The Communication Factor EDF and the Toxin–Antitoxin Module mazEF Determine the Mode of Action of Antibiotics

It was recently reported that the production of Reactive Oxygen Species (ROS) is a common mechanism of cell death induced by bactericidal antibiotics. Here we show that triggering the Escherichia coli chromosomal toxin–antitoxin system mazEF is an additional determinant in the mode of action of some antibiotics. We treated E. coli cultures by antibiotics belonging to one of two groups: (i) Inhibitors of transcription and/or translation, and (ii) DNA damaging. We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action. However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway. Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction

Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin–antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms

Synthesis and Activity of Biomimetic Biofilm Disruptors

Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis, to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus. An integrated approach involving structure–activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors

Analysis of adenoviral attachment to human platelets

<p>Abstract</p> <p>Background</p> <p>Systemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, <it>in vitro </it>analysis of Ad attachment to platelets is of interest.</p> <p>Methods</p> <p>We developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry.</p> <p>Results</p> <p>We found <it>in vitro </it>that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, αIIbβ3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets.</p> <p>Conclusion</p> <p>The flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful <it>in vitro </it>approach to investigate Ad interaction with platelets.</p

RETRACTED: A Self-Produced Trigger for Biofilm Disassembly that Targets Exopolysaccharide

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the authors. In this article, we reported that norspermidine is produced in aged biofilm cultures of Bacillus subtilis and that norspermidine could disassemble and inhibit B. subtilis biofilms. Both claims have been challenged by Hobley et al. (2014, Cell 156, 844–854). We have subsequently repeated the experiments and have found that the new results can no longer support our original conclusions. Therefore, the most appropriate course of action is to retract the article. We offer our apologies for these errors and for any inconvenience that they may have caused

Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.Organismic and Evolutionary Biolog

Norspermidine is not a self-produced trigger for biofilm disassembly

SummaryFormation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 μM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50–80 μM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 μM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species