李軍 馮世文 曾蕓 謝宇舟 彭昊 潘艷 賀會(huì)利 鐘舒紅 胡帥
摘要:【目的】分析大腸桿菌O157:H7氟苯尼考耐藥菌株與敏感菌株的差異表達(dá)蛋白,從蛋白組學(xué)層面揭示大腸桿菌O157:H7對(duì)氟苯尼考耐藥性的產(chǎn)生機(jī)制?!痉椒ā刻崛〈竽c桿菌O157:H7氟苯尼考耐藥菌株(RS2-256)和敏感菌株(RS2)的全菌蛋白,經(jīng)同位素標(biāo)記相對(duì)和絕對(duì)定量(iTRAQ)標(biāo)記后進(jìn)行液相色譜串聯(lián)質(zhì)譜(LC-MS/MS)分析,質(zhì)譜數(shù)據(jù)通過Mascot v2.2鑒定,并根據(jù)COGs數(shù)據(jù)庫對(duì)差異表達(dá)蛋白進(jìn)行GO功能注釋和分類?!窘Y(jié)果】RS2菌株獲得3385個(gè)肽段,RS2-256菌株獲得3955個(gè)肽段,合計(jì)7340個(gè)肽段,其中6975個(gè)肽段為特有肽段,分屬于1039個(gè)蛋白。與RS2菌株相比,RS2-256菌株有145個(gè)蛋白表達(dá)差異顯著(P<0.05),占檢測(cè)蛋白總數(shù)的13.96%,其中,上調(diào)表達(dá)蛋白有87個(gè)、下調(diào)表達(dá)蛋白有58個(gè)。表達(dá)差異蛋白按照功能進(jìn)行分類,可分為外排泵、轉(zhuǎn)運(yùn)蛋白、細(xì)胞膜形成、應(yīng)激調(diào)控、核糖體結(jié)構(gòu)、DNA復(fù)制、轉(zhuǎn)錄、翻譯、翻譯后修飾、能量產(chǎn)生和轉(zhuǎn)化、氨基酸轉(zhuǎn)運(yùn)和代謝、輔酶轉(zhuǎn)運(yùn)和代謝、糖轉(zhuǎn)運(yùn)和代謝、脂肪代謝、離子轉(zhuǎn)運(yùn)和代謝、細(xì)菌運(yùn)動(dòng)性、轉(zhuǎn)座及信號(hào)通路共18類;多藥物外排泵亞基AcrA和AcrB、外膜蛋白TolC、外膜蛋白X等52個(gè)蛋白的表達(dá)變化在3.00倍以上,占表達(dá)差異蛋白總數(shù)的35.86%?!窘Y(jié)論】外排泵AcrAB-TolC家族、ABC家族、外膜蛋白、毒性調(diào)節(jié)器B、饑餓蛋白等在大腸桿菌O157:H7對(duì)氟苯尼考耐藥性產(chǎn)生過程中發(fā)揮重要作用,同時(shí)涉及細(xì)菌糖代謝、氨基酸代謝、離子轉(zhuǎn)運(yùn)、DNA損傷與修復(fù)、生物膜及毒素與抗毒素系統(tǒng)等生命活動(dòng)。
關(guān)鍵詞: 大腸桿菌O157:H7;氟苯尼考;耐藥性;差異表達(dá)蛋白;iTRAQ技術(shù)
中圖分類號(hào): S852.612? ? ? ? ? ? ? ? ? ? ? ? ?文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2019)04-0875-08
Abstract:【Objective】This study was to identify differentially expressed proteins in Escherichia coli O157:H7 between florfenicol-resistant strain and florfenicol-sensitive strain, and reveal the resistance mechanism of E. coli O157:H7 against flunicol from protein level. 【Method】Complete bacterial proteins were extracted from florfenicol-resistant strain (RS2-256) and florfenicol-sensitive strain(RS2) of E. coli O157:H7, and labelled with isobaric tags for relative and absolute quantification(iTRAQ) reagent. Different expression proteins were identified and quantified with liquid chromatography tandem mass spectrometry(LC-MS/MS). The mass spectrometry data was identified and analyzed with Mascot v2.2. GO functional annotation and classification analysis of differentially expressed proteins were performed according to COGs database. 【Result】The RS2 strain obtained 3385 peptides, and the RS2-256 strain obtained 3955 peptides, a total of 7340 peptides, of which 6975 peptides were specific peptides belonging to 1039 proteins. Compared with RS2 strain, expressions of 145 proteins of RS2-256 strain were significantly different(P<0.05), accounting for 13.96% of the total protein detected, among which 87 proteins were up-regulated and 58 proteins were down-regulated. The differentially expressed proteins could be divided into 18 categories according to their functions:efflux pump, transporters, cell membrane biogenesis, stress control, the structure of the ribosome, DNA replication, transcription, translation, posttranslational modification, energy production and conversion, amino acid transport and metabolism, coenzyme transport and metabolism, sugar transport and metabolism, fat metabolism, ion transport and metabolism, bacterial motility, translocation, signaling pathways and metabolism. The expression of 52 proteins, including multidrug efflux pump subunits acrA and acrB, outer membrane protein TolC and outer membrane protein X, varied more than 3.00 times, accounting for 35.86% of the total number of differentially expressed proteins. 【Conclusion】The results show that various mechanisms such as the efflux pump AcrAB-TolC family, ABC family, outer membrane protein, toxicity regulator B, starvation protein play important roles in the development of resistance of E. coli O157:H7 to fluorbenicol. At the same time, it involves bacterial glucose metabolism, amino acid metabolism, ion transport, DNA damage and repair, biofilm, toxin and antitoxin systems and other life activities.
Key words: Escherichia coli O157:H7; florfenicol; disease resistance; differentially expressed protein; iTRAQ technique
0 引言
【研究意義】大腸桿菌O157:H7是一種人獸共患病原菌,致病力強(qiáng),通過食源性途徑感染人類后引起腹瀉或出血性腸炎,且極易繼發(fā)溶血性尿毒綜合癥和血栓性血小板減少性紫癜等并發(fā)癥(李軍等,2011)。氟苯尼考是獸醫(yī)臨床上治療細(xì)菌性疾病的一種專用抗菌藥物,主要通過與細(xì)菌70S核蛋白體50S亞基上的A位點(diǎn)結(jié)合,抑制細(xì)菌肽酰基轉(zhuǎn)移酶的轉(zhuǎn)肽反應(yīng),致使肽鏈無法延伸而抑制細(xì)菌蛋白合成,發(fā)揮抗菌作用(陳曉慧等,2006)。目前,獸醫(yī)臨床上尚無有效疫苗可用于大腸桿菌病防控,抗菌藥物仍是防治大腸桿菌的首選,但調(diào)查發(fā)現(xiàn)大腸桿菌已對(duì)氟苯尼考產(chǎn)生嚴(yán)重的耐藥性(馮世文等,2017;余波等,2017)。因此,研究大腸桿菌O157:H7對(duì)氟苯尼考產(chǎn)生耐藥的機(jī)制,對(duì)繼續(xù)發(fā)揮氟苯尼考的抗菌作用極其重要?!厩叭搜芯窟M(jìn)展】目前,科技人員主要從耐藥基因調(diào)節(jié)、接合轉(zhuǎn)移等層面揭示氟苯尼考耐藥性的產(chǎn)生機(jī)制。Tao等(2012)研究證實(shí),estDL136基因編碼的醋酸酯酶可水解氟苯尼考和氯霉素的酰胺鍵,使其失去抗菌活性。此外,以表達(dá)estDL136的質(zhì)粒轉(zhuǎn)化大腸桿菌DH5α感受態(tài)細(xì)胞可獲得對(duì)氟苯尼考和氯霉素的抗性,二者對(duì)大腸桿菌的最小抑菌濃度(MIC)由15 mg/L升高至128 mg/L(Tao et al.,2012)。floR和fexA基因是介導(dǎo)氟苯尼考耐藥的主要基因(Wu et al.,2006;Dai et al.,2010;李帆等,2018),其編碼的外排泵能特異性將氟苯尼考泵出體外(Kadlec et al.,2009;馮世文等,2014,2016);而ATP結(jié)合盒轉(zhuǎn)運(yùn)體家族中的AcrAB-ToLC多藥物外排系統(tǒng)可低水平泵出氟苯尼考,尤其敲除acrB和tolC基因后,細(xì)菌對(duì)氟苯尼考的敏感性明顯降低(Baucheron et al.,2004)。此外,細(xì)菌可通過改變藥物結(jié)合靶位點(diǎn)的結(jié)構(gòu)而對(duì)氟苯尼考產(chǎn)生耐藥性。已有研究證實(shí),cfr基因編碼的核糖體RNA甲基轉(zhuǎn)移酶作用于細(xì)菌70S核糖體50S亞基的23S rRNA核苷酸A2503位點(diǎn)和C2498位點(diǎn),在甲基化A2503位點(diǎn)的同時(shí)抑制C2498位點(diǎn)甲基化,促使氟苯尼考和氯霉素的結(jié)合位點(diǎn)發(fā)生構(gòu)型改變,而無法發(fā)揮其抑制蛋白合成的作用(Schwarz et al.,2000;Kehrenberg et al.,2005)。【本研究切入點(diǎn)】從蛋白組學(xué)層面進(jìn)行研究不僅可進(jìn)一步確定基因功能,還能挖掘到一些未知的功能基因。同位素標(biāo)記相對(duì)和絕對(duì)定量(isobaric tags for relative and absolute quantitation,iTRAQ)的定量效果好、重復(fù)性高,具有高通量、結(jié)果直觀、不限定樣本類型和數(shù)據(jù)可靠的優(yōu)勢(shì),在細(xì)菌學(xué)研究中主要用于尋找差異表達(dá)蛋白(Carvalhais et al.,2015),但至今鮮見采用iTRAQ技術(shù)從蛋白組學(xué)層面研究細(xì)菌對(duì)氟苯尼考的耐藥機(jī)制?!緮M解決的關(guān)鍵問題】利用iTRAQ技術(shù)結(jié)合液相色譜串聯(lián)質(zhì)譜(LC-MS/MS)分析大腸桿菌O157:H7氟苯尼考耐藥菌株與敏感菌株間的差異表達(dá)蛋白,篩選出可能與耐藥性產(chǎn)生相關(guān)的差異表達(dá)蛋白,為深入研究大腸桿菌O157:H7對(duì)氟苯尼考產(chǎn)生耐藥的機(jī)制提供參考依據(jù)。
1 材料與方法
1. 1 試驗(yàn)材料
大腸桿菌O157:H7氟苯尼考敏感菌株RS2(MIC為4 μg/mL)由廣西獸醫(yī)生物技術(shù)重點(diǎn)實(shí)驗(yàn)室分離鑒定并保存提供(李軍等,2011),大腸桿菌O157:H7氟苯尼考耐藥菌株RS2-256(MIC為256 μg/mL)為RS2菌株經(jīng)氟苯尼考誘導(dǎo)后獲得(馮世文等,2015)。氟苯尼考原粉(含量99.9%)由廣西大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院獸醫(yī)藥理與毒理學(xué)實(shí)驗(yàn)室提供;MHA和MHB培養(yǎng)基購自北京陸橋技術(shù)股份有限公司;蛋白提取試劑RIPA裂解液、丙酮、過硫酸銨和SDS等購自美國Bio-Rad公司;iTRAQ? Reagent 8 Plex Chemistry試劑盒和TEAB等試劑購自美國ABI公司;Triple TOF 5600質(zhì)譜儀購自美國AB Sciex公司。
1. 2 蛋白樣品制備
將RS2和RS2-256菌株復(fù)蘇后,挑取單一菌落接種于MHB培養(yǎng)基上,37 ℃培養(yǎng)6 h后,按培養(yǎng)基總體積的10%進(jìn)行接種。其中,RS2-256菌株處于氟苯尼考?jí)毫ο屡囵B(yǎng),培養(yǎng)液含64 μg/mL氟苯尼考。37 ℃下?lián)u床(200 r/min)培養(yǎng),每隔1 h用微量分光光度計(jì)測(cè)定培養(yǎng)基的OD600,繪制菌株生長曲線。于生長對(duì)數(shù)期分別取RS2和RS2-256菌株培養(yǎng)液,經(jīng)低溫離心后收集菌體,滅菌預(yù)冷PBS洗滌3次,棄上清液,收集沉淀。參照謝宇舟等(2012)的方法提取RS2和RS2-256菌株的總蛋白。
1. 3 蛋白酶解及iTRAQ標(biāo)記
每組蛋白樣品取100 μg,按20∶1(蛋白∶酶)的比例加入胰蛋白酶,37 ℃酶解4 h;重復(fù)1次,37 ℃再酶解8 h。真空凍干肽段后,加入含0.1% SDS的50% TEAB復(fù)溶。按iTRAQ? Reagent 8 Plex Chemistry試劑盒說明進(jìn)行肽段標(biāo)記,以iTRAQ 8 Plex 126標(biāo)記RS2-256菌株、iTRAQ 8 Plex 127標(biāo)記RS2菌株。
1. 4 標(biāo)記肽段SCX分離和LC-MS/MS分析
加入8倍體積的SCX-A液,調(diào)節(jié)pH至3.0。用清水清洗柱子,平衡,除鹽,用0.1% FA復(fù)溶,最后進(jìn)行LC-MS/MS分析。
1. 5 蛋白鑒定和定量分析
應(yīng)用Mascot v2.2進(jìn)行蛋白鑒定;依據(jù)蛋白豐度水平進(jìn)行判定,當(dāng)差異達(dá)2.00倍以上且顯著性檢驗(yàn)P小于0.05時(shí)視為差異表達(dá)蛋白。
1. 6 生物信息學(xué)分析
根據(jù)COGs數(shù)據(jù)庫對(duì)差異表達(dá)蛋白進(jìn)行GO功能注釋和分類分析。
2 結(jié)果與分析
2. 1 大腸桿菌O157:H7的生長曲線
RS2菌株培養(yǎng)1 h后,培養(yǎng)基的OD600開始上升,之后呈對(duì)數(shù)生長,培養(yǎng)5 h后隨時(shí)間推移,培養(yǎng)基的OD600緩慢上升并趨于穩(wěn)定(圖1)。RS2-256菌株接種于含氟苯尼考的MHB培養(yǎng)液中,在前3 h呈緩慢生長趨勢(shì),為適應(yīng)期;培養(yǎng)3~6 h呈快速生長,為對(duì)數(shù)期;之后隨時(shí)間推移,培養(yǎng)基的OD600緩慢上升,并趨于穩(wěn)定。根據(jù)大腸桿菌O157:H7的生長曲線,均在培養(yǎng)5 h時(shí)收集RS2和RS2-256菌株用于提取菌體蛋白。
2. 2 iTRAQ標(biāo)記蛋白圖譜整體分析結(jié)果
RS2和RS2-256菌株的蛋白樣品經(jīng)iTRAQ標(biāo)記后,采用Triple TOF 5600質(zhì)譜儀進(jìn)行LC-MS/MS分析,結(jié)果獲得48850張肽段圖譜,其中,RS2菌株獲得3385個(gè)肽段,RS2-256菌株獲得3955個(gè)肽段,合計(jì)7340個(gè)肽段。此外,有6975個(gè)肽段為特有肽段,分屬于1039個(gè)蛋白。
2. 3 表達(dá)差異蛋白分析結(jié)果
設(shè)定蛋白豐度水平差異在2.00倍以上且P<0.05,視為差異表達(dá)(Liu et al.,2013)。統(tǒng)計(jì)結(jié)果顯示,與RS2菌株相比,RS2-256菌株有145個(gè)蛋白表達(dá)差異顯著(P<0.05,下同),占檢測(cè)蛋白總數(shù)的13.96%,其中,上調(diào)表達(dá)蛋白有87個(gè)、下調(diào)表達(dá)蛋白有58個(gè)。表達(dá)差異蛋白按照功能進(jìn)行分類,可分為外排泵、轉(zhuǎn)運(yùn)蛋白、細(xì)胞膜形成、應(yīng)激調(diào)控、核糖體結(jié)構(gòu)、DNA復(fù)制、轉(zhuǎn)錄、翻譯、翻譯后修飾、能量產(chǎn)生和轉(zhuǎn)化、氨基酸轉(zhuǎn)運(yùn)和代謝、輔酶轉(zhuǎn)運(yùn)和代謝、糖轉(zhuǎn)運(yùn)和代謝、脂肪代謝、離子轉(zhuǎn)運(yùn)和代謝、細(xì)菌運(yùn)動(dòng)性、轉(zhuǎn)座及信號(hào)通路共18類(表1)。多藥物外排泵亞基AcrA和AcrB、外膜蛋白TolC、外膜蛋白X等52個(gè)蛋白的表達(dá)變化在3.00倍以上,占表達(dá)差異蛋白總數(shù)的35.86%(52/145)。其中,上調(diào)比例最顯著的為多藥物外排泵亞基AcrB,上調(diào)14.93倍;下調(diào)比例最顯著的為多糖輸出蛋白Wza,下調(diào)8.87倍(表2)。
3 討論
藥物外排泵是引起細(xì)菌對(duì)抗生素耐藥的重要機(jī)制,其中,AcrAB-TolC外排泵屬于耐藥結(jié)節(jié)細(xì)胞分化(RND)家族,由AcrA、AcrB和TolC 3部分組成(Yang et al.,2003)。AcrAB-TolC外排泵是大腸桿菌最主要的外排泵(Notka et al.,2002),當(dāng)大腸桿菌AcrAB-TolC外排泵過量表達(dá)時(shí),其對(duì)氯霉素、四環(huán)素、新霉素、紅霉素、氯比西林、氟喹諾酮類、β-內(nèi)酰胺類和利福平等藥物的耐藥性隨之增強(qiáng)(Zgurskaya et al.,1999;田亞凱,2018)。Baucheron等(2004)研究證實(shí),鼠傷寒沙門氏菌acrB和tolC基因被敲除后,其對(duì)氟苯尼考的敏感性明顯降低。本課題組前期研究表明,在主動(dòng)外排泵抑制劑CCCP存在的情況下,豬源大腸桿菌O157:H7氟苯尼考耐藥菌株對(duì)氟苯尼考的MIC從256 μg/mL下降至64 μg/mL(馮世文等,2016)。綜合大腸桿菌O157:H7氟苯尼考耐藥菌株的AcrB、AcrA和TolC蛋白較氟苯尼考敏感菌株分別上調(diào)14.93、8.05和4.05倍,推測(cè)AcrAB-TolC外排泵在大腸桿菌O157:H7對(duì)氟苯尼考耐藥性產(chǎn)生機(jī)制中發(fā)揮重要作用。
ABC家族是所有轉(zhuǎn)運(yùn)體家族中最龐大的一類轉(zhuǎn)運(yùn)體,通過偶聯(lián)ATP水解釋放的能量轉(zhuǎn)移底物(Davidson et al.,2008)。在細(xì)菌中,ABC轉(zhuǎn)運(yùn)體參與營養(yǎng)物質(zhì)的攝取或細(xì)菌毒素的分泌,以及將抗生素泵出細(xì)胞使其具有抗藥性,其中,MsbA外排系統(tǒng)與多藥抗性有關(guān)(Singh et al.,2016),YojI轉(zhuǎn)運(yùn)體負(fù)責(zé)小抗菌肽J25的外排(Socías et al.,2009),macAB系統(tǒng)則與大環(huán)內(nèi)酯類藥物外排有關(guān)(Lu and Zgurskaya,2012)。本研究結(jié)果顯示,大腸桿菌O157:H7氟苯尼考耐藥菌株的ABC轉(zhuǎn)運(yùn)ATP結(jié)合蛋白u(yù)up、YojI、YbiT,以及假定磷脂ABC轉(zhuǎn)運(yùn)結(jié)合蛋白MlaB、ABC轉(zhuǎn)運(yùn)周質(zhì)結(jié)合蛋白YtfQ、ABC轉(zhuǎn)運(yùn)精氨酸結(jié)合蛋白、假定ABC轉(zhuǎn)運(yùn)體ATP結(jié)合蛋白YcjV的表達(dá)上調(diào)2.22~3.45倍,提示大腸桿菌O157:H7的ABC轉(zhuǎn)運(yùn)體可能發(fā)揮著將氟苯尼考泵出細(xì)胞外的作用。
大腸桿菌外膜蛋白與其耐藥性密切相關(guān)(張麗芳和羅薇,2014),如外膜蛋白缺失或突變菌株對(duì)銀納米抗菌劑的抵抗力較野生菌菌株提高4.00~8.00倍(Radzig et al.,2012)。大腸桿菌重要的外膜蛋白有OmpA、OmpC、OmpF和PhoE等(Pagès et al.,2008;俱雄等,2017),其中,OmpC在抗生素進(jìn)入細(xì)菌發(fā)揮藥效的過程中發(fā)揮重要作用,在大腸桿菌多重耐藥菌株中OmpC mRNA和OmpC蛋白均呈高表達(dá)(董輝等,2016)。外膜蛋白OmpF是大腸桿菌的主要膜孔通道,也是抗生素進(jìn)入細(xì)菌的主要通道,當(dāng)OmpF基因缺失導(dǎo)致細(xì)菌表面的通透性降低而發(fā)揮耐藥作用(崔艷艷等,2013)。外膜蛋白X(OmpX)折疊成具有跨膜結(jié)構(gòu)域的結(jié)構(gòu),從細(xì)胞表面突出,而影響其與外部蛋白的結(jié)合(Choutko et al.,2011),并參與通道、抗生素抗性和信號(hào)轉(zhuǎn)導(dǎo)等(Meng et al.,2016)。本研究結(jié)果表明,與大腸桿菌O157:H7氟苯尼考敏感菌株相比,大腸桿菌O157:H7氟苯尼考耐藥菌株有9個(gè)外膜蛋白上調(diào)2.00~4.05倍,而有6個(gè)外膜蛋白下調(diào)2.00~3.52倍。其中,大腸桿菌O157:H7氟苯尼考耐藥菌株的OmpX和OmpA分別上調(diào)3.02和2.87倍,而OmpC和OmpF分別下調(diào)2.06和3.52倍。
在大腸桿菌中,Bam復(fù)合物由1個(gè)膜結(jié)合蛋白(Bama)和4個(gè)脂蛋白(Bamb、Bamc、Bamd和Bame)組成,Bam復(fù)合物催化組裝外膜蛋白(OPMs)的基本過程,但其更多的功能仍未知(Kim et al.,2011)。在本研究中,大腸桿菌O157:H7氟苯尼考耐藥菌株的外膜蛋白BamB、BamD和BamE上調(diào)2.00~3.16倍,說明這3個(gè)外膜蛋白可能通過影響大腸桿菌O157:H7外膜蛋白的形成及結(jié)構(gòu)變化,促進(jìn)菌株對(duì)氟苯尼考產(chǎn)生耐藥性。腸桿菌科的Rcs系統(tǒng)能調(diào)節(jié)細(xì)菌夾膜異多糖酸合成及細(xì)菌鞭毛基因、抗酸性基因等的表達(dá),其主要功能是調(diào)節(jié)細(xì)菌的毒力和應(yīng)激(葉立娜和瞿滌,2018)。外膜脂蛋白R(shí)csF是Rcs系統(tǒng)感受信號(hào)的重要輔助蛋白,在外膜缺陷信號(hào)傳導(dǎo)過程中發(fā)揮重要作用(Castanié-Cornet et al.,2006)。本研究發(fā)現(xiàn),大腸桿菌O157:H7氟苯尼考耐藥菌株的外膜脂蛋白R(shí)csF表達(dá)上調(diào)2.77倍,說明在氟苯尼考的壓力下,菌株誘發(fā)Rcs系統(tǒng)進(jìn)行信號(hào)轉(zhuǎn)導(dǎo),通過調(diào)節(jié)外膜蛋白和耐藥基因表達(dá)等方式而抵抗氟苯尼考。外膜蛋白Slp在宿主免疫逃避、營養(yǎng)物質(zhì)獲取及細(xì)菌應(yīng)激反應(yīng)的調(diào)節(jié)過程中發(fā)揮關(guān)鍵作用(Hooda et al.,2017)。SlyB是外膜的主要蛋白,有助于維持細(xì)胞膜的完整性(Plesa et al.,2006)。本研究發(fā)現(xiàn),大腸桿菌O157:H7氟苯尼考耐藥菌株的外膜脂蛋白Slp表達(dá)上調(diào)2.87倍,其中SlyB蛋白表達(dá)上調(diào)2.12倍,推測(cè)大腸桿菌O157:H7外膜蛋白在調(diào)節(jié)氟苯尼考應(yīng)激過程中發(fā)揮重要作用。
毒性調(diào)節(jié)器B(Modulator of drug activity B,MdaB)是NADPH氧化還原酶DT-黃素氧化還原蛋白家族的假定成員,能提供針對(duì)醌類化合物的細(xì)胞保護(hù)。據(jù)報(bào)道,在0.2~0.3 mmol/L甲萘醌作用下,MdaB在大腸桿菌胞質(zhì)部分中表達(dá)上調(diào)20.00倍以上(Hayashi et al.,1990)。攜帶編碼MdaB基因大腸桿菌的生長不受甲萘醌的影響,但MdaB或重組MdaB的過表達(dá)致使大腸桿菌耐受四環(huán)素、阿霉素、DMP 840和依托泊苷(Adams and Jia,2006)。本研究結(jié)果表明,大腸桿菌O157:H7氟苯尼考耐藥菌株的MdaB蛋白表達(dá)上調(diào)2.86倍,NADH泛醌氧化還原酶亞基G表達(dá)上調(diào)4.47倍,推測(cè)大腸桿菌O157:H7通過超表達(dá)MdaB和NADH泛醌氧化還原酶,促進(jìn)電子傳遞進(jìn)入呼吸鏈?zhǔn)箼C(jī)體處于低氧化狀態(tài),而增加對(duì)氟苯尼考的抗性。
饑餓蛋白主要負(fù)責(zé)細(xì)胞的饑餓存活和耐受性,如饑餓狀態(tài)下指數(shù)生長期大腸桿菌對(duì)熱刺激、氧化壓力、滲透壓、紫外線和抗生素等的耐受性提高。有研究發(fā)現(xiàn),在細(xì)菌饑餓早期加入氯霉素可阻遏蛋白合成,使細(xì)胞活性急劇降低;但在細(xì)菌進(jìn)入全面饑餓狀態(tài)后加入氯霉素,對(duì)細(xì)菌生長的影響甚微(Giard et al.,1996)。在抗生素加入早期,細(xì)菌生長受到抑制,即這一應(yīng)激條件與饑餓過程類似,致使細(xì)菌的基因表達(dá)和代謝發(fā)生改變,表現(xiàn)為有應(yīng)激蛋白產(chǎn)生甚至某些針對(duì)性基因發(fā)生突變,而產(chǎn)生耐藥性。本研究發(fā)現(xiàn),大腸桿菌O157:H7氟苯尼考耐藥菌株的饑餓蛋白B表達(dá)上調(diào)3.38倍,提示與菌株的氟苯尼考耐藥性密切相關(guān),但具體機(jī)制有待進(jìn)一步探究。
4 結(jié)論
外排泵AcrAB-TolC家族、ABC家族、外膜蛋白、毒性調(diào)節(jié)器B、饑餓蛋白等在大腸桿菌O157:H7對(duì)氟苯尼考耐藥性產(chǎn)生過程中發(fā)揮重要作用,同時(shí)涉及細(xì)菌糖代謝、氨基酸代謝、離子轉(zhuǎn)運(yùn)、DNA損傷與修復(fù)、生物膜及毒素與抗毒素系統(tǒng)等生命活動(dòng)。
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(責(zé)任編輯 蘭宗寶)