楊鳳環(huán), 田 芳, 陳華民, 何晨陽

(中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所, 植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室, 北京 100193)

病原細(xì)菌受體介導(dǎo)的c-di-GMP信號傳導(dǎo)及其調(diào)控機(jī)制

楊鳳環(huán), 田 芳, 陳華民, 何晨陽*

(中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所, 植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室, 北京 100193)

細(xì)菌第二信使環(huán)二鳥苷酸(c-di-GMP)信號網(wǎng)絡(luò)系統(tǒng)主要涉及信號代謝、識別、接受、傳遞、功能表達(dá)和調(diào)控。c-di-GMP胞內(nèi)水平受到鳥苷酸環(huán)化酶(DGC)和磷酸二酯酶(PDE)的控制。c-di-GMP信號受體類型多樣,包括轉(zhuǎn)錄調(diào)控因子、PilZ結(jié)構(gòu)域蛋白、退化的GGDEF和EAL結(jié)構(gòu)域蛋白、核糖體開關(guān)、多核苷酸磷酸化酶和新發(fā)現(xiàn)的蛋白激酶等。c-di-GMP受體接受信號后,可以在轉(zhuǎn)錄、翻譯以及翻譯后水平上對下游靶標(biāo)進(jìn)行調(diào)控,從而影響細(xì)菌的毒性、運(yùn)動性、生物膜形成、細(xì)胞分裂等生理生化過程。本文結(jié)合本實(shí)驗(yàn)室對水稻白葉枯病菌的研究結(jié)果,綜述了近年來國內(nèi)外在c-di-GMP信號受體介導(dǎo)的調(diào)控機(jī)制等方面的研究進(jìn)展。

c-di-GMP; 信號代謝; 受體; 傳導(dǎo); 調(diào)控

在復(fù)雜的生境中,細(xì)菌需要感知、轉(zhuǎn)導(dǎo)、傳遞來自細(xì)胞內(nèi)外的信號。第二信使小分子核苷酸是細(xì)菌細(xì)胞信號傳遞的重要載體,可將外界信號轉(zhuǎn)換成胞內(nèi)受體可感知的化學(xué)小分子[1]。環(huán)二鳥苷酸(c-di-GMP)是20世紀(jì)80年代在葡糖酸醋酸桿菌中發(fā)現(xiàn)的纖維素合成酶異構(gòu)激活因子[2]。隨后的研究表明,c-di-GMP是細(xì)菌中普遍存在的全新第二信使[3-6]。含有GGDEF結(jié)構(gòu)域的鳥苷酸環(huán)化酶(DGC)和含有EAL或者HD-GYP結(jié)構(gòu)域的磷酸二酯酶(PDE)分別控制了信號合成和降解[7]。c-di-GMP信號分子可通過與受體結(jié)合,從而調(diào)控細(xì)菌生物膜形成、運(yùn)動性和毒性等重要的生物學(xué)表型[3-6]。c-di-GMP信號代謝、受體感應(yīng)及調(diào)控機(jī)制的研究是病原細(xì)菌研究領(lǐng)域的熱點(diǎn)之一。近年來,在c-di-GMP受體介導(dǎo)的調(diào)控機(jī)制方面取得了重要的研究進(jìn)展。本文結(jié)合本實(shí)驗(yàn)室對水稻白葉枯病菌Xanthomonasoryzaepv.oryzaec-di-GMP受體功能的研究結(jié)果,綜述了近年來國內(nèi)外該領(lǐng)域的最新進(jìn)展。

1 c-di-GMP信號途徑的組成

c-di-GMP信號網(wǎng)絡(luò)主要包括4個(gè)組成部分：含有GGDEF結(jié)構(gòu)域的DGC、含有EAL或者HD-GYP結(jié)構(gòu)域的PDE、信號受體以及下游靶標(biāo)蛋白[3-5](圖1)。在c-di-GMP信號途徑中,DGC或PDE可感應(yīng)來自胞內(nèi)外的環(huán)境信號,影響其c-di-GMP代謝酶活性,進(jìn)而影響胞內(nèi)c-di-GMP水平,下游通過信號受體的感應(yīng)、實(shí)現(xiàn)c-di-GMP信號的輸出,即調(diào)控下游基因或靶標(biāo)蛋白的表達(dá)[6]。c-di-GMP信號網(wǎng)絡(luò)的4個(gè)組成部分在細(xì)菌中廣泛存在。因此,不同細(xì)菌中的調(diào)節(jié)過程也復(fù)雜多樣。

圖1 細(xì)菌c-di-GMP信號調(diào)控網(wǎng)絡(luò)模式[3-6]Fig.1 Bacterial c-di-GMP signaling pathway[3-6]

2 c-di-GMP信號代謝的調(diào)控網(wǎng)絡(luò)

在葡糖酸醋酸桿菌Acetobacterxylinum研究中發(fā)現(xiàn),c-di-GMP可作為纖維素合成酶的異構(gòu)激活因子[2]。在細(xì)菌體內(nèi)c-di-GMP的代謝水平主要受到DGC和PDE的控制[8]。DGC可將兩分子GTP合成一分子c-di-GMP,PDE將c-di-GMP降解為線性的二核苷酸pGpG、進(jìn)而降解為GMP。許多DGC或PDE具有REC、PAS和HAMP等感應(yīng)結(jié)構(gòu)域,可感應(yīng)來自環(huán)境的小分子、氧化還原、光、磷酸化、營養(yǎng)和抗生素等信號,從而調(diào)控胞內(nèi)c-di-GMP水平[9]。一般細(xì)菌可同時(shí)編碼多個(gè)GGDEF和EAL結(jié)構(gòu)域蛋白[7]。目前已經(jīng)鑒定出多個(gè)具有DGC和PDE酶活性的相關(guān)蛋白,例如新月柄桿菌Caulobactercrescentus的鳥苷酸環(huán)化酶PleD、銅綠假單胞菌Pseudomonasaeruginosa的鳥苷酸環(huán)化酶WspR、DgcP和磷酸二酯酶DipA、霍亂弧菌Vibriocholerae的磷酸二酯酶VieA等[10-14]。水稻白葉枯病菌PXO99A共有26個(gè)c-di-GMP代謝相關(guān)蛋白,14個(gè)蛋白具有信號感應(yīng)結(jié)構(gòu)域。其中含有GGDEF、EAL和REC結(jié)構(gòu)域蛋白PdeR具有PDE活性,與上游組氨酸激酶PdeK組成一對雙組分調(diào)控系統(tǒng),參與c-di-GMP代謝和病菌毒性的調(diào)節(jié)[15]。

3 c-di-GMP信號受體介導(dǎo)的調(diào)控機(jī)制

c-di-GMP通過與信號受體結(jié)合,改變受體空間構(gòu)象及其功能,影響下游基因表達(dá)或相關(guān)酶活性,從而調(diào)控細(xì)菌生物膜形成、細(xì)胞分裂、運(yùn)動性及毒性因子表達(dá)等生理生化過程[1,3-5]。已鑒定的信號受體包括轉(zhuǎn)錄調(diào)控因子、 PilZ結(jié)構(gòu)域蛋白、退化的GGDEF和EAL結(jié)構(gòu)域蛋白、 核糖體開關(guān)、多核苷酸磷酸化酶(PNPase)以及新發(fā)現(xiàn)的蛋白激酶六類。c-di-GMP受體接受信號后可以在轉(zhuǎn)錄、翻譯以及翻譯后水平對下游靶標(biāo)進(jìn)行調(diào)控[5, 16]。

3.1 轉(zhuǎn)錄調(diào)控因子受體介導(dǎo)的轉(zhuǎn)錄調(diào)控作用

轉(zhuǎn)錄調(diào)控因子可作為c-di-GMP受體,通過與調(diào)控序列的結(jié)合調(diào)控基因轉(zhuǎn)錄,形成c-di-GMP轉(zhuǎn)錄水平上的調(diào)控網(wǎng)絡(luò)。銅綠假單胞菌FleQ 蛋白是第一個(gè)被鑒定的、作為信號受體的轉(zhuǎn)錄調(diào)控因子。FleQ不僅通過激活鞭毛合成基因轉(zhuǎn)錄、調(diào)控鞭毛的產(chǎn)生,而且通過AAA結(jié)構(gòu)域與c-di-GMP結(jié)合,抑制了FleQ與調(diào)控胞外多糖產(chǎn)生的pel啟動子結(jié)合,從而調(diào)控細(xì)菌胞外多糖產(chǎn)生[17]。另外一種作為c-di-GMP受體的轉(zhuǎn)錄調(diào)控因子為CRP 家族蛋白。CRP/FNR家族蛋白C端具有cNMP結(jié)構(gòu)域和N端具有HTH結(jié)構(gòu)域。與FleQ作用方式不同,與c-di-GMP的結(jié)合可使CRP 蛋白行使轉(zhuǎn)錄因子活性,增強(qiáng)其與靶標(biāo)DNA的結(jié)合作用。大腸桿菌EscherichiacoliCRP蛋白、霍亂弧菌CRP家族蛋白VpsT以及克雷伯氏肺炎桿菌KlebsiellapneumoniaePliZ結(jié)構(gòu)域轉(zhuǎn)錄因子MrkH與c-di-GMP結(jié)合后行使轉(zhuǎn)錄調(diào)控活性[18-22]；新洋蔥伯克霍爾德菌BurkholderiacenocepaciaCRP/FNR家族蛋白成員Bcam139則通過與c-di-GMP結(jié)合,加強(qiáng)了與靶標(biāo)DNA的結(jié)合作用[23]。

水稻白葉枯病菌轉(zhuǎn)錄調(diào)控因子Clpxoo蛋白與cAMP受體CRP蛋白同源,可與c-di-GMP結(jié)合,參與細(xì)菌鞭毛運(yùn)動性、胞外多糖產(chǎn)生、H2O2抗性以及毒性的調(diào)控[24-25]。采用ChIP-seq方法,從病菌基因組中鑒定了78個(gè)與Clpxoo結(jié)合的基因啟動子,這些基因的功能涉及毒性因子產(chǎn)生、核苷酸代謝、蛋白分泌、信號感應(yīng)以及鞭毛合成等(待發(fā)表資料)。

3.2 PilZ蛋白受體介導(dǎo)的翻譯后調(diào)節(jié)作用

PilZ結(jié)構(gòu)域是最早發(fā)現(xiàn)可以與c-di-GMP結(jié)合的結(jié)構(gòu)域,隨后試驗(yàn)進(jìn)一步從銅綠假單胞菌中鑒定出來,命名為PilZ結(jié)構(gòu)域[26]。PliZ結(jié)構(gòu)域中保守殘基RXXXR和D/NXSXXG是與c-di-GMP結(jié)合的關(guān)鍵位點(diǎn)[26]。c-di-GMP 與PilZ 蛋白結(jié)合后會引起蛋白結(jié)構(gòu)發(fā)生改變,從而激活受體；通過蛋白與蛋白的互作、調(diào)控目標(biāo)基因表達(dá)或蛋白活性和功能。YcgR和BcsA是兩個(gè)最早證實(shí)作為c-di-GMP 受體的PliZ家族成員。大腸桿菌和沙門氏菌YcgR通過與鞭毛的啟動成分FliG和FliM 互相作用調(diào)控運(yùn)動性,而BcsA在與c-di-GMP結(jié)合的情況下具有調(diào)控纖維素合成的酶活性[27]。細(xì)菌基因組通常編碼一個(gè)或多個(gè)PilZ結(jié)構(gòu)域蛋白,不同蛋白在與c-di-GMP結(jié)合時(shí)調(diào)控不同的生物學(xué)功能。銅綠假單胞菌有7個(gè)PilZ結(jié)構(gòu)域蛋白,其中2個(gè)參與調(diào)控細(xì)菌運(yùn)動性,而Alg44參與調(diào)控海藻酸的產(chǎn)生[28-29]；霍亂弧菌存在5個(gè)PilZ結(jié)構(gòu)域蛋白,其中3個(gè)參與調(diào)控細(xì)菌的運(yùn)動性以及生物膜的形成[30]。

水稻白葉枯病菌有3個(gè)PilZ蛋白,其中PXO_00049和PXO_02374具有保守的PilZ結(jié)構(gòu)域，作為受體與c-di-GMP結(jié)合,而PXO_02715不能直接與c-di-GMP結(jié)合[31]。3個(gè)PilZ蛋白對病菌毒性和運(yùn)動性的調(diào)控功能也不盡相同。例如PXO_00049負(fù)向調(diào)控毒性、正向調(diào)控運(yùn)動性,PXO_02374負(fù)向調(diào)控毒性和運(yùn)動性,PXO_02715正向調(diào)控毒性、負(fù)向調(diào)控運(yùn)動性。然而,這些PilZ蛋白均不參與病菌EPS產(chǎn)生和生物膜形成的調(diào)控。PXO_00049 和PXO_02374定位細(xì)菌兩極和中間位置,而PXO_02715卻定位于細(xì)菌周身。PXO_02374可能通過與下游蛋白的互作,參與病菌毒性調(diào)控(待發(fā)表資料)。

3.3 退化的GGDEF/EAL蛋白受體介導(dǎo)的蛋白質(zhì)-蛋白質(zhì)互作

一些具有退化的GGDEF或EAL結(jié)構(gòu)域蛋白不具有DGC或者PDE活性,但可通過GGDEF結(jié)構(gòu)域中變構(gòu)的I位點(diǎn)(RXXD殘基)、或者EAL結(jié)構(gòu)域中的接觸位點(diǎn)與c-di-GMP結(jié)合,行使信號受體的功能。新月柄桿菌GGDEF結(jié)構(gòu)域蛋白PopA、霍亂弧菌GGDEF結(jié)構(gòu)域蛋白CdgG都可通過其I位點(diǎn)與c-di-GMP結(jié)合,參與調(diào)控細(xì)菌細(xì)胞周期進(jìn)程、菌體褶皺狀態(tài)、生物膜形成以及運(yùn)動性[32-33]。最新研究發(fā)現(xiàn),在大腸桿菌、沙門氏菌SalmonellaLignieres和克雷白氏桿菌Klebsiellapneumoniae的BcsE蛋白GIL結(jié)構(gòu)域中,RxGD殘基與GGDEF結(jié)構(gòu)域中的I位點(diǎn)類似,同樣可與c-di-GMP結(jié)合[34]。熒光假單胞菌PseudomonasfluorescensLapD、銅綠假單胞菌和柑橘潰瘍病菌Xanthomonascampestrispv.citriFimX都可通過退化的EAL結(jié)構(gòu)域與c-di-GMP結(jié)合；FimX還可與下游退化的PilZ結(jié)構(gòu)域蛋白互作,與菌毛合成相關(guān)蛋白形成復(fù)合體,從而影響運(yùn)動性[35-38]。

水稻白葉枯病菌Filp蛋白含有退化的GGDEF和EAL結(jié)構(gòu)域,不具有c-di-GMP代謝酶活性,但是可以通過EAL結(jié)構(gòu)域與c-di-GMP進(jìn)行結(jié)合。Filp可通過與下游PilZ結(jié)構(gòu)域蛋白PXO_02715互作,調(diào)控了病菌毒性和致敏性[39]。最新的蛋白組學(xué)分析發(fā)現(xiàn),Filp和PXO_02715共調(diào)控了100多個(gè)下游靶標(biāo)蛋白的表達(dá),包括雙組分調(diào)控系統(tǒng)激酶、c-di-GMP代謝相關(guān)蛋白以及TonB類受體等(待發(fā)表資料)。這些蛋白功能的解析將有助于闡明c-di-GMP受體介導(dǎo)的毒性調(diào)控機(jī)理。

3.4 核糖體開關(guān)受體介導(dǎo)的轉(zhuǎn)錄后調(diào)節(jié)作用

核糖體開關(guān)(riboswitch)是一類存在于mRNA中的非編碼部分,作為c-di-GMP非蛋白類受體在細(xì)菌中普遍存在[40]。核糖體開關(guān)可直接結(jié)合c-di-GMP,導(dǎo)致mRNA二級結(jié)構(gòu)發(fā)生改變,從而調(diào)控基因轉(zhuǎn)錄過程的mRNA元件。目前已發(fā)現(xiàn)來自霍亂弧菌的Class I和來自艱難梭狀芽胞桿菌Clostridiumdifficile的Class II兩類核糖體受體[41-43],這兩類核糖體開關(guān)與c-di-GMP結(jié)合的保守序列和結(jié)構(gòu)完全不同,其中Class I是細(xì)菌中c-di-GMP信號主要的核糖體開關(guān)受體[41-42]。c-di-GMP與核糖體開關(guān)的結(jié)合,不僅豐富了c-di-GMP所調(diào)控的基因種類,也實(shí)現(xiàn)了c-di-GMP對相關(guān)基因的轉(zhuǎn)錄后調(diào)控。

3.5 PNPase受體介導(dǎo)的RNA代謝催化作用

多核苷酸磷酸化酶(PNPase)在細(xì)菌體內(nèi)可行使3′多聚核糖核苷酸聚合酶活性,或者3′-5′的核糖核酸外切酶活性,參與mRNA反轉(zhuǎn)錄和核糖體RNA前體合成[44-45]。在大腸桿菌中,PNPase可與DosC(氧感應(yīng)DGC)和DosP(氧感應(yīng)PDE)形成復(fù)合體。當(dāng)環(huán)境中氧信號水平降低時(shí),PNPase從與DosC-DosP的復(fù)合體上解離,DosC呈現(xiàn)DGC酶活性，增加了胞內(nèi)的c-di-GMP水平,PNPase與c-di-GMP結(jié)合激活其核糖核酸外切酶活性；當(dāng)環(huán)境中氧信號水平上升時(shí),DosC與DosP相互結(jié)合,激活DosP的PDE酶活性,降低c-di-GMP水平,PNPase失去催化能力[43]。PNPase通過與c-di-GMP的結(jié)合,調(diào)控了細(xì)菌中依賴于氧信號的RNA加工。

3.6 蛋白激酶受體介導(dǎo)的細(xì)胞分化調(diào)控作用

蛋白激酶CckA是在新月柄桿菌中最新發(fā)現(xiàn)的c-di-GMP受體。c-di-GMP可直接與CckA結(jié)合,從而抑制后者的蛋白激酶活性,促進(jìn)磷酸酶活性,加速其從激酶到磷酸酶的過渡[46]。在細(xì)菌分化階段,c-di-GMP利用對CckA的空間調(diào)控設(shè)置未來子細(xì)胞復(fù)制的不對稱。新月柄桿菌雙組分系統(tǒng)中反應(yīng)調(diào)控蛋白PleD、感應(yīng)激酶DivL、反應(yīng)調(diào)控蛋白DivK都參與細(xì)菌的分化調(diào)控[47],使CckA在不同的調(diào)控模式間轉(zhuǎn)換。當(dāng)PleD定位于游動細(xì)胞時(shí),PleD和DivK處于脫磷酸化狀態(tài),CckA與DivL直接作用激活下游的CtrA,從而阻礙復(fù)制的開始；當(dāng)感應(yīng)激酶DivJ出現(xiàn)在柄狀細(xì)胞時(shí),PleD和DivK磷酸化,使CckA進(jìn)入磷酸酶模式,不能激活CtrA。c-di-GMP在柄桿菌細(xì)菌中是一個(gè)細(xì)胞周期蛋白相似分子,可協(xié)調(diào)細(xì)胞形態(tài)形成過程中染色體的復(fù)制。此外,在根癌農(nóng)桿菌Agrobacteriumtumefaciens中也存在c-di-GMP通過全局調(diào)控因子調(diào)控細(xì)菌毒性和生存的機(jī)制[48]?？梢奵-di-GMP對細(xì)菌周期及其分化的調(diào)控可能是一個(gè)普遍的調(diào)控機(jī)制。

4 結(jié)語

c-di-GMP信號作為在細(xì)菌中廣泛存在的第二信使,在生物學(xué)進(jìn)程的調(diào)控中發(fā)揮了至關(guān)重要的作用,已經(jīng)成為細(xì)菌中研究最為廣泛和深入的信號分子之一。盡管對c-di-GMP信號代謝及其與受體互作的分子機(jī)制已經(jīng)有了一定的認(rèn)識,但是對于c-di-GMP新受體的結(jié)構(gòu)與功能鑒定、c-di-GMP信號在不同水平上的調(diào)控差異和級聯(lián)效應(yīng)、c-di-GMP與其他信號系統(tǒng)的整合等都是未來需要解決的關(guān)鍵科學(xué)問題。本實(shí)驗(yàn)室多年來的研究表明,在水稻白葉枯病菌中存在一個(gè)c-di-GMP信號產(chǎn)生/降解、識別、接受和傳遞、表型和功能表達(dá)的調(diào)控途徑。3種不同類型的信號受體(Filp、PilZ和Clpxoo)的鑒定及其介導(dǎo)的毒性調(diào)控機(jī)理的解析,為揭示水稻白葉枯病菌c-di-GMP信號分子機(jī)制奠定了堅(jiān)實(shí)的基礎(chǔ)(圖2)。進(jìn)一步運(yùn)用組學(xué)等方法,對信號受體的全局性調(diào)控作用以及它們在c-di-GMP信號網(wǎng)絡(luò)中的交互和協(xié)同作用進(jìn)行解析,可為全面闡明揭示水稻白葉枯病菌c-di-GMP信號途徑及其對毒性表達(dá)的調(diào)控機(jī)制提供更多的科學(xué)依據(jù)。

圖2 水稻白葉枯病菌c-di-GMP受體介導(dǎo)的調(diào)控作用模式Fig.2 A working model for c-di-GMP receptor-mediated regulation in Xanthomonas oryzae pv. oryzae

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(責(zé)任編輯：田 喆)

Cyclic di-GMP signal receptor-mediated regulation of bacterial behaviors

Yang Fenghuan, Tian Fang, Chen Huamin, He Chenyang

(StateKeyLaboratoryforBiologyofPlantDiseasesandInsectPests,InstituteofPlantProtection,ChineseAcademyofAgriculturalSciences,Beijing100193,China)

The bacterial second message c-di-GMP signaling pathways mainly involve signal metabolism, signal recognition by receptor, reception and transduction, and phenotype expression and regulation. The intracellular level of c-di-GMP is precisely controlled by diguanylate cyclases (DGC) and phosphodiesterase (PDE) via biosynthesis or degradation, respectively. Several c-di-GMP receptors have been identified and characterized from various bacterial species, including transcription regulator, PilZ-domain protein, degenerate GGDEF or EAL domain protein, polynucleotide phosphorylase (PNPase), riboswitch and kinase, etc. The c-di-GMP receptors exert their regulatory functions at the transcription, translation, and post-translation levels, and regulate multiple bacterial properties including virulence, biofilm formation, motility and cell division. The recent progresses in c-di-GMP receptor-mediated regulation of bacterial behaviors were here reviewed, in combination with some of our findings on the bacterial blight pathogen of riceXanthomonasoryzaepv.oryzae.

c-di-GMP; signal metabolism; receptor; transduction; regulation

2016-12-18

2016-12-21

國家自然科學(xué)基金(31671990,31400117,31370160,31100947)

Q 78

A

10.3969/j.issn.0529-1542.2017.01.002

* 通信作者 E-mail:hechenyang@caas.cn