Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Abstract

Vancomycin-resistant enterococci (VRE) are common hospital pathogens that are resistant to most major classes of antibiotics. The incidence of VRE is increasing rapidly, to the point where over one-quarter of enterococcal infections in intensive care units are now resistant to vancomycin. The exact mechanism by which VRE maintains its plasmid-encoded resistance genes is ill-defined, and novel targets for the treatment of VRE are lacking. In an effort to identify novel protein targets for the treatment of VRE infections, we probed the plasmids obtained from 75 VRE isolates for the presence of toxin-antitoxin (TA) gene systems. Remarkably, genes for one particular TA pair, the mazEF system (originally identified on the Escherichia coli chromosome), were present on plasmids from 75/75 (100%) of the isolates. Furthermore, mazEF was on the same plasmid as vanA in the vast majority of cases (>90%). Plasmid stability tests and RT-PCR raise the possibility that this plasmidencoded mazEF is indeed functional in enterococci. Given this ubiquity of mazEF in VRE and the deleterious activity of the MazF toxin, disruption of mazEF with pharmacological agents is an attractive strategy for tailored antimicrobial therapy.antibiotics ͉ mazEF ͉ axe-txe ͉ relbE ͉ VRE E nterococci are the leading cause of surgical-site infections and the third leading cause of urinary tract and bloodstream infections (1, 2) and are implicated in bacterial endocarditis, intraabdominal infections, bacteremia, and meningitis (3). The intrinsic resistance of enterococci to cephalosporin and aminoglycoside antibiotics complicates treatment strategies. Historically, vancomycin has been effective in the management of enterococcal infections; however, after nearly 30 years of use, vancomycin-resistant enterococci (VRE) emerged in the mid-1980s. In a short time span, intensive care units in the U.S. have seen a dramatic increase in the percentage of enterococcal infections that are resistant to vancomycin, from 0. 4% in 1989 to 25% in 1999 (3). In fact, in a recent study, 60% of Enterococcus faecium clinical isolates were resistant to vancomycin (4).The genes encoding the elaborate protein systems that mediate vancomycin resistance generally reside on mobile genetic elements within the enterococci. In some cases, plasmids with the various vancomycin resistance gene clusters have been recovered from enterococci (5-8), although the prevalence of plasmid-encoded resistance in VRE has not been defined. Large plasmids often have intricate mechanisms by which they maintain themselves in the bacterial population. Some plasmids contain genes encoding postsegregational killing ''plasmid addiction'' systems (9, 10), in which the plasmid encodes a potent toxin and a labile antitoxin. Provided the plasmid is present, the antitoxin binds to and sequesters the toxin. However, if a plasmid-free daughter cell arises, the unstable antitoxin is degraded, and the toxic protein kills the bacterial cell from within.If toxin-antitoxin (TA) systems are prevalent and functional on ...

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

“…TA-driven RNA interference has been implicated in the regulation of virulence and antibiotic resistance in numerous pathogens including M. tuberculosis 16 , S. aureus 15,20 , Yersinia pestis 21 , Helicobacter pylori 22 , Streptococcus mutans 23 and C. difficile 17 , and also among enterococci 24 . However, the occurrence of this mechanism remains largely speculative, and the very attractive concept of global regulation of virulence factor expression by TA systems in pathogenic bacteria still awaits thorough experimental characterization.…”

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

“…The contribution of plasmids to antibiotic resistance in bacterial pathogens has led to the proposal that agents inhibiting the replication of these extrachromosomal DNAs, or activating their PSK systems, could be exploited to kill these organisms directly and selectively (11)(12)(13). Our work raises concerns against this approach in the case of R1 and its kis-kid TA pair.…”

“…The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5,(11)(12)(13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids.…”

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

“…Most proteic TA systems are characterized by two small (8)(9)(10)(11)(12)(13)(14)(15) proteins, a stable toxin and a labile antitoxin, and genes encoding these proteins have been identified on a wide range of bacterial chromosomes and plasmids [4,5]. If both proteins are actively being produced by the bacterial cell, the antitoxin binds the toxin and inhibits its toxic activity.…”

“…Given that a large percentage of genes that mediate resistance to antibiotics are found on extrachromosomal DNA such as plasmids [14,15], coupled with the ability of TA systems to stabilize plasmids, it has been speculated that pharmacological disruption of the toxin-antitoxin protein-protein interaction could unleash the toxin and be a novel antibacterial strategy [2,5,16,17]. It was not until recently, however, that it was discovered that TA systems are indeed ubiquitous on plasmids that reside in a common bacterial pathogen that is refractory to most antibiotics, vancomycin-resistant enterococci (VRE) [5].…”