Mechanisms of resistance to an amino acid antibiotic that targets translation

被引:37
作者
Ataide, Sandro F. [1 ]
Wilson, Shamise N. [2 ]
Dang, Sandy [1 ]
Rogers, Theresa E. [1 ]
Roy, Bappaditya [4 ]
Banerjee, Rajat [4 ]
Henkin, Tina M. [1 ,2 ,3 ]
Ibba, Michael [1 ,2 ,3 ]
机构
[1] Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA
[2] Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA
[3] Ohio State Univ, Ohio State RNA Grp, Columbus, OH 43210 USA
[4] Univ Calcutta, BC Guha Ctr Genet Engn & Biotechnol, Kolkata 700019, W Bengal, India
关键词
D O I
10.1021/cb7002253
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Structural and functional diversity among the aminoacyl-tRNA synthetases prevent infiltration of the genetic code by noncognate amino acids. To explore whether these same features distinguish the synthetases as potential sources of resistance against antibiotic amino acid analogues, we investigated bacterial growth inhibition by S-(2-aminoethyl)-L-cysteine (AEC). Wild-type lysyl-tRNA synthetase (LysRS) and a series of active site variants were screened for their ability to restore growth of an Escherichia coli LysRS null strain at increasing concentrations of AEC. While wild-type E. coli growth is completely inhibited at 5 mu M AEC, two LysRS variants, Y280F and F426W, provided substantial resistance and allowed E. coli to grow in the presence of up to 1 mM AEC. Elevated resistance did not reflect changes in the kinetics of amino acid activation or tRNA(Lys) aminoacylation, which showed at best 4-6-fold improvements, but instead correlated with the binding affinity for AEC, which was decreased similar to 50-fold in the LysRS variants. In addition to changes in LysRS, AEC resistance has also been attributed to mutations in the L box riboswitch, which regulates expression of the lysC gene, encoding aspartokinase. The Y280F and F426W LysRS mutants contained wild-type L box riboswitches that responded normally to AEC in vitro, indicating that LysRS is the primary cellular target of this antibiotic. These findings suggest that the AEC resistance conferred by L box mutations is an indirect effect resulting from derepression of lysC expression and increased cellular pools of lysine, which results in more effective competition with AEC for binding to LysRS.
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收藏
页码:819 / 827
页数:9
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