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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48948

Title: 三方組成轉運蛋白 VarABCDEF 賦予霍亂弧菌巨環內脂類藥物抗性之探討
VarABCDEF consists a tripartite pump to confer macrolide resistance in Vibrio cholerae
Authors: Shen, Yu-Chi
沈佑錡
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 霍亂弧菌;三方組成;ABC 轉運蛋白;TolC-依賴型;VarABCDEF
Vibrio cholerae;tripartite;ABC transporter;TolC-dependent;VarABCDEF
Date: 2015
Issue Date: 2018-08-22T06:39:36Z
Abstract: 霍亂是一種傳染性極強的疾病,感染之病患主要症狀為急性且嚴重的腹瀉,導致脫水現象,病況嚴重未能及時治療的病患會有致命的危險。目前主要治療霍亂的方式為利用靜脈注射補充流失體液並搭配抗生素。隨著臨床上發現越來越多具有抗藥性的霍亂弧菌產生,使得抗生素的治療受到了很大的限制。造成菌株具有抗生素抗藥性的方式很多,霍亂弧菌之 var operon 中包含 varG 及 varABCDEF,其中 VarG 推測為具有 beta-lactamase 活性蛋白,VarABCDEF 推測可形成一個橫跨霍亂弧菌雙層膜的 ABC 家族三方組成轉運蛋白,根據先前研究發現,當內膜蛋白 VarDEF 表現於 E. coli 藥敏性菌株 Kam3 中能賦予此菌株巨環內脂類藥物抗性,本次實驗將更進一步探討 VarDEF 所賦予 E. coli 菌株藥物抗性是否與次級主動外排系統有關和 VarABCDEF 完整存在於 E. coli 中是否能比只含內膜蛋白 VarDEF 提升更高的巨環內脂類藥物抗性。利用 H33342 累積測試,發現 VarDEF 於 E. coli Kam3 中能有效轉運染劑,而於 E. coli 外膜蛋白TolC缺失菌株TG1 則無法轉運,證實 VarDEF 轉運巨環內脂類藥物為 TolC-依賴型。利用次級主動外排幫浦抑制劑 PAβN 則發現菌株對於巨環內脂類藥物耐受性下降,染劑轉運能力也下降,表示 VarDEF 所賦予 E. coli的巨環內脂類藥物抗性有其他的次級主動外排系統協助。將 VarABCDEF 於 E. coli Kam3 菌株中表現能使其對於 azithromycin、clarithromycin、erythromycin 和 spiramycin 分別提升 16 倍、8 倍、8 倍 和 32 倍,比含 VarDEF 的 E. coli 皆有 2–4 倍之提升;VarABCDEF 於 E. coli TG1 (ΔtolC) 中能使上述藥物分別提升 2 倍、2 倍、8 倍 和 4倍,比只含 VarDEF 的 E. coli皆有 4–8 倍之提升。VarABCDEF 完整存在於 E. coli TG1 (ΔtolC) 時,能夠恢復菌株對於 H33342 的轉運能力,表示 VarA、VarB 和 VarC 的存在能夠替代 TolC,使得 E. coli TG1 恢復巨環內脂類藥物抗性。
Cholera is a highly contagious disease. The main symptoms of infection in patients with acute and severe diarrhea, leading to dehydeation. In clinical, the main way for the treatmeant of cholera involves aggressive fluid constitute replacement combined antibiotic treatment. There are several mechanisms that cause antibiotic resistance in bacteria, efflux pump is a part of it. E. coli outer membrane TolC could form a tripartite pump with inner membrane secondary active transporter AcrB and membrane fusion protein AcrA translocating various substrates from cytosol and periplasm to extracellular space. In V. cholerae, var operon consist of varG and varABCDEF, encoding a putative -lactamase VarG, and VarABCDEF, putatively froming a tripartite pump across the lipid bilayer of gram-negative bacteria. Our group previously indicated that overexpression of VarDEF could confer macrolide resistance in E. coli. In this study, the Hoechst 333242 (H33342) accumulation was decreased in the E. coli overexpressing VarDEF, suggesting the dye efflux by VarDEF, and the accumulation of H33342 could increase with the addition of ATPase inhibitor sodium orthovanadate. Intriguingly, VarDEF could not decrease H33342 accumulation in an outer membrane protein TolC-deletion E. coli strain, suggesting TolC was involved in the dye efflux. In addition, VarDEF failed to give macrolide resistance and H33342 efflux in E. coli in the presence of an secondary active transporter inhibitor PAβN, indicating that the efflux failure could be due to the AcrB inhibition. The IC50 against the macrolides azithromycin, clarithromycin, erythromycin and spiramycin of E. coli overexpressing VarABCDEF was 8 µg/ml, 16 µg/ml, 16 µg/ml and 276 µg/ml respectively, which was higher than the ones of the E. coli overexpressing VarDEF. Moreover, addition of PAβN and deletion of TolC protein did not disrupt the efflux of H33342 and macrolide resistance, suggesting that the dye and macrolide translocation to extracellular space by VarABCDEF was not affected by a blocked AcrB and absence of TolC. Our data suggested VarABCDEF could putatively form a tripartite pump to translocate the substrates from cytosol to extracellular space.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010232053.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48948
Appears in Collections:[食品科學系] 博碩士論文

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