此之外，hst6Δ 的交配效率僅有 0.0004%，相較於野生株的交配率 65.07% 有顯著減少，這意味著剔除 HST6 基因可能導致白色念珠球菌的a pheromone 分泌產生缺損，從而影響交配反應的進行。為探究 Hst6 運輸蛋白質除了影響費洛蒙誘導之細胞附著外，是否也會同時參與在傳統生物膜 (conventional biofilm) 的生成，因此針對 2 個與傳統生物膜相關的轉錄因子 (Tec1、Ndt80) 進行測試。實驗結果不論是生物膜乾重或是 TEC1 及 NDT80 基因的表現程度，在野生株與 hst6Δ 之間並沒有明顯的差異，顯示 Hst6 運輸蛋白質只會專一地參與於費洛蒙誘導之細胞附著，並不涉及傳統生物膜的發展。有趣的是，hst6Δ 的 white-to-opaque 型態轉換率 (1.95 ± 2.51%) 也遠低於野生株的轉換效率 (34.34 ± 20.16%)。然而於另一株 MTLa/a RBY717 菌株將 HST6 基因剔除後，並不影響其費洛蒙誘導之細胞附著與 white-to-opaque 表現型轉換，暗示著 Hst6 運輸蛋白質的調節作用可能具有菌株專一性。根據上述實驗結果，本次研究發現 MTLa/a 細胞中的 a pheromone 運輸蛋白質，Hst6，會參與在白色念珠球菌 P37005 菌株的費洛蒙誘導之細胞附著、交配反應以及 white-to-opaque 型態轉換之中，但對於傳統生物膜之生成不具影響力。

Candida albicans is a prevalent opportunistic human fungal pathogen that can cause vital systemic infections in immunocompromised patients. In order to adapt multiple niches, C. albicans develops a unique phenotypic transition between white and opaque phases. The most behavioral difference of these two cell forms is their sexual fertility. Opaque cells are the mating competent form that secrete cell type-specific (a or α) pheromones to activate mating responses in the opposite cell type. However, although white cells are incapable of mating, pheromones released from minority opaque cells could induce biofilm formation and cell adhesion of white cells. The array profiling has shown that MFA1 (encoded with an a-pheromone precursor) and HST6 (encoded with an a-pheromone transporter) in MTLa/a white cells were highly expressed when challenged with α-pheromone. We therefore hypothesized that MFA1 and HST6 are involved in the cell adhesion during the response to α-pheromone. Deletion of the HST6 gene, an a-type specific a-pheromone transporter, in MTLa/a P37005 strain resulted in the significant reduction of numbers in pheromone-induced cell adhesion (5.2 x 107) comparing with those of the wild-type strain (13.4 x 107), while deletion of the MFA1 gene exhibited no role in its cell adhesion. Preliminary studies have identified the transcriptional regulator of pheromone-induced cell adhesion as Cph1. Quantitative RT-PCR also showed that the expression of the HST6 is directly regulated by Cph1, Cek1 (MAPK) and Ste2 (receptor of α-pheromone). Additionally, hst6Δ showed an extremely low mating efficiency (0.004%) comparing to that of the wild-type strain (65.07%), suggesting that deletion of HST6 could cause a-pheromone peptide secretion defects in C. albicans. To understand if Hst6 involved in pheromone-induced cell adhesion also plays a role in conventional biofilm formation, two genes of biofilm-associated regulators were tested. Expression of TEC1 or NDT80 and the dry mass of biofilms between the wild-type strain and hst6Δ showed no significant differences, suggesting that Hst6 is specifically involved in pheromone-induced cell adhesion but not biofilm development. Interestingly, hst6Δ displayed a lower white-to-opaque switching frequency (1.95 ± 2.51%) than those of the wild-type (34.34 ± 20.16%). However, deletion of the HST6 gene in MTLa/a RBY717 strain exhibited no effects on pheromone-induced cell adhesion and white-to-opaque transition, which implied that the functions of Hst6 might be strain-specific. Taken together, our study has demonstrated that Hst6, an a-pheromone transporter in MTLa/a cells, is involved in pheromone-induced cell adhesion, mating and white-to-opaque transition in P37005 strain of C. albicans, but is dispensable for the formation of conventional biofilms.