The Relationship between Aminoglycosides' RNA Binding Proclivity and Their Antiplasmid Effect on an IncB Plasmid

Abstract

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

Ecology and Evolution as Targets: the Need for Novel Eco-Evo Drugs and Strategies To Fight Antibiotic Resistance

“…The exploitation of the mechanisms of plasmid incompatibility has been explored (37,139,153). In this case, the plasmid replication machinery is an obvious target, for instance using compounds able to reduce the intracellular levels of RepA replicase.…”

“…In this case, the plasmid replication machinery is an obvious target, for instance using compounds able to reduce the intracellular levels of RepA replicase. These levels are determined by its interaction with a small RNA, RNAI, whose conformation can be altered by drugs, such as apramycin, at least in incompatibility IncB systems (139). An apparent limitation to such an approach is the possible rapid emergence of plasmid elimination-resistant mutants by mutational changes in the Rep sequences.…”

Biosynthesis of 2-Deoxystreptamine by Three Crucial Enzymes in Streptomyces fradiae NBRC 12773

“…It has been shown that aminoglycosides specifically bind to the bacterial ribosomal RNA and inhibit the bacterial protein biosynthesis [1]. Based on the specific interaction with RNA molecules, many applications such as anti-HIV infection [2,3] and anti-plasmid [4,5], are continuously investigated [6ϳ8]. Thus, the creation of a library for structurally diverse aminoglycoside molecules is strongly desired.…”

Functional analysis of the plastid and nuclear encoded CbbX proteins of Cyanidioschyzon merolae

“…Introduction of the pET-28 (b) plasmid into the above E.coli strain reduced lacZ expression regardless of IPTG addition (Fig. 6, 5 th and 6 th bar) due to incompatibility as both plasmids, pDL and pET, harbor same replication origin (Thomas et al, 2005 (Fujita et al, 2008) suggesting that the increased amounts of CbbX (nuc) did not affect the transcription of RuBisCO operon. If CbbX (nuc) functions as a modifier of CbbX (pt) repressor in vivo in C. merolae, increased CO 2 concentration should affect expression of the RuBisCO operon on the plastid genome by modifying of CbbX (pt) repressor function.…”