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

Williams, Julia J.; Hergenrother, Paul J.

doi:10.1016/j.cbpa.2008.06.015

Cited by 80 publications

(65 citation statements)

References 71 publications

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“…Lastly, a single HigB residue modulates adenosine selectively at this third A-site position by a mode that is distinct from other ribosome-dependent toxins (21,22). Because toxin proteins play prominent roles in persister cell formation and represent novel antimicrobial targets (8,11,35,36), determining the molecular basis of mRNA substrate specificity may provide insights to subvert toxin activity.…”

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confidence: 99%

Defining the mRNA recognition signature of a bacterial toxin protein

Schureck

Dunkle

Maehigashi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosomedependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide. (1,3,4). This rapid inhibitory switch suppresses metabolite consumption and temporarily halts cell growth to promote bacterial survival until nutrients are readily available. Among the prosurvival genes regulated by (p)ppGpp production are toxin-antitoxin modules, which have additional roles in antibiotic resistance and tolerance, biofilm and persister cell formation, and niche-specific colonization (5-11). The critical roles toxin-antitoxin pairs play in bacterial physiology underscore the importance of understanding their molecular targets and modes of action.There are five different classes (I to V) of toxin-antitoxin systems defined by how the antitoxin represses toxin function (1). Type II toxin-antitoxin pairs form protein-protein complexes during exponential growth that serve two purposes: inhibition of toxin activity by antitoxin binding and transcriptional autorepression to limit toxin expression (12). Antitoxins are proteolytically degraded after (p)ppGpp accumulation, leading to derepression at the toxin-antitoxin promoter (8, 12). Liberated toxin proteins inhibit the replication or translation machinery by targeting DNA gyrase, initiator tRNA fMet , glutamyl-tRNA synthetase, EF-Tu, free mRNA, ribosome-bound mRNA, and the ribosome itself (13-20).Ribosome-dependent toxins cleave mRNAs on the ribosome between the second and third nucleotides of the aminoacyl (A)-site codon (21-23). Although collectively Escherichia coli ribosome-dependent toxins target a diverse range of codons, each individual toxin appears to have a strong codon preference and cleaves at defined positions along the mRNA (24-26). RelE cleaves at UAG stop codons and the CAG sense codon (all codons denoted in the 5′-3′ direction); YoeB cleaves at codons following a translational AUG start site and at the UAA stop codon; and YafQ cleaves a single AAA sense codon (16,24,(27)(28)(29). In contrast, Proteus vulgaris host inhibition of growth B (HigB) toxin degrades multiple codons encod...

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confidence: 99%

Defining the mRNA recognition signature of a bacterial toxin protein

Schureck

Dunkle

Maehigashi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…onjugative transfer of plasmid DNA is an important process that contributes to bacterial genome plasticity and adaption, particularly the spread of antibiotic resistance and virulence genes (1,2). The translocation of a conjugative plasmid relies on the coupling protein, the relaxosome complex, and a type IV secretion system (T4SS), known as the mating-pair formation (Mpf) apparatus (1).…”

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confidence: 99%

Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

et al. 2015

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The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus. Conjugative transfer of plasmid DNA is an important process that contributes to bacterial genome plasticity and adaption, particularly the spread of antibiotic resistance and virulence genes (1, 2). The translocation of a conjugative plasmid relies on the coupling protein, the relaxosome complex, and a type IV secretion system (T4SS), known as the mating-pair formation (Mpf) apparatus (1). For conjugation systems in Gram-negative bacteria, structural resolution of the outer membrane core complex and the inner membrane complex of the T4SS, as well as information about the pathway of DNA translocation across the T4SS in the donor cell, has been elucidated (3-11). Less is known about conjugation systems in Gram-positive bacteria, with our current understanding based primarily on comparisons to those in Gramnegative bacteria and with the systems in Gram-positive bacteria proposed to involve a "minimized" T4SS (3, 12-18).The Gram-positive, anaerobic pathogen Clostridium perfringens carries many disease-mediating toxin genes and antibiotic resistance genes on a unique family of large plasmids that have large regions of sequence identity (19)(20)(21)(22). Most of these plasmids carry the novel tcp conjugation locus (20, 23). The tcp locus has been identified on all known conjugative plasmids in C. perfringens, and proteins encoded by this locus have been shown to be required for conjugative transfer of the paradigm C. perfringens conjugative plasmid, pCW3 (20,(23)(24)(25)(26). The presence of the tcp genes on so many of these toxin plasmids suggests that the transfer of toxin genes by a shared conjugation mechanism in C. perfringens is important in disease dissemination. To this effect, several of these toxin plasmids have been shown experimentally to be conjugative (19,27,28).M...

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“…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.…”

Section: Kid Inhibits Cell Division In E Coli and Does Not Halt Protmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Pimentel

Nair

Bermejo-Rodríguez

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.PemK | mRNA interferase | parD | plasmid stability | RNase

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

Cited by 80 publications

References 71 publications

Defining the mRNA recognition signature of a bacterial toxin protein

Defining the mRNA recognition signature of a bacterial toxin protein

Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

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

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