MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa:: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719
MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa:: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719
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DOI：
10.1111/j.1365-2958.2004.04210.x
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发表时间：
2004-09-01
影响因子：
3.6
通讯作者：
Poole, K
中科院分区：
文献类型：
--
作者：
Cao, L;Srikumar, R;Poole, K
MexAB - OprM是一种多药外排系统，它导致铜绿假单胞菌具有固有和获得性多药耐药性，后者是由于mexAB - oprM操纵子的突变性过度表达所致。虽然外排基因过度表达通常是由相关的mexR阻遏基因的突变引起的，但在所谓的nalC突变体中也会独立于mexR突变发生，这些nalC突变体表现出比mexR菌株更适度的mexAB - oprM表达，因此具有更适度的多药耐药性。采用转座子插入诱变方法，筛选出nalC突变株，并鉴定出被破坏的基因PA3721。对先前分离的自发nalC突变体中该基因进行扩增和测序，结果显示在所有情况下都存在突变，因此，PA3721已被重新命名为nalC。PA3721（nalC）编码一种可能属于TetR/AcrR家族的阻遏物，位于一个明显的双基因操纵子PA3720 - PA3719的上游，PA3720 - PA3719的表达受PA3721负调控。因此，在转座子插入和自发nalC突变体中，PA3720 - PA3719过度表达。在一个自发nalC突变体中，PA3719（而非PA3720）表达缺失使MexAB - OprM表达降低至野生型水平，并损害了多药耐药性，这表明只有PA3719的过度表达对于nalC表型是必需的。实际上，将PA3719通过多拷贝质粒导入野生型铜绿假单胞菌中，足以促进MexAB - OprM表达升高以及出现nalC菌株所具有的多药耐药特征。因此，nalC（PA3721）突变仅用于增强PA3720 - PA3719的表达，PA3719（编码一种预测等电点为10.4的53个氨基酸的蛋白质）的表达直接或间接影响MexAB - OprM的表达。有趣的是，nalC菌株产生的稳定MexR蛋白水平显著升高，这表明PA3720 - PA3719的过度表达以某种方式调节MexR阻遏物的活性。然而，PA3720 - PA3719推导的产物与GenBank数据库中现有的序列没有同源性，因此无法提供关于这种情况如何发生的线索。
MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.