Jonathan A. Winkler

Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria

Silver enhances the activity of a wide range of antibiotics and broadens the spectrum of vancomycin, rendering it effective against Gram-negative bacteria. A Silver Spoon Makes the Medicine Go Down There is a growing need to enhance our antibacterial arsenal given the rising incidence of antibiotic resistance and the emergence of new virulent pathogens. Drug-resistant, difficult-to-treat Gram-negative bacterial infections have forced clinicians to revisit the use of older antimicrobials that have previously been discarded. Such is the case of silver, an intriguing compound that, despite its long-standing history as an antimicrobial (since 400 B.C.), has an unclear bactericidal mode of action. In their new study, Morones-Ramirez and his colleagues use a systems-based approach to show that silver disrupts multiple bacterial cellular processes, leading to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria. The authors harnessed these effects to potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as to restore antibiotic susceptibility to resistant bacterial strains. They show both in vitro and in vivo that (i) silver’s ability to induce oxidative stress can be harnessed to potentiate antibiotic activity; (ii) silver sensitizes Gram-negative bacteria to the Gram-positive–specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug; and (iii) silver enhances antibiotic activity against bacterial persister cells and biofilms. This new study provides a way to enhance the activity of existing antimicrobials and goes some way toward enlarging the dwindling armamentarium of drugs to fight bacterial diseases. A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive–specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production

The ever-increasing incidence of antibiotic-resistant infections combined with a weak pipeline of new antibiotics has created a global public health crisis. Accordingly, novel strategies for enhancing our antibiotic arsenal are needed. As antibiotics kill bacteria in part by inducing reactive oxygen species (ROS), we reasoned that targeting microbial ROS production might potentiate antibiotic activity. Here we show that ROS production can be predictably enhanced in Escherichia coli, increasing the bacterias susceptibility to oxidative attack. We developed an ensemble approach of genome-scale, metabolic models capable of predicting ROS production in E. coli. The metabolic network was systematically perturbed and its flux distribution analyzed to identify targets predicted to increase ROS production. Targets that were predicted in silico were experimentally validated and further shown to confer increased susceptibility to oxidants. Validated targets also increased susceptibility to killing by antibiotics. This work establishes a systems-based method to tune ROS production in bacteria and demonstrates that increased microbial ROS production can potentiate killing by oxidants and antibiotics.

Hydroxyurea Induces Hydroxyl Radical-Mediated Cell Death in Escherichia coli

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. Although HU inhibition of RNR is well recognized, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death, we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability. Continued HU stress activates iron uptake and toxins MazF and RelE, whose activity causes the synthesis of incompletely translated proteins and stimulation of envelope stress responses. These effects alter the properties of one of the cells terminal cytochrome oxidases, causing an increase in superoxide production. The increased superoxide production, together with the increased iron uptake, fuels the formation of hydroxyl radicals that contribute to HU-induced cell death.

Central role for RNase YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria.

BackgroundConceptual parallels exist between bacterial and eukaryotic small-RNA (sRNA) pathways, yet relatively little is known about which protein may recognize and recruit bacterial sRNAs to interact with targets. In eukaryotes, Argonaute (AGO) proteins discharge such functions. The highly conserved bacterial YbeY RNase has structural similarities to the MID domain of AGOs. A limited study had indicated that in Sinorhizobium meliloti the YbeY ortholog regulates the accumulation of sRNAs as well as the target mRNAs, raising the possibility that YbeY may play a previously unrecognized role in bacterial sRNA regulation.ResultsWe have applied a multipronged approach of loss-of-function studies, genome-wide mRNA and sRNA expression profiling, pathway analysis, target prediction, literature mining and network analysis to unravel YbeY-dependent molecular responses of E. coli exposed to hydroxyurea (HU). Loss of ybeY function, which results in a marked resistance to HU, had global affects on sRNA-mediated gene expression. Of 54 detectable E. coli sRNAs in our microarray analysis, 30 sRNAs showed a differential expression upon HU stress, of which 28 sRNAs displayed a YbeY-dependent change in expression. These included 12 Hfq-dependent and 16 Hfq-independent sRNAs. We successfully identified at least 57 experimentally inferred sRNA-mRNA relationships. Further applying a ‘context likelihood of relatedness’ algorithm, we reverse engineered the YbeY-dependent Hfq-dependent sRNA-mRNA network as well as YbeY-dependent Hfq-independent sRNA-mRNA network.ConclusionYbeY extensively modulates Hfq-dependent and independent sRNA-mRNA interactions. YbeY-dependent sRNAs have central roles in modulating cellular response to HU stress.

Identification and Characterization of Programmed Cell Death Markers in Bacterial Models

In eukaryotic organisms facing terminal stress, activation of genetically encoded cell death pathways underlies fundamental changes in core cellular processes and functional modification of critical biomolecules. These physiological alterations manifest themselves as phenotypic hallmarks during programmed cell death, and are markers of the particular mode of death initiated. A growing volume of work has illustrated that prokaryotes too are capable of exhibiting hallmarks of programmed cell death, albeit without the multiple, tight regulatory layers which control these events in higher order organisms.This chapter describes how methods and materials which have been used to assay for hallmarks of programmed cell death in eukaryotic models are transferrable to prokaryotic models. In particular, we describe the applicability of these methods to the study of post-antibiotic effects on bacteria, notably the biochemical changes induced by the interaction of drug molecules and targets, including oxidative stress, that accompany and ensure cell death. Specifically we discuss techniques for detecting DNA fragmentation, chromosomal condensation, phosphatidylserine exposure, membrane depolarization, and caspase substrate peptide binding, thereby providing a launchpoint for the study of the evolution of these physiological events in bacteria.