Exposing plasmids as the Achilles' heel of drug-resistant bacteria

Abstract

Many multidrug-resistant bacterial pathogens harbor large plasmids that encode proteins conferring resistance to antibiotics. Although the acquisition of these plasmids often enables bacteria to survive in the presence of antibiotics, it is possible that plasmids also represent a vulnerability that can be exploited in tailored antibacterial therapy. This review highlights three recently described strategies designed to specifically combat bacteria harboring such plasmids: inhibition of plasmid conjugation, inhibition of plasmid replication, and exploitation of plasmid-encoded toxin-antitoxin systems.

Fig. 1

Three approaches to exploit plasmids in antibacterial therapy. 1. Plasmids are transferred between bacteria through conjugation. Inhibition of the relaxase enzyme (blue oval) has been proposed as an antibacterial strategy, and several relaxase inhibitors have been identified. 2. Plasmids replicate to maintain themselves in the bacterial population. Strategies have been devised and small molecules have been identified that inhibit plasmid replication, thus resulting in the elimination of the plasmid from the bacterial population and re-sensitizing the bacteria to antibiotics. 3) Large plasmids rely on elaborate mechanisms to ensure their faithful segregation to daughter cells after cell division. One of the most common plasmid maintenance systems is the toxin-antitoxin (TA) post-segregational killing mechanism. In this mechanism, if a plasmid-free daughter cell arises, the labile antitoxin is degraded and the toxin induces cell death. TA genes are ubiquitous in clinical isolates of certain drug-resistant bacteria, and it has been postulated that compounds that disrupt the toxin-antitoxin interaction could free the toxin to kill the bacterial cell.

Fig. 2

The identification of inhibitors of plasmid conjugation. (A) To screen for inhibitors of plasmid conjugation, a donor cell harboring an F plasmid derivative with the lux gene under control of the lac promoter is utilized. This cell also harbors a second plasmid, a non-mobilizable plasmid containing lacI, encoding the lac repressor protein LacI; LacI binds lacO, repressing the expression of lux. Transfer of the F plasmid derivative to the recipient cell results in luciferase production and light emission. (B) This screen was used to identify two conjugation inhibitors: DHCA and linoleic acid.

Fig. 3

Targeting relaxase as a strategy to inhibit plasmid conjugation. (A) Overview of the relaxase mechanism. The relaxase domain (shown as the blue circle) domain of TrwC binds the oriT (1) of the T-strand. Tyrosine-19 (shown as an orange circle) of relaxase catalyzes a cleavage at the nic site and forms a covalent phosphotyrosyl intermediate with the 5′ terminus of oriT (2). Rolling circle replication using the uncleaved strand as a template displaces the T-strand (3) and Y26 cleaves at the T-strand nic site (4). The T-strand is re-ligated and transferred to the recipient cell. Figure adapted from Llosa et al. [72] and Llosa and de la Cruz [73]. (B) Certain bisphosphonates will inhibit relaxase and prevent plasmid conjugation. The bisphosphonates shown are inhibitors of F plasmid TraI relaxase; these compounds prevent plasmid conjugation and induce plasmid- and relaxase-dependent cell death.

Fig 4

“Antiplasmid” antibiotics induce plasmid elimination from the bacterial cell population, resensitzing bacteria to antibiotics. A) Plasmid replication control by RepA. In IncB systems, an intramolecular pseudoknot forms between SLI and SLIII of the RepA mRNA, allowing for ribosome binding and Rep protein translation (left). Rep protein translation and thus plasmid replication is inhibited by the reversible binding of RNA I (middle) or a small molecule (right, orange hexagon) to SLI. Figure adapated from Thomas et al. [19]. B) The compound apramycin was discovered to bind to SLI in vitro, and induce the elimination of an IncB plasmid from bacterial cells in culture.

Fig. 5

Activation of the toxin of a toxin-antitoxin system, and subsequent cell death. At least two mechanisms for toxin activation are possible. In pathway 1 (left), the small molecule acts to directly or indirectly to inhibit transcription and/or translation of the TA locus, preventing the replenishment of the labile antitoxin, which is quickly degraded. This frees the toxin to kill the cell. This half of figure adapted from Engelberg-Kulka et al. [22,23,74] In pathway 2 (right), the small molecule directly disrupts the toxin-antitoxin protein-protein interaction, freeing the toxin to kill the cell.

Fig. 6

The genes for toxin-antitoxin systems have different functions, depending on their location. A) Chromosomally-encoded TA systems are possibly used by the cell to respond to stress. Stress causes toxin activation and reversible inhibition of cell growth; if the “point of no return” is reached, cell death occurs. B) Plasmid-encoded TA systems function as a “post-segregational killing” system, resulting in the elimination of plasmid-free bacterial cells.