The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: HM RLS AM. Performed the experiments: HM MG RLS AM. Analyzed the data: HM RLS AM. Contributed reagents/materials/analysis tools: AM. Wrote the paper: HM AM.
The authors have declared that no competing interests exist.
Most genomes of bacteria contain toxin–antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in
Most bacteria harbor toxin–antitoxin (TA) systems, in which a bacterial toxin is rendered inactive under resting conditions by its antitoxin counterpart. Under conditions of stress, however, the antitoxin is degraded, freeing the toxin to attack its host bacterium. One such TA system, PezAT, has been difficult to study in the past because the PezT toxin is so toxic without its antitoxin counterpart that bacteria die before any useful measurements can be made. Here, we use a truncated version of PezT that kills bacteria more slowly than normal, allowing us to examine the mechanisms of how this TA system operates. We find that zeta toxins convert an essential building block of bacterial cell walls (known as UNAG) into a form that prevents normal cell wall growth, causing distortions in bacterial shape that leave the bacteria vulnerable to the hydrostatic pressure of its contents. Consequently, the bacteria burst, similar to what happens when they are treated with penicillin. These results may serve useful for designing new antibiotics. Additionally, our results support the hypothesis that activation of PezT during bacterial infections may be a method by which rapidly growing bacteria can instigate a suicide program, which would promote the release of virulence factors that facilitate spread of infections.
Almost all prokaryotic genomes encode toxin–antitoxin (TA) systems
Members of the epsilon/zeta TA family have been shown to stabilize resistance plasmids in major human pathogens such as
PezAT systems are found encoded on pneumococcal pathogenicity islands that support their host with virulence factors and resistance to different antibiotics
Here, we reveal the mechanism used by zeta toxins to induce programmed cell death in bacteria. Since expression of wild-type zeta toxins leads to either instantaneous cell death or spontaneous mutation of the open reading frame
Genetic manipulations of the full-length zeta or homologous PezT toxins without the cognate antitoxin are unfeasible because of the high toxicity of the proteins. Moreover, toxin variants that can be isolated from surviving clones are generally inactive because of spontaneous mutations
(A) Phase contrast image of fixed
Lysis through bulge formation implied that cells poisoned by PezT suffered from defects in their cell wall integrity. Bacterial growth and binary fission demand a tightly controlled balance between murein synthesis and degradation. Perturbations of this balance are known to cause uncontrolled peptidoglycan degradation by murein hydrolases following lysis because of the intracellular turgor pressure
To further corroborate our hypothesis that PezT targets cell wall synthesis, we probed whether toxin expression resembles β-lactam treatment in general. We found that the onset of cell death caused by PezT expression was preceded by a strong rise in membrane permeability, by measuring influx kinetics of the membrane-impermeable dye propidium iodide (
In summary, we concluded that the toxic mechanism of PezT causes inhibition of cell wall synthesis that eventually provokes bacterial autolysis. Nevertheless, the identity of the molecular target of PezT remained enigmatic, since inhibition of cytosolic steps of bacterial cell wall synthesis can occur on a multitude of levels
Given the structural similarity of PezT and zeta toxins with phosphotransferases
(A) Samples containing 0.25 mM UNAG, 5 mM MgCl2, 1 mM ATP, and 1 µM PezTΔC242 (red) or additionally 1 µM PezA antitoxin (black) were analyzed by anion exchange chromatography after 1 h of incubation at 25°C. The asterisk indicates the retention volume of the product formed by PezTΔC242 in the absence of the antitoxin PezA. (B) Analysis of the equivalent reaction using 1 µM epsilon/zeta complex from
We investigated next whether PezT indeed phosphorylates UNAG. To this end, we performed electrospray ionization spectrometry experiments, which showed that the product of the PezT toxin and UNAG differ by the mass of a phosphoryl group (Δ
Site directed mutagenesis studies of PezT and zeta toxins yielded several inactive toxin variants that have been linked with binding of the at-that-time-unknown substrate
UNAG binds to a deep cleft at the molecular surface of the zeta toxin (
A transparent molecular surface representation of zeta toxin with a ribbon representation shown underneath. Residues of the zeta toxin that are important for substrate binding are depicted as a stick model. UNAG shown as a stick model is embedded in a deep cleft. Hydrogen bonds relevant for substrate binding are illustrated as yellow dashed lines.
In accordance with our in vitro results, we also showed that UNAG-3P is the main product of the PezT toxin activity in vivo. In fact, we identified enriched UNAG-3P in low-molecular-weight-metabolite pools of PezT-poisoned cells obtained by aqueous acetonitrile extraction and HPLC (
In vivo extracts of metabolites from cells after 1 h of PezTΔC242 (red) or PezTΔC242 (D66T) (blue) expression. The mixture was analyzed by HPLC using a strong anion exchange column. The chromatogram of authentic standards is shown in the top panel (black). Note that individual concentrations of isolated small molecules cannot be compared quantitatively, since concentrations of individual runs were adjusted to similar absorbance at 260 nm. Furthermore, some species may have been partially degraded during extraction.
Modification of the amino sugar 3′-hydroxyl group by PezT and zeta toxins suggests several possible scenarios by which UNAG-3P can interfere with peptidoglycan synthesis. One of these is inhibition of the conserved enolpyruvyl transferase MurA, which catalyzes the initial step of muramic acid synthesis. Subsequent to the hexosamine biosynthesis pathway, MurA modifies the amino sugar 3′-hydroxyl group of UNAG (
(A) MurA performs the first step of UDP-muramic acid biosynthesis. After sequential binding of UNAG and phosphoenolpyruvate (PEP), a tetrahedral intermediate is formed that yields enolpyruvyl-UNAG after cleavage of inorganic phosphate. UNAG-3P most likely mimics this tetrahedral intermediate and thereby inhibits MurA catalysis by competitive inhibition. (B) MurA is able to transfer the enolpyruvyl moiety from phosphoenolpyruvate (1 mM) to UNAG (black) but not UNAG-3P (red). MurA enzyme kinetics were followed by coupling the reaction to phosphate-dependent cleavage of fluorescent 7-methylguanosine by nucleoside phosphorylase, resulting in a decrease in fluorescence at 400 nm (λexc = 300 nm). (C) Determination of the
MurA catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate to the 3′-hydroxyl group of
During the native reaction of MurA, a negatively charged tetrahedral adduct is formed
Most antibiotics that target peptidoglycan synthesis are known to rapidly kill bacteria during exponential growth but fail to kill slowly growing and stationary cells
(A) Cell growth monitored by optical density measurements of parallel cultures after PezTΔC242 induction (red arrows) at different optical density. Growth in fresh LB medium (solid) and nutritionally deprived LB (dashed) is shown before (black) and after induction (red) with IPTG at the optical densities indicated. (B) Cells grown in exhausted medium after 180 min of PezTΔC242 expression observed by phase contrast microscopy after fixation or after fluorescent live/dead staining (inset). Note that the culture was induced at an optical density at which cell growth continues upon PezT expression.
The mechanism of how epsilon/zeta TA systems kill their host cell has remained a mystery to date
The synthesis of muramic acids is regulated by a negative feedback loop in which MurA is inhibited by its downstream product UDP-
UNAG is an abundant metabolite in bacteria
The pneumococcal PezT toxin has been suggested to support virulence of its pathogenic host during infection, since strains in which the PezT gene has been deleted are attenuated in mouse models of infection
Stress conditions lead to release of the UNAG kinase PezT via degradation of the antitoxin PezA. PezT converts the cellular pool of UNAG to UNAG-3P, which leads to inhibition of peptidoglycan synthesis and competes with the synthesis of other glycoconjugates. Metabolically silent persister cells as well as slowly dividing cells will survive PezT release. In contrast, cells that require fully functional murein synthesis, such as rapidly dividing cells, will undergo lysis and release cytosolic pneumolysin, a major virulence factor of
Release of PezT toxin from PezA antitoxin might appear under conditions that lead to either prolonged down-regulation of protein biosynthesis or enhanced PezA degradation. Similar to other TA systems, down-regulation of PezA biosynthesis could occur, for instance, during antibiotic treatment or amino acid starvation, when general transcription or translation is impaired
The ability of TA systems to induce cell lysis or cell stasis has also been linked to biofilm and persister cell formation in pathogens
UNAG-3P is a suicide antibiotic, because bacteria are harmed by their self-inflicted enzymatic activity depending on environmental conditions. This is in contrast to common antibiotics that bacteria produce and target against other species. Additionally, UNAG-3P is a naturally derived lead compound for the development of novel antibiotics. Our results imply that either activation or inhibition of epsilon/zeta and PezAT systems will interfere with the fate of the host bacteria and thus make them a potent Achilles' heel for microbes.
For microscopy, cultures of
To measure time-resolved breakdown of the osmotic barrier, cells were grown in LB medium at 37°C to an OD600 = 0.2 in absence of any inducing agent. Next, 75 µl of culture was diluted with an equal volume of LB containing 1 mM IPTG and 20 µM propidium iodide. Control cultures expressing PezT (D66T) were additionally supplemented with ampicillin (50 µg/ml) or tetracycline (15 µg/ml). Samples for baseline correction were not inoculated with bacteria. All samples were transferred to a black 96-well microtiter plate (Corning) and were incubated at 37°C on a Thermomixer comfort (Eppendorf) equipped with a MTP sample holder. Breakdown of the osmotic barrier and staining of cytosolic DNA/RNA was monitored by measuring fluorescence at 620 nm in a Varioskan Flash (Thermo Scientific) with fluorescence excitation set to 520 nm at 20-min intervals. The optical density was recorded from the same samples in absorbance mode. Between measurements, the microtiter plates were covered with an AirPore tape sheet (Qiagen). For each time series, fluorescence intensities were baseline corrected and averaged (
The kinase activity of PezTΔC242 and its inhibition by PezA were investigated by incubating 1 µM recombinant toxin in buffer R (25 mM HEPES-NaOH [pH 7.5], 100 mM NaCl, 5 mM MgCl2) supplemented with 1 mM ATP and 0.25 mM of the nucleotide sugars UNAG, UDP-
The epsilon/zeta complex was purified as described previously
The enolpyruvyl activity of
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We thank M. Cryle, T. Barends, B. Loll, B. Lunde, and C. C. Yeo for helpful discussions. We especially thank J. Graf (University of Heidelberg) for his expert collection and assignment of NMR spectra. We acknowledge I. Vetter and C. Roome for support of the crystallographic software and IT, M. Müller and A. Helal for technical help, the Dortmund-Heidelberg team for data collection, and the PXII staff for their support in setting up the beamline. Diffraction data were collected at the Swiss Light Source, beamline X10SA, Paul Scherrer Institute, Villigen, Switzerland. We are grateful to I. Schlichting for continuous encouragement and support. A. M. is a member of CellNetworks, Heidelberg, Germany.
high pressure liquid chromatograpy
Luria broth
nuclear magnetic resonance
toxin–antitoxin
uridine diphosphate
uridine diphosphate-
uridine diphosphate-