郭東東，岳慧珍，魏羽佳，黃廣華

1 貴州醫(yī)科大學(xué)附屬醫(yī)院，貴州 貴陽(yáng) 550004

2 中國(guó)科學(xué)院微生物研究所 真菌學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100101

白念珠菌生物被膜形成的遺傳調(diào)控機(jī)制

郭東東1*，岳慧珍2*，魏羽佳1，黃廣華2

1 貴州醫(yī)科大學(xué)附屬醫(yī)院，貴州 貴陽(yáng) 550004

2 中國(guó)科學(xué)院微生物研究所 真菌學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100101

白念珠菌是人體重要的條件性致病真菌。形態(tài)的多樣性和可塑性是白念珠菌典型的生物學(xué)特征，這與它的致病性、宿主適應(yīng)能力以及有性生殖過(guò)程密切相關(guān)。白念珠菌生物被膜(Biofilm)是由不同形態(tài)細(xì)胞(包括酵母型、菌絲和假菌絲)以及胞外基質(zhì)組成的致密結(jié)構(gòu)，也是毒性和耐藥性形成的重要因子。生物被膜對(duì)抗真菌藥物、宿主免疫系統(tǒng)和環(huán)境脅迫因子等都表現(xiàn)出較強(qiáng)的抵抗力和耐受性，是臨床上病原真菌感染防治的重大挑戰(zhàn)。隨著基因表達(dá)譜和遺傳操作技術(shù)的發(fā)展，白念珠菌生物被膜的形成及其耐藥性的獲得所依賴的遺傳調(diào)控通路和分子調(diào)控機(jī)制越來(lái)越清楚。主要包括MAPK和cAMP介導(dǎo)的信號(hào)途徑以及Bcr1和Tec1等因子介導(dǎo)的轉(zhuǎn)錄調(diào)控。此外，白念珠菌生物被膜的形成與形態(tài)轉(zhuǎn)換和有性生殖之間存在密切的聯(lián)系。文中綜述了白念珠菌生物被膜形成的遺傳調(diào)控機(jī)制，重點(diǎn)介紹了細(xì)胞壁相關(guān)蛋白、轉(zhuǎn)錄因子和交配型對(duì)該過(guò)程的調(diào)控以及生物被膜的耐藥機(jī)制。

白念珠菌，生物被膜，調(diào)控機(jī)制，MTL，耐藥性

1 前言

生物被膜是由微生物細(xì)胞(細(xì)菌或真菌)及其胞外分泌物(Extracellular polymeric substances，EPS)聚集于生物或非生物體表面而形成的一種復(fù)雜群體結(jié)構(gòu)。EPS主要包括多糖、蛋白、核酸和脂類等高分子化合物。生物被膜中的細(xì)胞與浮游型細(xì)胞具有明顯不同的表型和基因表達(dá)特征。微生物形成生物被膜后能夠增強(qiáng)對(duì)宿主免疫攻擊、營(yíng)養(yǎng)限制和抗菌藥物等有害因子的耐受性[1-2]。人體病原菌可以粘附和定殖在惰性材料、有機(jī)聚合物和醫(yī)療器材表面，并逐步形成成熟的生物被膜；隨后部分病原菌細(xì)胞脫離成熟的生物被膜，進(jìn)行擴(kuò)散和傳播[3-4]。在臨床上，患者內(nèi)置的導(dǎo)尿管、心臟瓣膜、起搏器和假牙等醫(yī)用材料都為病原菌生物被膜的形成提供了良好的環(huán)境和場(chǎng)所，同時(shí)為病原微生物的繁殖和傳播創(chuàng)造了有利的條件[5-6]。病原菌生物被膜的形成對(duì)人類健康構(gòu)成了嚴(yán)重的威脅，在淺表的粘膜感染和深度系統(tǒng)感染中都起著重要作用[7-9]。2012年美國(guó)國(guó)立衛(wèi)生研究院(NIH)研究顯示，生物被膜在所有病原微生物感染中所占的比例已超過(guò)80%[10]。因此，生物被膜的形成已成為臨床病原感染中極具有挑戰(zhàn)性的醫(yī)療健康問(wèn)題[5-6]。

白念珠菌Candida albicans是人體中常見的共生菌，同時(shí)也是重要的機(jī)會(huì)性致病真菌[11]。它能夠長(zhǎng)期地定殖于口腔、胃腸道和泌尿生殖道等部位而不引發(fā)疾??；但當(dāng)宿主免疫能力下降、體內(nèi)微生物菌群結(jié)構(gòu)失衡或體內(nèi)環(huán)境發(fā)生重大改變時(shí)，則能夠引發(fā)不同程度的念珠菌感染(例如皮膚感染、鵝口瘡和陰道炎等淺表和粘膜感染，以及心內(nèi)膜炎、腦膜炎和敗血癥等系統(tǒng)或深部器官感染)[12]。白念珠菌容易感染免疫功能低下的患者(如艾滋病患者或免疫抑制藥物治療者)及植入醫(yī)療器械的健康人群[12-13]。這種感染進(jìn)一步增加了臨床上疾病治療的風(fēng)險(xiǎn)和成本。白念珠菌對(duì)宿主不同環(huán)境的適應(yīng)能力和致病能力依賴于其獨(dú)特的生物學(xué)特性，即形態(tài)多樣性和可塑性。該菌能夠在多種形態(tài)之間相互轉(zhuǎn)換。酵母-菌絲型轉(zhuǎn)換是白念珠菌常見的一種形態(tài)轉(zhuǎn)換模式[14]。酵母型細(xì)胞呈游離態(tài)，菌落表面光滑；能夠定殖在皮膚和黏膜的表面，并隨血液循環(huán)進(jìn)行傳播引發(fā)宿主系統(tǒng)性感染[15-16]。菌絲是較長(zhǎng)的多細(xì)胞結(jié)構(gòu)絲狀體，菌落表面易形成皺褶或侵入固體培養(yǎng)基內(nèi)；它有利于白念珠菌侵入和穿透宿主組織細(xì)胞[15-16]。White-opaque轉(zhuǎn)換是白念珠菌另一典型的形態(tài)轉(zhuǎn)換系統(tǒng)[17]。White細(xì)胞較小，呈球形或橢球形，其菌落為白色，表面光滑。Opaque細(xì)胞較大，呈長(zhǎng)橢球形或柱形，并常有大液泡結(jié)構(gòu)；其菌落在含有熒光桃紅染料(Phloxine B)的固體培養(yǎng)基上呈紅色，表面較為粗糙。此外，white和opaque對(duì)宿主組織細(xì)胞具有不同的侵染和破壞能力；white細(xì)胞更容易粘附人體上皮細(xì)胞并侵入組織，opaque細(xì)胞則易于定殖和破壞皮膚和黏膜組織[18-19]。生物被膜是白念珠菌重要的毒力因子。在常規(guī)實(shí)驗(yàn)條件下，white細(xì)胞比 opaque細(xì)胞更容易形成生物被膜。因此，對(duì)白念珠菌形態(tài)轉(zhuǎn)換和生物被膜形成機(jī)制的研究將有助于認(rèn)識(shí)念珠菌病的發(fā)病機(jī)理。

白念珠菌生物被膜的形成與酵母和菌絲型細(xì)胞的發(fā)育存在密切的關(guān)系[20-21]。其中，細(xì)胞壁及粘附相關(guān)蛋白(例如Als蛋白、Hwp1、Eap1、Ywp1、Ece1和Csh1)不僅是白念珠菌酵母型或菌絲型細(xì)胞發(fā)育的重要因子，在細(xì)胞粘附和生物被膜形成調(diào)控中也發(fā)揮重要作用[21-22]。另外，由不同的轉(zhuǎn)錄因子(例如 Bcr1、Efg1、Tec1、Gat2、Ndt80 和 Rob1，以及 Zap1、Ume6、Adh1 和 Chk1)介導(dǎo)的遺傳調(diào)控網(wǎng)絡(luò)通過(guò)響應(yīng)不同的環(huán)境信號(hào)分子對(duì)白念珠菌生物被膜形成的各階段進(jìn)行嚴(yán)格的調(diào)控，從而確保了生物被膜有條不紊地建成和發(fā)育[23-24]。White-opaque形態(tài)轉(zhuǎn)換以及MTL(Mating type like locus，交配型基因座)也參與調(diào)控白念珠菌生物被膜的形成[25-29]。根據(jù)交配基因型的不同，白念珠菌能形成兩種不同的生物被膜：一種是具有致病性特征的MTL-雜合型生物被膜，另一種是具有促進(jìn)交配特性的MTL-純合型生物被膜[30-31]。這兩種不同類型的生物被膜分別受 cAMP/PKA信號(hào)通路和性信息素介導(dǎo)的MAPK信號(hào)途徑的調(diào)控[28]。耐藥性是生物被膜獨(dú)特的生物學(xué)功能，同時(shí)也是白念珠菌研究的熱點(diǎn)[32]。對(duì)白念珠菌生物被膜的遺傳調(diào)控機(jī)制的深入研究，將有助于進(jìn)一步揭示其耐藥性的分子調(diào)控機(jī)制，并為臨床上預(yù)防和治療生物被膜引發(fā)的病原感染提供理論基礎(chǔ)。

2 白念珠菌生物被膜的形成及遺傳調(diào)控

白念珠菌生物被膜結(jié)構(gòu)復(fù)雜，由多種不同形態(tài)的細(xì)胞(包括酵母、菌絲和假菌絲型細(xì)胞)和胞外基質(zhì)組成[10,33-34]。生物被膜的形成是一個(gè)連續(xù)的過(guò)程，主要包括粘附、起始、成熟、脫離和擴(kuò)散幾個(gè)階段[23](圖1)。首先，酵母型細(xì)胞通過(guò)粘附作用定殖在生物或非生物體表面，粘附細(xì)胞進(jìn)行分裂和增殖形成一個(gè)緊密的微菌落群體，即生物被膜的基層[21,23]。隨后，酵母型細(xì)胞開始伸長(zhǎng)并形成大量的菌絲或假菌絲，同時(shí)伴有胞外基質(zhì)的產(chǎn)生[21,35]。胞外基質(zhì)能夠有效地保護(hù)白念珠菌逃避宿主免疫細(xì)胞的吞噬、阻止抗菌藥及有害物質(zhì)的殺傷，并維持生物被膜內(nèi)部營(yíng)養(yǎng)物質(zhì)的穩(wěn)定[36-37]。最后，酵母型細(xì)胞自發(fā)地脫離成熟的生物被膜菌體，擴(kuò)散至其他部位并引發(fā)新生生物被膜的形成和發(fā)育[8]。白念珠菌生物被膜的形成、發(fā)育和擴(kuò)散過(guò)程復(fù)雜多變，隨著近年來(lái)基因組測(cè)序、分子生物學(xué)和遺傳學(xué)技術(shù)的改進(jìn)，相關(guān)調(diào)控機(jī)制的研究取得了很大的進(jìn)展，大量研究表明很多細(xì)胞壁及粘附相關(guān)蛋白、轉(zhuǎn)錄調(diào)控因子、MTL基因座和激酶等都參與了這些過(guò)程的調(diào)控[38](表1)。

圖1 白念珠菌生物被膜形成的四個(gè)階段(粘附、起始、成熟和擴(kuò)散)Fig.1 Four steps of biofilm development inC.albicans.

2.1 細(xì)胞壁及粘附相關(guān)蛋白

粘附是白念珠菌成功定殖在黏膜及其他物體表面的第一步，同時(shí)也是生物被膜形成并引發(fā)病原感染的開始[39]。粘附蛋白，即粘附素，是細(xì)胞粘附和定殖過(guò)程中的關(guān)鍵誘導(dǎo)因子，它們?cè)谏锉荒さ男纬蛇^(guò)程中發(fā)揮著重要的作用[40]。粘附是病原菌細(xì)胞與宿主表面的一種相互作用，因此，病原菌細(xì)胞壁結(jié)構(gòu)、宿主細(xì)胞表面受體以及外部環(huán)境等都能調(diào)控粘附過(guò)程的發(fā)生[38]。

2.1.1 Als(Agglutinin-like sequence，凝集素樣序列)蛋白家族

Als蛋白家族是一類細(xì)胞壁糖蛋白，也是白念珠菌中研究最多的一類調(diào)控細(xì)胞粘附作用的蛋白分子。它由8個(gè)具有相同 N-端分泌性信號(hào)序列和 C-端 GPI錨定結(jié)構(gòu)域的蛋白組成，即Als1-7和Als9[41]。Als蛋白家族中，Als1、Als3和Als5在白念珠菌與宿主細(xì)胞相互粘附過(guò)程中發(fā)揮著重要作用；其中，Als1和Als3蛋白主要與宿主內(nèi)皮細(xì)胞和上皮細(xì)胞發(fā)生粘附作用，而 Als5蛋白主要與宿主細(xì)胞胞外基質(zhì)蛋白相結(jié)合[42]。

ALS1是白念珠菌ALS基因家族中第一個(gè)被鑒定的基因，它與釀酒酵母細(xì)胞表面粘附蛋白 α-凝集素具有高度相似的氨基酸序列，并由此推測(cè)Als1是白念珠菌中重要的細(xì)胞粘附蛋白[43]。Als3蛋白在白念珠菌生物被膜形成過(guò)程中起著關(guān)鍵作用，當(dāng)基因ALS3敲除后，突變株als3/als3在體外生物材料上無(wú)法形成正常的生物被膜[44-45]。有意思的是，格氏鏈球菌Streptococcus gordonnii還可以通過(guò)與 Als3蛋白結(jié)合促進(jìn)口腔黏膜中白念珠菌生物被膜的形成[46]。此外，Als1和Als3在不同形態(tài)細(xì)胞中的蛋白表達(dá)水平存在差異；Als1在酵母和菌絲型細(xì)胞中均能夠表達(dá)，而Als3是菌絲細(xì)胞特異性表達(dá)蛋白[47]。

2.1.2 Hwp1、Rbt1、Eap1和 Ywp1

Hwp1、Rbt1、Eap1和 Ywp1都是 GPI(Glycosyl phosphatidyll inositol，糖基磷脂酰肌醇)-錨定細(xì)胞壁蛋白。Hwp1是菌絲特異性細(xì)胞壁蛋白，它能調(diào)控生物被膜中細(xì)胞芽管和菌絲的形成，并促進(jìn)白念珠菌與宿主細(xì)胞的接觸和粘附；所以 Hwp1也是白念珠菌重要的致病因子[48-49]。另一重要的GPI-錨定型細(xì)胞壁蛋白 Rbt1與Hwp1具有相似的結(jié)構(gòu)和功能，兩者具有相同的C-端結(jié)構(gòu)域[50]。RBT1也是菌絲特異性細(xì)胞壁蛋白編碼基因，其表達(dá)水平在菌絲型細(xì)胞生長(zhǎng)過(guò)程中明顯上調(diào)。當(dāng)HWP1或RBT1基因敲除以后，其突變株hwp1/hwp1和rbt1/rbt1生物被膜形成能力均受到抑制；而當(dāng)兩者同時(shí)敲除后，其雙突變株對(duì)生物被膜形成的抑制作用明顯增強(qiáng)，這說(shuō)

明HWP1和RBT1在生物被膜形成過(guò)程中具有一定的協(xié)同作用[50]。此外，Hwp1和Rbt1還能通過(guò)響應(yīng)性信息素來(lái)調(diào)控生物被膜與細(xì)胞交配的過(guò)程[51-52]。Eap1是較早發(fā)現(xiàn)的細(xì)胞壁粘附蛋白，它在酵母型和菌絲型細(xì)胞中均能夠表達(dá)[53]。白念珠菌eap1/eap1突變株不僅大大減弱了對(duì)聚苯乙烯的粘附能力，它在宿主體內(nèi)及體外的生物被膜形成能力也受到了抑制[54]。同樣，白念珠菌Eap1在釀酒酵母中能夠異源表達(dá)并賦予釀酒酵母粘附能力[53]，這也進(jìn)一步表明了Eap1是生物被膜形成過(guò)程中重要的細(xì)胞壁粘附蛋白。Ywp1是酵母特異性細(xì)胞壁蛋白，它是酵母型細(xì)胞生物被膜形成的抑制因子。過(guò)表達(dá)Ywp1使酵母細(xì)胞失去粘附能力；而ywp1/ywp1突變株能增強(qiáng)酵母細(xì)胞的粘附能力、增加生物被膜的厚度，并減少生物被膜細(xì)胞的脫離和擴(kuò)散[55-56]。

表1 參與白念珠菌生物被膜形成的相關(guān)蛋白Table1 Selected proteins involved in biofilm development inC.albicans

2.1.3 Ece1和Csh1

ECE1最早被鑒定為菌絲特異性表達(dá)基因，但其生物學(xué)功能卻不清楚[57]。直到2006年，Nobile等發(fā)現(xiàn)Ece1可以促進(jìn)生物被膜的形成，這可能是由于Ece1能使細(xì)胞表面粘附素暴露從而增強(qiáng)了細(xì)胞的粘附能力[45]。Csh1是一種細(xì)胞表面疏水性蛋白[58]。疏水蛋白與細(xì)胞的粘附性密切相關(guān)，Csh1能增強(qiáng)細(xì)胞粘附并促進(jìn)生物被膜的形成[59-60]。此外，Csh1還能調(diào)控成熟生物被膜胞外基質(zhì)的產(chǎn)生，而這一作用機(jī)制主要受控于轉(zhuǎn)錄因子Zap1的表達(dá)[61]。

2.2 轉(zhuǎn)錄調(diào)控因子和蛋白激酶

信號(hào)轉(zhuǎn)導(dǎo)途徑和轉(zhuǎn)錄因子對(duì)白念珠菌形態(tài)發(fā)生起著重要的調(diào)控作用，它們同樣能調(diào)控白念珠菌生物被膜的形成。轉(zhuǎn)錄因子和蛋白激酶通過(guò)激活或抑制下游相關(guān)基因的表達(dá)以實(shí)現(xiàn)對(duì)生物被膜形成和發(fā)育的調(diào)控作用。

2.2.1 正調(diào)控因子

Bcr1、Efg1和Tec1在白念珠菌形態(tài)發(fā)生過(guò)程中起著重要的調(diào)控作用，同樣也是生物被膜形成的關(guān)鍵轉(zhuǎn)錄調(diào)控因子。BCR1編碼蛋白具有典型的C2H2型鋅指結(jié)構(gòu)，它參與調(diào)控細(xì)胞粘附和生物被膜的形成[45]。轉(zhuǎn)錄因子Bcr1的下游靶標(biāo)基因主要為細(xì)胞壁粘附蛋白編碼基因ALS1、ALS3和HWP1，但它卻不參與調(diào)控菌絲的生長(zhǎng)，這說(shuō)明Bcr1主要在生物被膜形成的初始階段起調(diào)控作用[45,48]。另外，Bcr1的下游靶標(biāo)基因RBT5、PGA10和CSA1均為胞外基質(zhì)蛋白編碼基因，它們可通過(guò)表面受體或粘附作用參與調(diào)控生物被膜的形成[62-63]。Efg1是白念珠菌生物被膜形成的另一重要轉(zhuǎn)錄因子，它對(duì)細(xì)胞的粘附作用和菌絲生長(zhǎng)均有調(diào)控作用[64]。Efg1通過(guò)調(diào)控下游基因Eap1的表達(dá)來(lái)參與調(diào)控細(xì)胞的粘附作用[53]，此外它還參與轉(zhuǎn)錄因子 Tec1的調(diào)控通路[65]。Tec1屬于TEA/ATTS轉(zhuǎn)錄因子家族[66]，它對(duì)白念珠菌致病性和菌絲生長(zhǎng)具有重要調(diào)控作用[45]。轉(zhuǎn)錄因子Efg1-Tec1共同參與調(diào)控生物被膜的菌絲發(fā)育過(guò)程[65]。有意思的是，轉(zhuǎn)錄因子Bcr1的表達(dá)也依賴于Tec1的調(diào)控作用。Bcr1位于 Efg1-Tec1調(diào)控的菌絲發(fā)育信號(hào)途徑的下游，通過(guò)賦予菌絲型細(xì)胞粘附特性再次參與調(diào)控生物被膜的形成[67]。

Gat2(也稱Brg1)、Ndt80和Rob1是最近發(fā)現(xiàn)的參與調(diào)控白念珠菌生物被膜形成的轉(zhuǎn)錄因子[23]。Gat2蛋白具有GAGA型鋅指結(jié)構(gòu)域，其突變株gat2/gat2表現(xiàn)出菌絲生長(zhǎng)缺陷、粘附能力下降的特點(diǎn)，并失去生物被膜形成能力[68]。NDT80是白念珠菌菌絲發(fā)育和毒力表現(xiàn)的關(guān)鍵基因，同時(shí)也是生物被膜形成的重要轉(zhuǎn)錄因子[23,69-70]。它不僅能調(diào)控細(xì)胞壁蛋白Als3、Hwp1和Ece1的表達(dá)，同時(shí)對(duì)轉(zhuǎn)錄因子Tec1和Ume6的表達(dá)具有間接調(diào)控作用[69]。ROB1缺失株rob1/rob1不能形成生物被膜，但其調(diào)控機(jī)制卻仍不清楚[23]。Bcr1、Efg1、Gat2、Ndt80、Rob1和Tec1六個(gè)主要轉(zhuǎn)錄因子形成一個(gè)相互調(diào)控的環(huán)路，并調(diào)控一系列生物被膜相關(guān)基因，是生物被膜形成的調(diào)控核心[65]。

2.2.2 負(fù)調(diào)控因子

除了以上正調(diào)控轉(zhuǎn)錄因子外，Zap1、Ume6、Adh1和 Chk1等在白念珠菌生物被膜的形成過(guò)程中起負(fù)調(diào)控作用。Zap1是鋅感應(yīng)型轉(zhuǎn)錄因子，抑制生物被膜胞外基質(zhì)的產(chǎn)生，并在生物被膜成熟階段發(fā)揮作用[61]。β-1,3葡聚糖是生物被膜胞外基質(zhì)的重要成分，Zap1下游靶標(biāo)基因CSH1、IFD6、GCA1和ADH5能調(diào)控β-1,3葡聚糖產(chǎn)生[61]。其中，CSH1和IFD6在Zap1調(diào)控下能抑制胞外基質(zhì)的形成；而GCA1、GCA2和ADH5在Zap1調(diào)控下能夠促進(jìn)胞外基質(zhì)的形成和積累[61]。

在生物被膜形成過(guò)程中，酵母型細(xì)胞的脫離和擴(kuò)散是誘導(dǎo)新生生物被膜形成的關(guān)鍵因素[71]。Ume6是白念珠菌菌絲延伸和生長(zhǎng)的轉(zhuǎn)錄調(diào)控因子，它能抑制酵母型細(xì)胞脫離生物被膜，并對(duì)新生生物被膜的形成和發(fā)展具有負(fù)調(diào)控作用[71-73]。此外，PES1和NRG1是調(diào)控菌絲-酵母形態(tài)轉(zhuǎn)換和酵母型細(xì)胞生長(zhǎng)的重要基因，它們能促進(jìn)成熟生物被膜酵母型細(xì)胞的脫離和擴(kuò)散[71,74]。

法尼醇(Farnesol)是白念珠菌重要的群感效應(yīng)分子，它能抑制菌絲的生長(zhǎng)和生物被膜的形成[75-77]。CHK1是組蛋白激酶編碼基因，其突變株chk1/chk1能解除法尼醇對(duì)白念珠菌菌絲生長(zhǎng)及生物被膜形成的抑制作用[78]。因此，轉(zhuǎn)錄因子Chk1可能參與調(diào)控法尼醇介導(dǎo)的生物被膜的形成。2006年，Mukherjee等發(fā)現(xiàn)乙醇能抑制白念珠菌生物被膜的形成，這一負(fù)調(diào)控作用與乙醇脫氫酶編碼基因ADH1密切相關(guān)[79]。在乙醇存在條件下，adh1/adh1突變株具備生物被膜形成能力[79]，但Adh1調(diào)控生物被膜形成的分子機(jī)制仍需進(jìn)一步探索。

2.2.3 蛋白激酶Mkc1和Cek1

MAPK(Mitogen-activated protein kinase，絲裂原激活蛋白激酶)信號(hào)途徑是第一個(gè)與白念珠菌形態(tài)發(fā)生相關(guān)的信號(hào)調(diào)控通路，并在不同物種的進(jìn)化中具有高度保守性[80]。Mkc1是一種MAPK蛋白激酶，它是白念珠菌細(xì)胞壁完整結(jié)構(gòu)的一部分，并參與調(diào)控細(xì)胞表面接觸反應(yīng)[81-82]。表面接觸反應(yīng)是細(xì)胞進(jìn)行菌絲侵入性生長(zhǎng)和生物被膜形成的前提條件[82]。MKC1基因敲除以后，mkc1/mkc1突變株形成大量具有菌絲缺陷的生物被膜，而且此異常生物被膜對(duì)氟康唑的敏感性增強(qiáng)[24,82]。Cek1是一種胞外信號(hào)調(diào)節(jié)激酶，它也是 MAPK家族的重要成員并在細(xì)胞表面接觸反應(yīng)過(guò)程起作用[83]。與MKC1一樣，CEK1也是白念珠菌形成正常生物被膜的重要調(diào)節(jié)基因，但Mkc1和Cek1對(duì)生物被膜形成的具體調(diào)控機(jī)制卻不清楚[83]。

3 交配型基因座MTL對(duì)生物被膜形成的調(diào)控作用

白念珠菌通常以二倍體形式存在，有3種不同交配型（MTLa/α,MTLa/a 或MTLα/α）。MTLa/α交配型細(xì)胞形成MTL-雜合型生物被膜，MTLa/a或MTLα/α則形成MTL-純合型生物被膜。這兩種生物被膜具有相似的形態(tài)和結(jié)構(gòu)特征，但兩者卻受控于不同的信號(hào)傳遞通路，并在細(xì)胞交配和耐藥能力方面存在明顯差異[29-31]。

3.1 “病原性”和“交配性”生物被膜的形成

自然界中分離得到的白念珠菌大部分為MTLa/α型菌株，由此推測(cè)，宿主體內(nèi)以及內(nèi)置醫(yī)用導(dǎo)管表面所形成的生物被膜主要為MTL-雜合型生物被膜。MTL-雜合型生物被膜似乎保留了病原菌固有的致病特性，它能夠阻止1.6–112 kDa分子量的物質(zhì)的滲透、抵御宿主多形核白細(xì)胞的侵襲，并對(duì)氟康唑表現(xiàn)出較強(qiáng)的耐藥性，因此也稱它為“病原性”生物被膜[84-86]。

MTLa/α型菌株第5號(hào)染色體經(jīng)過(guò)純合化后產(chǎn)生MTLa/a或MTLα/α型細(xì)胞，MTL-純合型細(xì)胞能夠從酵母型white細(xì)胞轉(zhuǎn)變?yōu)榫哂懈咝Ы慌淠芰Φ膐paque細(xì)胞[25-26]。與MTLa/α型細(xì)胞一樣，MTLa/a和MTLα/α型的white細(xì)胞也能形成生物被膜；然而，MTLa/a和MTLα/α型的opaque細(xì)胞卻無(wú)法形成生物被膜[27,87]。少量的(<10%)MTL-純合型 opaque細(xì)胞(MTLa/a和MTLα/α等比例混合)通過(guò)釋放性信息素能夠增強(qiáng)MTL-純合型 white細(xì)胞生物被膜的厚度，這也進(jìn)一步說(shuō)明 white-opaque形態(tài)轉(zhuǎn)換能夠調(diào)控細(xì)胞生物被膜的形成；然而，過(guò)多(>40%)MTL-純合型 opaque細(xì)胞反而會(huì)影響MTL-純合型white細(xì)胞生物被膜的整體結(jié)構(gòu)而不利于其厚度的增加[27]。MTL-純合型 white細(xì)胞能夠自發(fā)地低頻率轉(zhuǎn)變成opaque細(xì)胞，這些opaque細(xì)胞可以通過(guò)旁分泌系統(tǒng)產(chǎn)生與自身相反交配型的性信息素，從而刺激自身生物被膜的形成[28]。與MTL-雜合型生物被膜相比，MTL-純合型生物被膜致病性的特征并不明顯；低或高分子量的物質(zhì)以及宿主免疫細(xì)胞均能滲入MTL-純合型生物被膜內(nèi)，并對(duì)抗菌藥物表現(xiàn)出敏感性[29]。有意思的是，誘導(dǎo)MTL-純合型 white細(xì)胞生物被膜形成的性信息素同樣能促進(jìn)其少量存在的opaque細(xì)胞進(jìn)行交配[28]；因此，MTL-純合型 white生物被膜也被稱為“交配性”生物被膜。

3.2 交配基因型MTL對(duì)生物被膜形成的調(diào)控

cAMP/PKA和MAPK信號(hào)途徑是調(diào)控白念珠菌形態(tài)發(fā)生的主要信號(hào)傳遞途徑[28,88-90]。MTL-雜合型生物被膜形成的信號(hào)調(diào)控通路是 Ras1-cAMP/PKA信號(hào)途徑[28]。其中，cAMP依賴性蛋白激酶編碼基因TPK2是MTL-雜合型生物被膜形成的必要基因[28,91-92]，而Efg1、Tec1和Bcr1則是MTL-雜合型生物被膜維持正常生長(zhǎng)所必需的轉(zhuǎn)錄因子[45]。轉(zhuǎn)錄因子Tec1不僅能直接調(diào)控Bcr1的表達(dá)，同時(shí)還直接參與調(diào)控MTL-雜合型生物被膜的菌絲形成過(guò)程[28,45,93]。然而，Tec1同樣也是MTL-純合型生物被膜菌絲形成的關(guān)鍵轉(zhuǎn)錄因子，這說(shuō)明Tec1可能參與調(diào)控生物被膜基本構(gòu)造的形成，而轉(zhuǎn)錄因子Bcr1則可能參與MTL-雜合型調(diào)控生物被膜胞外基質(zhì)的產(chǎn)生。

MTL-純合型 white細(xì)胞生物被膜的形成與細(xì)胞交配均受控于性信息素介導(dǎo)的MAPK信號(hào)途徑，但 MAPK信號(hào)途徑通過(guò)激活下游不同的轉(zhuǎn)錄因子來(lái)調(diào)控兩種生物過(guò)程的發(fā)生[28,94-97]。其中，轉(zhuǎn)錄因子 Tec1能夠與 white細(xì)胞特異的性信息素響應(yīng)元件結(jié)合并觸發(fā)與生物被膜形成相關(guān)基因的表達(dá)[93]，而 Cph1是調(diào)控 opaque細(xì)胞交配的關(guān)鍵轉(zhuǎn)錄因子[98-99]。

4 生物被膜與耐藥性形成的關(guān)系

白念珠菌生物被膜對(duì)抗真菌藥物具有一定的耐藥性[8,84-85]。生物被膜耐藥性形成機(jī)制復(fù)雜多樣，這與生物被膜的不同發(fā)展階段密切相關(guān)。其中，藥物外排泵(Drug efflux pumps)的正調(diào)控作用、胞外基質(zhì)(Extracellular matrix)的產(chǎn)生以及持留細(xì)胞(Persister cells)的存在是造成白念珠菌生物被膜耐藥性的重要原因[32]。

4.1 外排泵的正調(diào)控作用

藥物的外排作用是導(dǎo)致耐藥性產(chǎn)生的主要原因。在白念珠菌中，與藥物外排泵相關(guān)的兩大類基因分別為 ATP結(jié)合區(qū)轉(zhuǎn)運(yùn)子基因(ATP-binding cassette transporter，CDR1和CDR2)和主要易化子基因(Major faciliator，MDR1)[100-102]。當(dāng)抗菌藥物存在時(shí)，浮游型細(xì)胞外排泵的表達(dá)水平明顯升高；有意思的是，生物被膜外排泵的表達(dá)卻并不取決于抗菌藥物，它在細(xì)胞發(fā)生粘附后幾個(gè)小時(shí)便開始持續(xù)上調(diào)[23,102-106]。由此可見，外排泵的自主高表達(dá)作用促成了生物被膜高度耐藥的生物學(xué)特性。

4.2 胞外基質(zhì)與耐藥性

胞外基質(zhì)的分泌是生物被膜形成耐藥性的另一關(guān)鍵原因。生物被膜在成熟階段會(huì)釋放出大量的胞外基質(zhì)，它作為天然的物理屏障可以阻擋藥物的滲入，并維持和保護(hù)生物被膜結(jié)構(gòu)的完整性[35-36,107-108]。由于胞外基質(zhì)的主要成分難以鑒定，這使得深入了解其耐藥機(jī)制的研究更加具有挑戰(zhàn)性。最近研究發(fā)現(xiàn)，胞外基質(zhì)主要是由大分子物質(zhì)蛋白和糖蛋白(55%)、碳水化合物(25%)、脂質(zhì)(15%)和核酸(5%)構(gòu)成[109]。其中，蛋白質(zhì)已鑒定出500多種，大部分為酶類；由此推測(cè)，胞外基質(zhì)可能通過(guò)酶解外源分子保護(hù)自身并為生物被膜的發(fā)展提供營(yíng)養(yǎng)物質(zhì)。多糖是胞外基質(zhì)第二大組成成分，主要為甘露聚糖復(fù)合體[109]。目前已知，β-1,3葡聚糖是胞外基質(zhì)發(fā)揮耐藥作用的重要分子；當(dāng)用β-1,3葡聚糖酶處理生物被膜時(shí)，生物被膜對(duì)氟康唑的敏感性明顯增加；而且，外源添加β-1,3葡聚糖也能夠增強(qiáng)浮游型細(xì)胞對(duì)氟康唑的抗藥性[107,110]。另外，胞外DNA在生物被膜抵抗藥物的殺傷過(guò)程中也表現(xiàn)出間接的促進(jìn)作用[111]。有研究報(bào)道，脫氧核糖核酸酶能夠增強(qiáng)卡泊芬凈和兩性霉素 B對(duì)成熟期生物被膜的破壞和殺傷能力[112]。然而，β-1,3葡聚糖和胞外DNA只是胞外基質(zhì)組成的一小部分，這更充分說(shuō)明胞外基質(zhì)并不單單是生物被膜所釋放的細(xì)胞壁組分，它還具備獨(dú)特的生物學(xué)功能。

4.3 持留細(xì)胞

持留細(xì)胞(Persister cells)是生物被膜隨機(jī)產(chǎn)生的少量處于休眠狀態(tài)的酵母型細(xì)胞。它們也是白念珠菌生物被膜形成耐藥性的關(guān)鍵因子，對(duì)抗真菌藥物具有較強(qiáng)的耐受性[113]。雖然生物被膜中持留細(xì)胞的形成和作用機(jī)制尚不清楚，但它的耐藥性卻不依賴于細(xì)胞壁的構(gòu)成和外排泵的表達(dá)，而與持留細(xì)胞所處的休眠代謝狀態(tài)有關(guān)[113-114]。持留細(xì)胞對(duì)生物被膜耐藥性的形成至關(guān)重要，但它的形成和調(diào)控機(jī)制卻仍有待進(jìn)一步深入研究。

5 結(jié)論與展望

生物被膜不僅是白念珠菌形態(tài)多樣性的表現(xiàn)，更是對(duì)宿主環(huán)境變化的一種適應(yīng)性策略。生物被膜的形成為白念珠菌的定殖和侵染提供了更加有利的條件，同時(shí)也為臨床上預(yù)防和治療白念珠菌病帶來(lái)了困難和挑戰(zhàn)。近年來(lái)，白念珠菌生物被膜的研究越來(lái)越深入，尤其在生物被膜的形成和分子調(diào)控機(jī)制、MTL依賴性生物被膜的信號(hào)調(diào)控網(wǎng)絡(luò)以及耐藥性形成的分子機(jī)制等方面取得了重大進(jìn)展，但該領(lǐng)域仍存在很多問(wèn)題亟待進(jìn)一步研究。例如，菌絲發(fā)育和 white-opaque形態(tài)轉(zhuǎn)換怎樣調(diào)控生物被膜的形成？MTL-純合型生物被膜胞外基質(zhì)的產(chǎn)生是如何被調(diào)控的？其他病原真菌中是否存在類似于白念珠菌中不同功能的“病原性”和“交配性”生物被膜？生物被膜胞外基質(zhì)的組成成分還有哪些？其生物學(xué)功能是否與耐藥性有關(guān)？這些研究將有助于加深人們對(duì)白念珠菌生物被膜的生物學(xué)特性及分子調(diào)控機(jī)制的認(rèn)識(shí)和了解，為臨床上防治白念珠菌引起的感染提供理論依據(jù)，并為新型抗真菌藥物的研發(fā)提供新思路。

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(本文責(zé)編 郝麗芳)

Genetic regulatory mechanisms ofCandida albicansbiofilm formation

Dongdong Guo1*, Huizhen Yue2*, Yujia Wei1, and Guanghua Huang2
1Affiliated Hospital of Guizhou Medical University,Guiyang550004,Guizhou,China
2State Key Laboratory of Mycology,Institute of Microbiology,Chinese Academy of Sciences,Beijing100101,China

Candida albicansis an important opportunistic fungal pathogen of humans.Phenotypic plasticity is a typical biological feature ofC.albicans, which is associated with pathogenicity, host adaptation, and sexual reproduction.BiofilmofC.albicansis a complex community formed by different morphological types of cells(yeast, hyphae and pseudohyphae)and secreted extracellular matrix.C.albicansbiofilms are intrinsically resistant to antifungal drugs, the host immune system, and environmental stresses.Biofilm is an important virulence factor and a major clinical challenge.With the development of new technologies in global gene expression profiles and genetic manipulation, the regulatory mechanisms that governC.albicansbiofilm development and drug resistance become more and more clear.Major regulatory mechanisms involve the MAPK and cAMP signaling pathways and transcriptional regulators such as Bcr1 and Tec1.In addition, morphological transitions and sexual reproduction are also involved in the regulation of biofilm development.In this review, we focus on the genetic regulatory mechanisms of biofilm including the roles of cell-wall related proteins,transcription factors, and theMTLlocus.In the last section, we also summarize the mechanisms of drug resistance of biofilm inC.albicans.

Candida albicans, biofilm, regulatory mechanism,MTL, drug resistance

March28,2017;Accepted:July19,2017

s:Yujia Wei.Tel: +86-851-86774009, E-mail: weiyujia1@sina.com Guanghua Huang.Tel: +86-10-64806133, E-mail: huanggh@im.ac.cn

郭東東, 岳慧珍, 魏羽佳, 等.白念珠菌生物被膜形成的遺傳調(diào)控機(jī)制.生物工程學(xué)報(bào),2017,33(9):1567–1581.

Guo DD, Yue HZ, Wei YJ, et al.Genetic regulatory mechanisms ofCandida albicansbiofilm formation.Chin J Biotech,2017,33(9):1567–1581.

Supported by:the Special Foundation of Governor for Talents in Science and Technology and Education of Guizhou Province(No.[2011]28).

*These authors contributed equally to this work.

貴州省優(yōu)秀科技教育人才省長(zhǎng)專項(xiàng)資金(No.[2011]28號(hào))資助。

時(shí)間：2017-08-24

http://kns.cnki.net/kcms/detail/11.1998.Q.20170824.0944.002.html

魏羽佳 貴州醫(yī)科大學(xué)附屬醫(yī)院主任醫(yī)師、皮膚科副主任。長(zhǎng)期從事真菌性皮膚病和性病的中西醫(yī)結(jié)合治療研究，擅長(zhǎng)各種性傳播疾病、皮膚濕疹類皮膚病及癬菌病的診治。近年來(lái)，致力于臨床皮膚及侵襲性真菌致病機(jī)理及真菌耐藥機(jī)理研究。

黃廣華 中國(guó)科學(xué)院微生物研究所研究員、博士生導(dǎo)師。2012年入選中科院“百人計(jì)劃”，2016年獲國(guó)家自然科學(xué)基金委員會(huì)“杰出青年科學(xué)基金”資助。近年來(lái)主要從事人體致病真菌白念珠菌形態(tài)發(fā)生、有性生殖和分子致病機(jī)理等方面研究，在PLoS Biology、PLoS Genetics和Molecular Microbiology等國(guó)際著名雜志上發(fā)表了一系列文章。