Stenotrophomonas maltophilia 的SmeYZ多重藥物輸出幫浦之特性分析

Characterization of SmeYZ Multidrug Efflux Pump in Stenotrophomonas maltophilia

林卉蓉(Hui-Jung Lin)

指導教授 ： 梁有志

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2013年)

摘要

Stenotrophomonas maltophilia 為一具有多重抗藥性之臨床致病菌株。 文獻指出，革蘭氏陰性菌之nodulation division (RND) efflux system過度表現將導致其產生多重抗藥性。經由生物資訊結果得知，S. maltophilia基因體中含有八套之RND-type多重藥物輸出幫浦。本論文將探討其中之SmeYZ多重藥物輸出幫浦與 S. maltophilia多重抗藥性之關係以及smeYZ 基因上游之smeSy, smeRy two-component regulatory system (TCS)所扮演的調控角色。為了瞭解SmeYZ多重藥物輸出幫浦與 S. maltophilia多重抗藥性的關係，構築且利用KJΔSmeYZ突變株與原生菌株 KJ進行抗生素感受性試驗。結果顯示，相較於原生菌株KJ ，突變菌株KJΔSmeYZ之amikacin、gentamicin 和 kanamycin MIC值有明顯下降的趨勢，然而在chloramphenicol、nalidixic acid、macrolide 和tetracyclines類抗生素之MIC值則不變。證實SmeYZ多重藥物輸出幫浦對aminoglycosides 類抗生素之抗性有抗性。接著構築smeYZ 基因上游之 smeSy-smeRy two-component regulatory system 突變株，KJΔSmeSRy，以釐清其對smeYZ表現所扮演之調控角色。qRT-PCR結果發現，smeSR基因之缺陷除了使smeZ之表現量減少外，亦導致 smeE、smeF之表現量上升。抗生素感受性試驗結果顯示，當smeSRy基因缺陷時，除了預期中之SmeYZ 輸出幫浦的受質aminoglycosides 類抗生素 MIC 值下降之外， 意外發現KJΔSmeSRy 突變株對 macroide 類抗生素(erythromycin 、leucomycin) 之MIC 值有4~16 倍的上升； 而chloramphenicol、 nalidixic acid 及 tetracycline 類抗生素其MIC 值則有約2~4 倍的些微上升，此類抗生素已知為SmeDEF pump 之Substrate。SmeT 此一型之轉錄調控因子是為一repressor 會抑制其下游smeDEF operon 之表現，最後並以啟動子報導基因(promoter fusion assay)的策略證實ΔSmeSRy會降低smeT 基因之表現量，經由上述之結果說明，smeSRy TCS 在 smeYZ 和smeT 的表現扮演正調控的角色，推測藉此TetR 型之轉錄調控因子SmeT 表現量的改變，進而調控smeDEF operon 之表現。

關鍵字

多重藥物輸出幫浦

並列摘要

Stenotrophomonas maltophilia is an important opportunistic human pathogen characterized by the phenotype of multidrug resistance (MDR). The resistance nodulation cell division (RND)-type multidrug efflux systems play a critical role in multidrug resistance (MDR), especially in gram-negative bacteria. Genome sequence analysis reveals that S. maltophilia harbors as many as eight RND efflux systems. In this study, SmeYZ, one of the RND-type efflux pumps, and the smeSRy two-component regulatory system (TCS), located upstream of the smeYZ, were characterized. The role of SmeYZ pump in antimicrobials resistance was assessed by the construction of smeYZ isogenic mutant and the susceptibility test between wild-type KJ and KJΔSmeYZ. The MICs of KJΔSmeYZ to aminoglycoside significantly decreased, compared with those of wiild-type KJ. However, the MICs to chloramphenicol, nalidixic acid, macrolide and tetracyclines were comparable between KJ and KJΔSmeYZ. To assess the regulatory role of SmeSRy TCS, a smeSRy deletion isogenic mutant, KJΔSmeSRy, was constructed. The RNA transcripts quantification of RND-efflux pump-associated genes and the susceptibility test were comparatively determined between KJ and KJΔSmeYZ. SmeSRy inactivation decreased the expression of smeZ but increased the expression of smeE and smeF. In the meanwhile, the susceptibility test demonstrated that compared with wild-type KJ, KJΔSmeSRy showed the decreased MICs to aminoglycosides, which have known to be extruded by SmeYZ pump, and the increased MICs to chloramphenicol, nalidixic acid, and tetracycline, which are the known substrates of SmeDEF pump. SmeT, a TetR-type regulator, has known to act as a repressor for the expression of smeDEF operon. The linkage between SmeSRy TCS and SmeT was assessed by promoter transcriptional fusion assay. The expression of smeT was decreased in the ΔSmeSRy background, indicating that SmeSRy TCS positively regulates the smeT expression. These results concluded that smeSRy TCS positively regulates the expression of smeYZ operon and smeT gene. In the case of smeSRy inactivation, the SmeYZ pump-mediated resistance was compromised and the decreased expression in smeT gene, in turn, increased the SmeDEF pump-mediated resistance.

並列關鍵字

Multidrug efflux pump

參考文獻

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