寧年智，王慧

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王慧 軍事醫(yī)學(xué)研究院微生物流行病研究所研究員，博士生導(dǎo)師。主要從事病原微生物進(jìn)化、致病機(jī)理與防治基礎(chǔ)研究，先后主持和承擔(dān)國(guó)家傳染病重大專項(xiàng)、國(guó)家新藥創(chuàng)制重大專項(xiàng)、國(guó)家973計(jì)劃、國(guó)家重點(diǎn)研發(fā)計(jì)劃、國(guó)家自然科學(xué)基金課題20余項(xiàng)。在高致病病原新毒力因子發(fā)現(xiàn)、病原菌適應(yīng)性進(jìn)化以及感染防治技術(shù)方面取得重要進(jìn)展。研究成果在、、、、等國(guó)內(nèi)外學(xué)術(shù)期刊發(fā)表論文80余篇。獲得中國(guó)發(fā)明專利17項(xiàng)，獲得美國(guó)、日本、澳大利亞、歐洲等國(guó)際發(fā)明專利5項(xiàng)?，F(xiàn)兼任全軍科學(xué)技術(shù)委員會(huì)生物技術(shù)專業(yè)委員會(huì)常委。

細(xì)菌自然轉(zhuǎn)化的機(jī)制及影響因素

寧年智，王慧

軍事科學(xué)院軍事醫(yī)學(xué)研究院 微生物流行病研究所 病原微生物生物安全國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100071

寧年智, 王慧. 細(xì)菌自然轉(zhuǎn)化的機(jī)制及影響因素. 生物工程學(xué)報(bào), 2018, 34(8): 1297–1305.Ning NZ, Wang H. Mechanism and influencing factors of natural transformation in bacteria. Chin J Biotech, 2018, 34(8): 1297–1305.

水平基因轉(zhuǎn)移對(duì)耐藥基因傳播、編碼毒素基因質(zhì)粒的擴(kuò)散和毒力島的轉(zhuǎn)移等過程具有重要的生物學(xué)意義。自然轉(zhuǎn)化是指具有感受態(tài)的細(xì)菌從外界攝取并整合裸露DNA，是水平基因轉(zhuǎn)移的方式之一。細(xì)菌發(fā)生自然轉(zhuǎn)化極大地促進(jìn)了耐藥基因在不同細(xì)菌間的播散，導(dǎo)致細(xì)菌對(duì)抗生素耐藥，給臨床治療帶來極大的困難。許多細(xì)菌具備自然轉(zhuǎn)化能力，但不同細(xì)菌自然轉(zhuǎn)化過程存在著差異。細(xì)菌自然感受及轉(zhuǎn)化的誘發(fā)及效率亦受到多種因素的影響。文中著重于闡述不同細(xì)菌的自然轉(zhuǎn)化機(jī)制及其影響因素。

自然感受態(tài)，自然轉(zhuǎn)化，Ⅳ型菌毛，耐藥基因

細(xì)菌間水平基因轉(zhuǎn)移主要有3種機(jī)制，包括接合 (Conjugation)、轉(zhuǎn)導(dǎo) (Transduction) 和自然轉(zhuǎn)化 (Natural transformation)。自然轉(zhuǎn)化是指處于感受態(tài)的細(xì)菌從外界攝取并整合裸露DNA (比如噬菌體、質(zhì)粒和染色質(zhì)) 的過程。自然感受 (Natural competence) 是自然轉(zhuǎn)化過程中的重要一環(huán)，是指細(xì)菌在自然狀態(tài)下能夠從外界獲取裸露DNA并轉(zhuǎn)運(yùn)至細(xì)胞質(zhì)中。細(xì)菌中普遍存在著自然感受和轉(zhuǎn)化現(xiàn)象，并且已經(jīng)有許多文章在這方面進(jìn)行論述[1-3]。自然轉(zhuǎn)化與臨床細(xì)菌感染存在密切關(guān)系。首先細(xì)菌可通過自然轉(zhuǎn)化途徑獲取毒力因子。在1928年，Griffith等在肺炎鏈球菌的毒性轉(zhuǎn)化實(shí)驗(yàn)中發(fā)現(xiàn)了自然轉(zhuǎn)化現(xiàn)象的存在，無毒性的肺炎鏈球菌菌株可轉(zhuǎn)化有毒株的DNA從而可獲取毒力因子[4]。Tribble等研究證明牙齦卟啉單胞菌間主要通過自然感受及轉(zhuǎn)化進(jìn)行DNA交換，形成細(xì)菌基因多樣性，有助于該菌在人體內(nèi)生存并逃避機(jī)體免疫[5]。其次細(xì)菌可通過自然轉(zhuǎn)化途徑獲取耐藥基因。鮑曼不動(dòng)桿菌是我國(guó)醫(yī)院內(nèi)感染的主要致病菌之一，主要引起獲得性肺炎尤其是呼吸機(jī)相關(guān)性肺炎、菌血癥、尿路感染、繼發(fā)性腦膜炎等。近年來，鮑曼不動(dòng)桿菌的耐藥率逐年上升，甚至出現(xiàn)了泛耐藥或者全耐藥菌株[6-8]。多數(shù)耐藥鮑曼不動(dòng)桿菌含有耐藥質(zhì)粒，對(duì)耐藥質(zhì)粒進(jìn)行測(cè)序后發(fā)現(xiàn)大部分質(zhì)粒缺少質(zhì)粒接合所需要的轉(zhuǎn)移基因和移動(dòng)基因，表明鮑曼不動(dòng)桿菌部分耐藥質(zhì)粒不是通過接合作用進(jìn)行播散，而可能是通過自然轉(zhuǎn)化途徑[9]。鑒于自然轉(zhuǎn)化與細(xì)菌進(jìn)化的密切關(guān)系，越來越多研究者投身于細(xì)菌自然轉(zhuǎn)化的研究當(dāng)中。本綜述的主要目的是介紹細(xì)菌自然轉(zhuǎn)化機(jī)制及其影響因素的研究進(jìn)展。

1 致病菌的自然轉(zhuǎn)化現(xiàn)象

霍亂弧菌是人類霍亂的病原體，可引起烈性腸道傳染病。研究發(fā)現(xiàn)其在浮游生物的幾丁質(zhì)外骨骼表面生長(zhǎng)時(shí)可從外界攝取DNA并重組至基因組中[10]?；魜y弧菌可通過自然轉(zhuǎn)化獲取毒力因子，Blokesch等的實(shí)驗(yàn)表明霍亂弧菌O1亞型獲取O抗原基因簇后可轉(zhuǎn)變?yōu)镺139亞型[11]。實(shí)驗(yàn)條件下霍亂弧菌A1552菌株的自然轉(zhuǎn)化效率可達(dá)2×10–4[12]。另一種具備自然轉(zhuǎn)化能力的常見致病菌是腦膜炎奈瑟菌[13]，該菌是一種人類共生菌及病原菌，在人體缺乏細(xì)菌抗體時(shí)可導(dǎo)致腦膜炎或菌血癥[14]。不動(dòng)桿菌屬sp.細(xì)菌是條件致病菌，常見于醫(yī)院內(nèi)感染，被認(rèn)為均具有自然感受能力[15]。近年來，不動(dòng)桿菌由于出現(xiàn)多重耐藥甚至是全耐藥菌株而受到廣泛關(guān)注。研究不動(dòng)桿菌的自然感受與轉(zhuǎn)化時(shí)，大部分實(shí)驗(yàn)是以不動(dòng)桿菌BD413菌株 (BD413曾用名稱是草酸鈣不動(dòng)桿菌BD413[16]) 作為代表菌。不動(dòng)桿菌BD413作為研究常用的對(duì)象是因?yàn)槠渚哂泻芨叩淖匀晦D(zhuǎn)化效率，大約為0.1%–0.7%[17]，同時(shí)該菌攝取的裸露DNA無序列特異性，DNA以單鏈形式進(jìn)入細(xì)胞[18-19]。相反地，已有的研究表明鮑曼不動(dòng)桿菌A118菌株對(duì)外源質(zhì)粒 (pDsRed) 或外源gDNA的自然轉(zhuǎn)化效率大約為2×10–8[20]；鮑曼不動(dòng)桿菌M2菌株對(duì)外源質(zhì)粒 (pGEM) 的自然轉(zhuǎn)化效率約為2×10–7[21]。顯而易見，鮑曼不動(dòng)桿菌的自然轉(zhuǎn)化率顯著低于不動(dòng)桿菌BD413的自然轉(zhuǎn)化率。Wilharm等對(duì)10株臨床分離的鮑曼不動(dòng)桿菌進(jìn)行了自然轉(zhuǎn)化研究，發(fā)現(xiàn)其轉(zhuǎn)化gDNA的效率均顯著高于轉(zhuǎn)化質(zhì)粒的效率[22]。Ramirez等還發(fā)現(xiàn)鮑曼不動(dòng)桿菌A118菌株對(duì)5種不同質(zhì)粒的轉(zhuǎn)化率有明顯差異，轉(zhuǎn)化效率為7.4×102–26.9×102CFU/μg DNA。與大腸桿菌相比，經(jīng)過長(zhǎng)期傳代培養(yǎng)后被轉(zhuǎn)化質(zhì)粒在鮑曼不動(dòng)桿菌A118菌株內(nèi)更不容易丟失，具備更強(qiáng)的穩(wěn)定性，從而增加了基因被整合至細(xì)菌染色質(zhì)的概率[23]。

2 影響自然轉(zhuǎn)化的基因

2.1 Ⅳ型菌毛參與外源DNA攝取

自然轉(zhuǎn)化過程的第一步是外源裸露DNA從細(xì)胞表面穿過細(xì)胞外膜進(jìn)入到細(xì)胞周間質(zhì)中。Ⅳ型菌毛是細(xì)菌表面常見的附屬結(jié)構(gòu)，現(xiàn)有研究表明施氏假單胞菌、霍亂弧菌、肺炎鏈球菌、淋病奈瑟菌和腦膜炎奈瑟菌等細(xì)菌的Ⅳ型菌毛系統(tǒng)參與細(xì)胞攝取外源DNA的過程[24-28]。比如在腦膜炎奈瑟菌中，Ⅳ型菌毛從位于外膜上的分泌蛋白pilQ的孔徑中穿過，菌毛在延伸與收縮的過程中，使pilQ蛋白打開通道，外源DNA可進(jìn)入該通道并與通道中相應(yīng)受體結(jié)合后被轉(zhuǎn)運(yùn)進(jìn)細(xì)胞周間質(zhì)[28-29]。但是幽門螺桿菌則是由Ⅳ型分泌系統(tǒng)介導(dǎo)外源DNA轉(zhuǎn)運(yùn)穿越細(xì)胞外膜，菌毛直接與細(xì)菌表面的DNA結(jié)合，通過旋轉(zhuǎn)等方式直接將DNA轉(zhuǎn)運(yùn)至細(xì)胞周間質(zhì)[30]。在鮑曼不動(dòng)桿菌中，外源DNA如何穿過細(xì)胞外膜進(jìn)入細(xì)胞周間質(zhì)？2007年Smith等對(duì)鮑曼不動(dòng)桿菌ATCC 17978菌株進(jìn)行全基因組測(cè)序后發(fā)現(xiàn)，在該菌株中存在多個(gè)與轉(zhuǎn)化相關(guān)的基因，并預(yù)測(cè)這些基因能夠編碼類Ⅳ型菌毛系統(tǒng)的結(jié)構(gòu)蛋白[31]。另外，2011年有研究對(duì)已發(fā)表的鮑曼不動(dòng)桿菌基因組進(jìn)行分析，發(fā)現(xiàn)在鮑曼不動(dòng)桿菌基因組中存在著許多預(yù)測(cè)為Ⅳ型菌毛蛋白的基因，這些基因編碼的蛋白與其他細(xì)菌中的Ⅳ型菌毛蛋白具有同源性[32]。同年，Eijkelkamp等報(bào)道鮑曼不動(dòng)桿菌ATCC 17978菌株在缺乏鐵離子的培養(yǎng)環(huán)境中失去了蹭動(dòng) (Twitching motility) 功能 (需Ⅳ型菌毛參與)，其原因是一系列疑似Ⅳ型菌毛蛋白的編碼基因表達(dá)量輕微下調(diào)[33]。隨后的研究表明，鮑曼不動(dòng)桿菌臨床菌株可能普遍存在Ⅳ型菌毛結(jié)構(gòu)。在Eijkelkamp等的研究中，所有國(guó)際克?、裥图安糠中驴寺⌒王U曼不動(dòng)桿菌菌株均表現(xiàn)出蹭動(dòng)能力，表明這些菌株可能產(chǎn)生了Ⅳ型菌毛[34]；Ramirez等確認(rèn)一株分離自血液的鮑曼不動(dòng)桿菌A118具備自然轉(zhuǎn)化能力，但并未研究其自然轉(zhuǎn)化機(jī)制[23]。直到2013年，Harding等[21]和Wilharm等[22]分別通過實(shí)驗(yàn)證明了Ⅳ型菌毛參與鮑曼不動(dòng)桿菌自然轉(zhuǎn)化。Ⅳ型菌毛是細(xì)菌表面的一種多蛋白附屬結(jié)構(gòu)，可見于多種革蘭氏陰性桿菌[35-36]。Ⅳ型菌毛通常處于動(dòng)態(tài)平衡狀態(tài)，可快速地聚合與解聚合，參與細(xì)菌的自然轉(zhuǎn)化、蹭動(dòng)和粘附 (Adherence) 等生物過程。基因編碼Ⅳ型菌毛解聚合所需的ATPase，淋病奈瑟菌和銅綠假單胞菌的基因缺失后，Ⅳ型菌毛無法收縮和解聚合，在細(xì)菌表面形成多菌毛狀態(tài)從而可利用顯微鏡直接觀察其結(jié)構(gòu)[37-38]。對(duì)鮑曼不動(dòng)桿菌的基因進(jìn)行缺失突變，致使Ⅳ型菌毛失去功能，可在菌體表面觀察到菌毛結(jié)構(gòu)，同時(shí)菌株也失去了自然轉(zhuǎn)化能力，表明Ⅳ型菌毛參與鮑曼不動(dòng)桿菌的自然轉(zhuǎn)化過程[21-22]。Harding等的研究還證明鮑曼不動(dòng)桿菌M2菌株的Ⅳ型菌毛的主體亞單位是由基因所編碼，當(dāng)基因失活后，鮑曼不動(dòng)桿菌M2表面的菌毛結(jié)構(gòu)消失；、和基因均參與了Ⅳ型菌毛的合成與裝配[21]。

2.2 ComEC通道參與DNA穿越細(xì)胞內(nèi)膜進(jìn)入細(xì)胞質(zhì)

在細(xì)胞周間質(zhì)內(nèi)的DNA穿越細(xì)胞內(nèi)膜進(jìn)入細(xì)胞質(zhì)是自然轉(zhuǎn)化的第2個(gè)步驟。研究發(fā)現(xiàn)，在所有自然感受細(xì)菌中這一過程均由高度保守的細(xì)胞內(nèi)膜通道蛋白ComEC介導(dǎo)[28,39]。與外膜轉(zhuǎn)運(yùn)DNA的方式不同，由于細(xì)胞內(nèi)膜參與形成并保持細(xì)菌與外界環(huán)境的離子濃度差，所以細(xì)胞內(nèi)膜不能通過松散的通道進(jìn)行DNA轉(zhuǎn)運(yùn)?？莶菅堪麠U菌的ComEC膜通道被研究得比較透徹，其ComEC蛋白的感受結(jié)構(gòu)域高度保守，形成的通道被水填充，DNA可通過該通道進(jìn)入細(xì)胞質(zhì)[39]。在不同細(xì)菌種屬中，ComEC蛋白其他區(qū)域會(huì)發(fā)生細(xì)菌種屬特異性變化[39]。研究發(fā)現(xiàn)，基因參與不動(dòng)桿菌BD413的自然轉(zhuǎn)化，而基因的氨基酸序列與枯草芽胞桿菌的基因的氨基酸序列具有高度同源性，表明基因可能參與了不動(dòng)桿菌BD413將外源DNA從細(xì)胞周間質(zhì)轉(zhuǎn)運(yùn)至細(xì)胞內(nèi)部的過程[40]；全基因組測(cè)序發(fā)現(xiàn)具有自然轉(zhuǎn)化能力的鮑曼不動(dòng)桿菌A118菌株亦攜帶基因[41]；Wilharm等將多株鮑曼不動(dòng)桿菌臨床菌株的基因敲除后，未能檢測(cè)到缺失突變株在半固體培養(yǎng)基生長(zhǎng)時(shí)轉(zhuǎn)化外源質(zhì)粒[22]。遺憾的是，目前仍未有報(bào)道闡述鮑曼不動(dòng)桿菌ComEC通道轉(zhuǎn)運(yùn)外源DNA的具體機(jī)制。外源DNA穿越細(xì)胞內(nèi)膜前，不同細(xì)菌通過不同的方式在細(xì)胞周間質(zhì)內(nèi)轉(zhuǎn)運(yùn)DNA。在霍亂弧菌中，進(jìn)入細(xì)胞周間質(zhì)內(nèi)的外源DNA首先與DNA結(jié)合蛋白ComEA結(jié)合，ComEA蛋白推動(dòng)DNA以單鏈形式 (ssDNA) 穿越ComEC通道[25,42]。幽門螺桿菌則不依賴于ComEA蛋白，該菌在攝取外源DNA時(shí)，由Ⅳ型分泌系統(tǒng)完成細(xì)胞周間質(zhì)內(nèi)的DNA轉(zhuǎn)運(yùn)，最終通過ComEC通道進(jìn)入細(xì)胞質(zhì)[43]。

2.3 DprA基因參與自然轉(zhuǎn)化

在自然轉(zhuǎn)化過程中，進(jìn)入細(xì)胞質(zhì)內(nèi)的ssDNA需要多種蛋白輔助其重組至染色質(zhì)上。比較基因組學(xué)研究發(fā)現(xiàn)所有具備轉(zhuǎn)化能力的細(xì)菌都存在著基因[44]?；蛟诖蟛糠旨?xì)菌的自然轉(zhuǎn)化過程中均起到重要作用：實(shí)驗(yàn)表明該基因缺失后，細(xì)菌自然轉(zhuǎn)化效率發(fā)生顯著變化，變化幅度與細(xì)菌種類和DNA底物種類相關(guān)[13,45-49]。細(xì)菌通過同源重組將基因整合至染色質(zhì)的過程依賴于基因。Quevillon-Cheruel等研究發(fā)現(xiàn)DprA蛋白與RecA蛋白相互作用是肺炎鏈球菌發(fā)生轉(zhuǎn)化的基礎(chǔ)，同時(shí)自然轉(zhuǎn)化的效率與DprA蛋白和RecA相互作用并形成穩(wěn)定復(fù)合體的能力密切相關(guān)[50]。DprA蛋白還與ssDNA結(jié)合，避免其被細(xì)胞內(nèi)的核酸酶降解[51]。在枯草芽胞桿菌中，依賴于的染色質(zhì)轉(zhuǎn)化和非依賴于的質(zhì)粒轉(zhuǎn)化途徑都需要基因的參與。該菌在發(fā)生染色質(zhì)轉(zhuǎn)化時(shí)，細(xì)胞質(zhì)內(nèi)的單鏈結(jié)合蛋白SsbB和SsbA首先結(jié)合至ssDNA上并解除其二級(jí)結(jié)構(gòu)，同時(shí)阻止RecA蛋白裝配至ssDNA上。DprA蛋白存在時(shí)，其可結(jié)合至ssDNA并使單鏈結(jié)合蛋白SsbB和SsbA脫離，促發(fā)RecA介導(dǎo)的DNA鏈交換，導(dǎo)致同源重組；在轉(zhuǎn)化質(zhì)粒時(shí)，DprA蛋白介導(dǎo)質(zhì)粒單鏈退火形成完整的環(huán)狀質(zhì)粒結(jié)構(gòu)[52]。

3 影響自然轉(zhuǎn)化的外部因素及調(diào)控

3.1 營(yíng)養(yǎng)補(bǔ)給提升不動(dòng)桿菌的自然轉(zhuǎn)化

早期研究發(fā)現(xiàn)，不動(dòng)桿菌BD413菌株的自然感受與轉(zhuǎn)化能力與環(huán)境中的營(yíng)養(yǎng)狀態(tài)呈正相關(guān)。Palmen等發(fā)現(xiàn)剛剛進(jìn)入生長(zhǎng)對(duì)數(shù)期時(shí)，細(xì)菌變成感受狀態(tài)，直到進(jìn)入生長(zhǎng)平臺(tái)期數(shù)小時(shí)，即營(yíng)養(yǎng)貧乏后，自然感受能力才逐漸下降[18]。其他研究者也得出相同的結(jié)論，當(dāng)不動(dòng)桿菌BD413菌株的過夜培養(yǎng)物被接種至新鮮培養(yǎng)基時(shí)，細(xì)菌的自然轉(zhuǎn)化效率達(dá)到最高峰[53]?；蚝突蚴遣粍?dòng)桿菌自然轉(zhuǎn)化的必需基因，但研究發(fā)現(xiàn)這兩個(gè)基因的轉(zhuǎn)錄水平與自然轉(zhuǎn)化并非正相關(guān)[53-54]?；虻谋磉_(dá)水平在培養(yǎng)的靜止末期達(dá)到高峰[53]；基因的轉(zhuǎn)錄隨著生長(zhǎng)周期而改變并在靜止末期達(dá)到高峰[54]。綜合以上數(shù)據(jù)，研究者認(rèn)為細(xì)菌在靜止末期已經(jīng)具備自然轉(zhuǎn)化的基礎(chǔ)；將細(xì)菌接種至新鮮培養(yǎng)基可為其提供足夠的能量，從而發(fā)生自然轉(zhuǎn)化[53-54]。對(duì)于鮑曼不動(dòng)桿菌而言，提升營(yíng)養(yǎng)亦可誘發(fā)其自然感受與轉(zhuǎn)化。在研究鮑曼不動(dòng)桿菌自然轉(zhuǎn)化時(shí)，研究者發(fā)現(xiàn)鮑曼不動(dòng)桿菌M2菌株和鮑曼不動(dòng)桿菌A118菌株在富營(yíng)養(yǎng)狀態(tài)下成為感受狀態(tài)并發(fā)生自然轉(zhuǎn)化[20-21]，但其具體機(jī)制尚未見報(bào)道。在1993年，Palmen等發(fā)現(xiàn)培養(yǎng)環(huán)境中的Ca2+離子濃度可影響不動(dòng)桿菌BD413菌株的自然轉(zhuǎn)化。此后雖然缺乏其他菌種的實(shí)驗(yàn)驗(yàn)證，但這一結(jié)論被認(rèn)為適用于整個(gè)不動(dòng)桿菌屬。直到2016年，Traglia等研究發(fā)現(xiàn)提升培養(yǎng)環(huán)境中的Ca2+離子濃度或添加適量牛血清白蛋白 (0.2%) 可提升鮑曼不動(dòng)桿菌的自然轉(zhuǎn)化效率；此時(shí)鮑曼不動(dòng)桿菌的基因及基因表達(dá)量都顯著上調(diào)[20]。

3.2 幾丁質(zhì)能誘發(fā)霍亂弧菌自然轉(zhuǎn)化

霍亂弧菌常見于河流、湖泊等水域環(huán)境中的甲殼浮游生物的表面，并可降解甲殼和利用其中的幾丁質(zhì)作為碳源進(jìn)行生命活動(dòng)[55]。2005年，Meibom等首先報(bào)道了霍亂弧菌的自然轉(zhuǎn)化現(xiàn)象，霍亂弧菌在幾丁質(zhì)表面生長(zhǎng)時(shí)可從外界攝取裸露的DNA并整合至基因組上[10]。幾丁質(zhì)可被降解為可溶性的幾丁質(zhì)低聚體，低聚體可被霍亂弧菌的組氨酸激酶感受器ChiS識(shí)別，從而促進(jìn)和基因的表達(dá)?；蚓幋a一類小調(diào)控RNA，該RNA可促進(jìn)tofX mRNA的翻譯，產(chǎn)物蛋白能誘導(dǎo)自然轉(zhuǎn)化相關(guān)基因的表達(dá)[3,56]。有證據(jù)表明幾丁質(zhì)誘發(fā)的自然轉(zhuǎn)化與霍亂毒素質(zhì)粒的播散密切相關(guān)[57]。

3.3 DNA損傷促進(jìn)幽門螺桿菌的自然轉(zhuǎn)化

幽門螺桿菌是一種感染率非常高的人體致病菌，在所有生長(zhǎng)周期均表現(xiàn)出固有的自然轉(zhuǎn)化能力[58]。幽門螺桿菌的基因重組頻率非常高，導(dǎo)致其具備明顯的基因多樣性[59]。目前唯一被證實(shí)能促進(jìn)幽門螺桿菌自然轉(zhuǎn)化的因素是DNA損傷。環(huán)丙沙星是一種常用抗生素，可導(dǎo)致細(xì)菌DNA雙鏈斷裂。幽門螺桿菌的自然轉(zhuǎn)化依賴于T4SS 系統(tǒng)，Dorer等發(fā)現(xiàn)細(xì)菌經(jīng)環(huán)丙沙星處理后，部分T4SS基因的轉(zhuǎn)錄顯著上調(diào)，同時(shí)自然轉(zhuǎn)化效率提升4–5倍[60]?？股睾妥贤饩€導(dǎo)致的DNA損傷亦可誘發(fā)嗜肺軍團(tuán)菌的自然轉(zhuǎn)化[61]。

3.4 營(yíng)養(yǎng)匱乏促進(jìn)流感嗜血桿菌自然轉(zhuǎn)化

流感嗜血桿菌感染人體時(shí)，其定植部位多為上呼吸道，該部位可供細(xì)菌利用的營(yíng)養(yǎng)和能量源比較貧乏。流感嗜血桿菌的自然轉(zhuǎn)化誘發(fā)或許與缺乏理想的碳源相關(guān)，早有文獻(xiàn)報(bào)道該菌在生長(zhǎng)進(jìn)入靜止期或者被接種至乏養(yǎng)培養(yǎng)基時(shí)可誘發(fā)自然轉(zhuǎn)化[62-63]?；蚴橇鞲惺妊獥U菌自然轉(zhuǎn)化的上游調(diào)控因子，該基因與其他感受態(tài)細(xì)菌的基因具有同源性[63]。在營(yíng)養(yǎng)匱乏狀態(tài)下，該基因的表達(dá)上調(diào)，從而導(dǎo)致下游處于13個(gè)操縱子控制下的共26個(gè)感受態(tài)基因 (比如) 的轉(zhuǎn)錄顯著提高[62,64-66]。

3.5 影響細(xì)菌自然轉(zhuǎn)化的其他因素

淋病奈瑟菌的自然轉(zhuǎn)化效率在接近人體溫度 (37 ℃) 時(shí)達(dá)到最大值，但是其調(diào)控機(jī)制目前尚不清楚[67]。Wilharm等對(duì)28株鮑曼不動(dòng)桿菌臨床菌株進(jìn)行自然轉(zhuǎn)化研究，發(fā)現(xiàn)有10株菌可在含0.5%瓊脂的半固體培養(yǎng)基生長(zhǎng)時(shí)發(fā)生自然轉(zhuǎn)化，但是當(dāng)瓊脂的濃度提升到1.5%后，未能檢測(cè)到細(xì)菌發(fā)生自然轉(zhuǎn)化[22]。最近，Traglia等系統(tǒng)地研究了影響鮑曼不動(dòng)桿菌自然轉(zhuǎn)化的因素。研究發(fā)現(xiàn)，溫度和滲透壓并不能影響鮑曼不動(dòng)桿菌A118菌株的自然轉(zhuǎn)化；相反pH值對(duì)自然轉(zhuǎn)化有顯著影響，當(dāng)實(shí)驗(yàn)pH值靠近血液的pH值 (pH 7.35–7.45) 時(shí)自然轉(zhuǎn)化效率最高，自然轉(zhuǎn)化效率隨著pH值的降低而降低[20]。

4 小結(jié)與展望

自然轉(zhuǎn)化在細(xì)菌獲取耐藥基因及毒力因子的過程中起到重要作用。在自然轉(zhuǎn)化過程中，大部分細(xì)菌是通過Ⅳ型菌毛系統(tǒng)將外源DNA從細(xì)菌表面轉(zhuǎn)運(yùn)至細(xì)胞周間質(zhì)，然后通過ComEC通道將位于細(xì)胞周間質(zhì)內(nèi)的外源DNA繼續(xù)轉(zhuǎn)運(yùn)至細(xì)胞質(zhì)內(nèi)，最終外源DNA在DprA蛋白的作用下在細(xì)菌內(nèi)獨(dú)立表達(dá)或者整合至染色質(zhì)中。細(xì)菌的自然轉(zhuǎn)化受到營(yíng)養(yǎng)條件、外界物理與化學(xué)因素的影響。

細(xì)菌的自然轉(zhuǎn)化是一個(gè)復(fù)雜的過程，需要多系統(tǒng)協(xié)調(diào)共同參與，不同細(xì)菌的自然轉(zhuǎn)化機(jī)制具有差異。目前細(xì)菌自然轉(zhuǎn)化的機(jī)制研究多限于抗生素敏感菌株，尚缺少合適的技術(shù)手段對(duì)多重耐藥菌株的自然轉(zhuǎn)化進(jìn)行研究。自然轉(zhuǎn)化導(dǎo)致細(xì)菌耐藥，甚至形成多重耐藥，多重耐藥菌株可能具有更強(qiáng)自然轉(zhuǎn)化能力，對(duì)這類細(xì)菌進(jìn)行研究往往更具有臨床意義。探究細(xì)菌感受態(tài)的誘發(fā)因素及作用機(jī)制是未來研究的重要方向，可為臨床實(shí)踐中控制細(xì)菌耐藥發(fā)展提供借鑒；外源DNA穿越細(xì)胞內(nèi)膜時(shí)以ssDNA的形式進(jìn)入，在DNA上共價(jià)結(jié)合額外基團(tuán)可阻斷該步驟，導(dǎo)致無法直接觀察該過程，因此dsDNA如何被加工成ssDNA并轉(zhuǎn)運(yùn)進(jìn)細(xì)胞質(zhì)的過程仍有待探究；Ⅳ型菌毛合成與裝配機(jī)制仍有待研究。解決這些問題可加強(qiáng)我們對(duì)細(xì)菌自然轉(zhuǎn)化機(jī)制的了解。

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(本文責(zé)編 陳宏宇)

Mechanism and influencing factors of natural transformation in bacteria

Nianzhi Ning, and Hui Wang

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Academy of Military Medical Sciences, Academy of Military Sciences, Beijing100071, China

Horizontal gene transfer contributes to the spread of antibiotic-resistance cassettes, the distribution of toxin-encoding phages and the transfer of pathogenicity islands. Natural transformation, which is the process of competent cells to uptake free DNA from environment and to recombine this DNA into the chromosome, is a mode of horizontal gene transfer. Natural transformation promotes the spread of antibiotic-resistance cassettes among different bacteria, resulting in the emergence of antibiotic resistant bacteria. The emergence of antibiotic resistant pathogens poses an enormous threat to the treatment of infections. Natural transformation could occur in many bacteria, but the mechanism many be different in different bacteria. Also, the inducer and efficiency of natural transformation in different bacteria are influenced by various factors. This review focuses on the mechanism and influencing factors of natural transformation in bacteria.

natural competence, natural transformation, type Ⅳ pilus, antibiotic resistant genes

January 4, 2018;

April 9, 2018

National Basic Research Program of China (973 Program) (No. 2015CB554202).

Hui Wang. Tel/Fax: +86-10-66948587; E-mail: geno0109@vip.sina.com

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃 (973計(jì)劃) (No. 2015CB554202) 資助。

10.13345/j.cjb.180005