Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro
Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro
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DOI：
10.1016/j.tube.2011.05.002
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发表时间：
2011-09-01
期刊：
影响因子：
3.2
通讯作者：
Garcia, George A.
中科院分区：
文献类型：
--
作者：
Gill, Sumandeep K.;Garcia, George A.
结核分枝杆菌（MTB）感染全球900多万人，每年夺去约200万人的生命。利福平（Rif）是一线抗结核药物之一，其通过与原核RNA聚合酶（RNAP）的β亚基（由rpoB基因编码）结合来抑制转录。原核生物B亚基中高度保守的81个碱基对核心区含有大部分导致利福霉素耐药（RifR）突变的点突变，其中大部分临床相关MT B RifR突变由以下三种氨基酸之一的氨基酸取代引起：β Asp 435、β His 445和β Ser 450（MT B编号）。在这项研究中，为了确定利福霉素对MTB RNAP的直接作用，构建了共过表达载体以共表达MTB RNAP的野生型和RifR突变体的核心亚基。通过定点诱变，将上述三种氨基酸各自突变为在MTB临床分离株中发现的rpoB基因中最普遍的取代（Asp 435 Val、His 445 Tyr、Ser 450 Leu）。在通过两步柱层析纯化后，通过滚环转录测定评估野生型和RifR突变体MTB RNAP的体外活性。测定了三种关键利福霉素（利福平（Rif）、利福昔明（Rbn）和利福昔明（Rfx））的表观IC 50值，这些结果表明突变体RNAP相对于野生型MTB RNAP表现出约10（3）倍或更大的利福霉素亲和力损失。沿着MTB RNAP，还评估了利福霉素对大肠杆菌RNAP对应物的抑制。以前，据报道，革兰氏阳性菌（特别是分枝杆菌）比革兰氏阴性菌对利福霉素更敏感。在我们的实验条件下，利福霉素对MTB和E.大肠杆菌RNAP（野生型和相应的突变体）是非常相似的，因此，对利福霉素的敏感性的差异不在于RNAP本身。使用野生型E. coli菌株（TG 2和DH 5 α）和突变型E.大肠杆菌EC2880，tolC（-）/imp（-）。EC 2880菌株的MIC显著降低，这与以前的报道一致，即MTB和E.大肠杆菌对利福霉素的敏感性与RNAP无关，而与E.杆菌未来的工作将集中在阐明这些MTB RifR突变体与利福霉素的分子相互作用，为新型利福霉素的设计提供见解。(C)2011爱思唯尔有限公司保留所有权利。
Mycobacterium tuberculosis (MTB) infects over 9 million people globally and claims approximately 2 million lives annually. Rifampin (Rif) is one of the first-line anti-tuberculosis drugs that inhibits transcription by binding to the beta subunit (encoded by the rpoB gene) of the prokaryotic RNA polymerase (RNAP). A highly conserved 81 base pair core region among the b subunit of prokaryotes harbors most of the point mutations leading to rifamycin-resistant (RifR) mutations, where the majority of the clinically relevant MTB RifR mutations result from amino acid substitutions of one of the following three amino acids: beta Asp435, beta His445, and beta Ser450 (MTB numbering). In this study, to determine the direct effect of rifamycins on the MTB RNAP, co-overexpression vectors were constructed to co-express the core subunits of wild-type and RifR mutants of MTB RNAP. The three aforementioned amino acids were each mutated to the most prevalent substitution found in the MTB clinical isolates (Asp435Val, His445Tyr, Ser450Leu) in the rpoB gene via site-directed mutagenesis. After purification via two-step column chromatography, the in vitro activity of the wild-type and RifR mutant MTB RNAPs was assessed via rolling circle transcription assay. The apparent IC50 values for three key rifamycins (rifampin (Rif), rifabutin (Rbn), and rifaximin (Rfx)) were determined and these results indicate that the mutant RNAPs demonstrate approximately 10(3)-fold or greater loss of affinities for rifamycins relative to wild-type MTB RNAP.Along with the MTB RNAPs, rifamycin inhibition of the Escherichia coli RNAP counterparts was also assessed. Previously, it has been reported that Gram-positive bacteria (particularly mycobacteria) are more sensitive to rifamycins than Gram-negative bacteria. Under our experimental conditions, the rifamycin IC(50)s for wild-type and RifR mutants of MTB and E. coli RNAPs (wild-type and corresponding mutants) were very similar; therefore, the difference in sensitivity toward rifamycins does not reside in the RNAP itself. The correlation between the sensitivity of rifamycins and permeability into cells was evaluated using the wild-type E. coli strains (TG2 and DH5 alpha) and a mutant E. coli strain with efflux pump defects (EC2880, tolC(-)/imp(-)). The MICs were drastically lower in the EC2880 strain, consistent with previous reports that the differential sensitivity of MTB and E. coli to rifamycins is not related to the RNAP, but rather has to do with efflux pumps in E. coli. Future work will focus on the elucidation of the molecular interaction of these MTB RifR mutants with rifamycins to provide insight to the design of novel rifamycins. (C) 2011 Elsevier Ltd. All rights reserved.