枯草桿菌對氨茴環(huán)霉素的抗性

1、    枯草桿菌對氨茴環(huán)霉素的抗性        【 摘要 】目的研究枯草桿菌對氨茴環(huán)霉素(ANC)的抗性特征并克隆抗性片段。方法用遞增濃度方法在LB平板上篩選枯草桿菌菌株1831對ANC的自發(fā)抗性突變株，用鳥槍法在枯草桿菌BD224菌株原生質(zhì)體中克隆抗性重組質(zhì)粒，提取重組質(zhì)粒后再次轉(zhuǎn)化敏感菌株以證實(shí)克隆片段的抗性功能。結(jié)果對ANC強(qiáng)抗性突變株DM104對化學(xué)特性不相關(guān)的化合物如溴化乙錠和諾丹明產(chǎn)生交叉抗性。鈣通道阻斷劑異搏定使突變株失去對氨茴環(huán)霉素的抗性?；蚩寺～@得了4個

2、帶有DM104染色體上不同片段的重組質(zhì)粒，只有1個片段與已知的枯草桿菌bmr基因同源。用重組質(zhì)粒再次轉(zhuǎn)化敏感菌株證實(shí)克隆片段的抗性功能。 結(jié)論枯草桿菌對ANC的抗性呈多重藥物抗性特征，這種抗性可能與染色體上多個位點(diǎn)有關(guān)。【 主題詞 】枯草桿菌氨茴環(huán)霉素抗性多重藥物抗性Resistance of Bacillus subtilis to anthracyclinesTANG Xinyun  Edda De Rossi  Orio Ciferri.Bioengineering Department, Anhui Agricultural University, Hefei 23

3、0036【 Abstract 】ObjectiveTo explore the resistance of Bacillus subtilis to anthracycline and clone the fragment responsible for resistance. MethodsSpontaneous mutants of strain 1831 of Bacillus subtilis resistant to anthracyclines (ANC) were isolated by selection on LB plates containing increasing c

4、oncentrations of doxorubicin (up to 100g/ml). ResultsThese mutants were cross-resistant towards chemically-unrelated compounds such as ethidium bromide and rhodamine. In the presence of a calcium channel blocker, verapamil, mutant DM104 became as sensitive to inhibition by doxorubicin as the parenta

5、l strain. Four recombinant plasmids containing different fragments of DM104 DNA were obtained by direct gene cloning in protoplasts of B. subtilis BD224 strain, with only one fragment homologous to known bmr gene of B. subtilis. The sensitive strain BD224 became resistant to ANC after retransformed

6、by the recombinant plasmids. ConclusionAll data indicate that the mutants display a multidrug resistance mechanism and the resistance may involve more loci in B. subtilis DNA.【 Subject words 】Anthracycline resistanceMultidug resistanceBacillus subtilis在對腫瘤細(xì)胞株、實(shí)驗(yàn)?zāi)[瘤及化療患者使用氨茴環(huán)霉素(anthracyclines, ANC)一個以

7、上療程后，一般總是出現(xiàn)多重藥物抗性現(xiàn)象(multidrug resistance, MDR)1 。這種抗性的特征是腫瘤細(xì)胞對很多化學(xué)性質(zhì)不相關(guān)的細(xì)胞毒性藥物加快排出，降低攝入。很多報(bào)告證實(shí)這種抗性的出現(xiàn)與mdr 1基因擴(kuò)增有關(guān)，該基因編碼細(xì)胞表面一個磷酸化糖蛋白P-1702 。Neyfakh3 報(bào)道了Bacillus subtilis突變株對諾丹明6G、溴化乙錠、氯霉素、嘌呤霉素及其它結(jié)構(gòu)上不相關(guān)的藥物產(chǎn)生多重抗性。這種抗性也與1個編碼多重藥物外向運(yùn)輸?shù)鞍椎幕騜mr的擴(kuò)增有關(guān)。經(jīng)比較發(fā)現(xiàn)，B.subtilis的這種蛋白類似Staphylococcus aureus的氟喹酮外向運(yùn)輸?shù)鞍? 和G

8、-細(xì)菌中的四環(huán)素外向運(yùn)輸?shù)鞍? 。Miyauchi等在古細(xì)菌Haloferax volcanii也發(fā)現(xiàn)了類似MDR蛋白。本文報(bào)道B.subtilis對氨茴環(huán)霉素的抗性突變株的篩選，突變株的抗性規(guī)律及抗性基因克隆。材料與方法1.菌株、質(zhì)粒及培養(yǎng)條件：B.subtilis 1831菌株(trpC2，phe1)對氨茴環(huán)霉素(anthracyclines,ANC)敏感，由意大利帕維亞大學(xué)遺傳學(xué)和微生物學(xué)系A(chǔ).Galizzi博士惠贈。B.subtilis BD224菌株(trpC2，thr5，recE4)、穿梭質(zhì)粒pCB20(Ampr, Emr)(7595bp)和質(zhì)粒pBSBMR2(后一質(zhì)粒是帶有bmr

9、基因的pBluescript KS+質(zhì)粒，bmr是B.subtilis BD170的多重藥物抗性基因)，由Neyfakh博士惠贈3 。質(zhì)粒pCBMR為本實(shí)驗(yàn)用pCB20克隆pBSBMR2中bmr基因構(gòu)建獲得。pTE06、pTE10、pTSD1、pTSD6為本實(shí)驗(yàn)中用pCB20克隆突變株染色體上抗性片段獲得的重組質(zhì)粒。菌株一般在LB液體或固體培養(yǎng)基37培養(yǎng)。2.抗生素和抗性測定：阿霉素(doxorubicin, DXR)、表阿霉素(epirubicin, EPR)、碘阿霉素(iododoxorubicin, IDX)、柔紅霉素(daunorubicin, DNR)和去甲柔紅霉素(4-demeth

10、ydaunorubicin或idarubicin, IDR)由意大利Pharmacia-Farmitalia Carlo Erba公司化學(xué)實(shí)驗(yàn)室提供，其它藥物為市售商品。定量稱取抗生素，溶于無菌水中，-20貯存。在LB平板或液體中測定各菌株對抗生素的抗性。3.突變株篩選：制備含有不同濃度抗生素的LB平板，在平板上涂上約5×108個對數(shù)后期細(xì)胞，篩選B.subtilis 1831菌株對DXR、EPR和溴化乙錠的自發(fā)抗性突變株，然后逐步增加藥物濃度，從自發(fā)突變株中篩選強(qiáng)抗性突變株。4.遺傳操作技術(shù)：用堿性方法提取B.subtilis質(zhì)粒7 ，用原生質(zhì)體和感受態(tài)細(xì)胞法轉(zhuǎn)化B.subtili

11、s BD224菌株8，9 。根據(jù)文獻(xiàn)10進(jìn)行質(zhì)粒的CsCl梯度離心純化、瓊脂糖凝膠電泳、Southern blotting、bmr片段的32P標(biāo)記和雜交。5.DNA制備和基因克隆：用標(biāo)準(zhǔn)方法10 從突變株DM104中提取染色體DNA。用Sau 3AI酶部分降解DNA。質(zhì)粒pCB20用BamH酶切開，用小牛腸道堿性磷酸酶去磷酸化。用T4DNA連接酶連接部分降解的DNA片段和載體。以上各酶均購自Promega或Sigma公司。用制備好的基因表達(dá)文庫轉(zhuǎn)化BD224菌株原生質(zhì)體，用下列方法篩選抗ANC克?。喊艳D(zhuǎn)化后的原生質(zhì)體涂在含有10g/ml紅霉素(pCB20標(biāo)記)的DM3再生培養(yǎng)基上，23天后將菌

12、落復(fù)印轉(zhuǎn)移至含有2g/ml的DXR的LB平板上；或把轉(zhuǎn)化的原生質(zhì)體直接涂在含有2g/ml DXR的DM3再生培養(yǎng)基上。結(jié)果與討論1.自發(fā)突變率：1831菌株在含有10g/ml DXR和EPR的平板上的自發(fā)抗性突變率約為2×10-8，在20g/ml溴化乙錠平板上的自發(fā)抗性突變率約為10-8。2.突變株的抗性特征：如表1所示，在DXR平板上篩選到抗DXR突變株DM104對EPR和DNR也產(chǎn)生很強(qiáng)抗性，不同突變株間抗性存在差異。同樣，在EPR平板上篩選到的突變株也對DXR和DNR產(chǎn)生很強(qiáng)抗性。但是這些突變株對ANC的衍生物IDX和IDR只產(chǎn)生微弱抗性(數(shù)據(jù)略)，與MDR在腫瘤細(xì)胞中的表現(xiàn)相

13、似，可能是親脂的衍生物可快速透過細(xì)胞膜而減弱了細(xì)胞的泵出效應(yīng)11 。表1突變株對不同藥物的抗性(g/ml)Table 1. Resistance level to different drugs of mutant strainsStrainsSelected as resistance toDoxorubicinEpirubicinDaunorubicinEthidium bromideRhodamin 1231831None111216DM058Doxorubicin10018136128DM101Doxorubicin5024-3064DM102Doxorubicin5018102064

14、DM104Doxorubicin100501236128EP201Epirubicin100501052128EB02Ethidium 1820-3064EB03Bromide10050-100128-：Not tested所獲抗ANC突變株還對溴化乙錠和諾丹明123產(chǎn)生交叉抗性(表1)。而從含溴化乙錠平板上篩選到的抗性菌株也對諾丹明和ANC類抗生素產(chǎn)生交叉抗性。所獲菌株對氯霉素、四環(huán)素、鏈霉素、梭鏈孢酸、放線菌素D和利福霉素不產(chǎn)生抗性(數(shù)據(jù)略)。在培養(yǎng)基中加10g/ml的鈣通道阻斷劑異搏定(verapamil)后，突變株DM104和其出發(fā)菌株1831一樣對DXR敏感(1)，和其他生物中的MD

15、R的抑制特征相同1，2，6 。1阿霉素和異搏定對出發(fā)菌株與突變株DM104生長的影響Fig 1. Effect of doxorubicin and verapamil on the growth of the parental strain and the anthracycline-resistant mutant DM104Growth conditions as reported under materials and methods. When present, doxorubicin and Verapamil were added at 0 time3.基因克?。河胮CB20作為

16、載體，在BD224菌株原生質(zhì)體中直接進(jìn)行基因克隆，獲得4個抗性較強(qiáng)的重組質(zhì)粒，其上所帶DNA片段長度在1.57.0kb之間(2)。用這些重組質(zhì)粒再次轉(zhuǎn)化BD224菌株的抗性特征見表2。從表2可見，帶有這些質(zhì)粒的菌株對ANC類抗生素的抗性是對照菌株(不帶質(zhì)?；蛑粠лd體)的1020倍，高于pCBMR。這些重組質(zhì)粒還使得細(xì)胞對其它藥物產(chǎn)生交叉抗性，并且其抗性受到異搏定的明顯抑制(表3)，表現(xiàn)出和DM104菌株相同的MDR特征。2重組質(zhì)粒用Acc和EcoR酶切瓊脂糖凝膠電泳譜Fig 2. The agarose gel electropherogram of recombinant plasmids

17、digested with Acc and EcoR 1.pCB20； 2.pTE06； 3.pTE10；4.pTSD1；5.pTS06；6.DNA/Hind+EcoR marker；7.DNA/Hind marker表2BD224菌株用不同重組質(zhì)粒轉(zhuǎn)化后對藥物的抗性(g/ml)Table 2. Resistance to different drugs of BD224 strain transformed with recombinant plasmidsDrugsPlasmids-pCB20pCBMRpTE06pTE10pTSD1pTSD6Doxorubicin0.20.224244Ep

18、irubicin0.40.40.620.622Daunorubicin0.20.20.420.422Ethidium bromide221210121012Rhodamin 1231815100181002530-：Not plasmid表3異搏定對帶重組質(zhì)粒BD224菌株抗性的抑制Table 3. Reversal by verapamil of resistance of BD224 strain with different recombinant plasmidsDrugsPlasmidspCBMRpTE06pTE10pTSD1pTSD6Doxorubicin+Doxorubicin+

19、Verapamil±±±±+Ethidium bromide+Ethidium bromide+Verapamil+-Effects of verapamil were assessed in LB liquid. Verapamil: 25g/ml; doxorubicin:2 to 4g/ml; ethidium bromide: 4 to 8g/ml. Being quantitatively inoculated, cultures were grown for 8h and then measured spectrophotometricall

20、y at 660nm, and inhibition was expressed as follows:+, growth equal to 20% of the control; ±，less than 10% of the control; -， no growth.用pTE06重新轉(zhuǎn)化BD224菌株原生質(zhì)體后，從含紅霉素的DM3再生平板上隨機(jī)挑取40個菌落接到含DXR的平板上，發(fā)現(xiàn)有2個菌落不長，提取質(zhì)粒電泳后發(fā)現(xiàn)這兩個菌落細(xì)胞中的質(zhì)粒丟失了原攜帶的克隆片段，而其它菌落中的細(xì)胞都帶有完整重組質(zhì)粒，說明重組質(zhì)粒中的DNA片段與抗性確實(shí)有關(guān)。丟失克隆片段的現(xiàn)象在枯草桿菌中是存在的7

21、 。用pCBMR上的bmr基因3 作探針進(jìn)行雜交試驗(yàn)，發(fā)現(xiàn)只有pTE10上所帶的DNA片段與bmr雜交，其它質(zhì)粒不與bmr雜交。從表2也可看出，pTE10和 pCBMR一樣對溴化乙錠和諾丹明具有較強(qiáng)抗性，而對ANC類抗生素抗性較弱。這些數(shù)據(jù)說明pTE10上帶有和bmr相同或同源片段，而其它重組質(zhì)粒上分別帶有不同的抗性片段?？莶輻U菌突變株DM104對ANC類抗生素產(chǎn)生較強(qiáng)抗性(因逐步加大藥物濃度，多次突變結(jié)果)，并對其他藥物產(chǎn)生交叉抗性，其抗性受到異博定的抑制，表現(xiàn)典型的MDR型抗性特征?；蚩寺～@得4個較強(qiáng)抗性重組質(zhì)粒，分別帶有不同片斷，分子雜交結(jié)果說明只有pTE10所帶片斷與bmr同源。用這

22、些重組質(zhì)粒重新轉(zhuǎn)化敏感菌株BD224后獲得的大量菌落都表現(xiàn)比BD224高1020倍的抗性，本實(shí)驗(yàn)中自發(fā)突變率為2×10-8，而且克隆片斷的丟失也導(dǎo)致抗性的喪失，這些都說明重新轉(zhuǎn)化的菌株的抗性不可能是染色體上基因突變的結(jié)果。因?yàn)榭寺〉蕉鄠€不同片斷，說明DM104的抗性涉及多重機(jī)理或染色體上多個位點(diǎn)。帶有不同重組質(zhì)粒的BD224菌株的抗性比突變株DM104明顯要低，有2個原因。一是BD224比DM104的出發(fā)菌株1831對ANC要敏感5倍(表1、2)。二是DM104是經(jīng)過多次增加藥物濃度篩選獲得，涉及多次突變，或多個位點(diǎn)突變，而每個重組質(zhì)粒只帶有部分片斷，因而只產(chǎn)生較低抗性。在E.col

23、i染色體上已發(fā)現(xiàn)多個與MDR現(xiàn)象有關(guān)的基因或位點(diǎn)，涉及不同機(jī)理12 。非特異性多重藥物抗性可能在細(xì)菌中是普遍現(xiàn)象，本文數(shù)據(jù)初步證實(shí)在B.subtilis中也存在類似情況。有關(guān)這些位點(diǎn)的位置、不同基因編碼的蛋白質(zhì)的性質(zhì)及作用機(jī)理有待進(jìn)一步實(shí)驗(yàn)加以探討。作者單位：唐欣昀230036合肥安徽農(nóng)業(yè)大學(xué)生物工程系(獲國家教委資助赴意大利訪問進(jìn)修Edda De RossiOrio Ciferri意大利帕維亞大學(xué)遺傳學(xué)和微生物學(xué)系參考文獻(xiàn)1Gottesman MM, Pastan I. Resistance to multiple chemotherapeutic agents in human cance

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