A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis

Abstract

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 Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics.

Figure 1. Phenotype of PezTΔC₂₄₂ expression in E. coli.

(A) Phase contrast image of fixed E. coli cells bearing pET28b(pezTΔC₂₄₂) grown in liquid cultures before and 30 min and 60 min after toxin expression. The insets show representative unfixed cells that were examined after live/dead fluorescence staining. (B) Phase contrast image of adherently growing E. coli cell 1 h after induction of PezTΔC₂₄₂ (left) or PezTΔC₂₄₂ (D66T) expression (right). (C) Time correlation between cell growth/lysis and the breakdown of the osmotic barrier. Growth kinetics were monitored by measuring the optical density (upper panel) of cultures expressing PezTΔC₂₄₂ (solid red line) or nontoxic PezTΔC₂₄₂ (D66T) (solid black line). The influx of membrane-impermeable propidium iodide present in the medium was measured in parallel by the increase in fluorescence at 620 nm (lower panel). Additional cultures expressing nontoxic PezTΔC₂₄₂ (D66T) were treated with either 50 µg/ml ampicillin (dashed red line) or 15 µg/ml tetracycline (dashed blue line).

Figure 2. PezT from S. pneumoniae and zeta from S. pyogenes phosphorylate UNAG.

(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 S. pyogenes after 1 h (dark red) and 24 h (red) of incubation. The asterisk indicates the retention volume of the product formed by zeta. Note that UNAG turnover was slow because of the presence of stoichiometric amounts of the antitoxin epsilon. However, in contrast to the PezAT system, zeta is not entirely inhibited. This difference is most probably caused by the different TA affinities in the two TA systems ,. (C) Schematic reaction mechanism of UNAG-3P formation by zeta toxins.

Figure 3. UNAG bound to zeta toxin from S. pyogenes.

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.

Figure 4. UNAG-3P accumulates in cells expressing PezTΔC₂₄₂.

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.

Figure 5. Mechanism of MurA inhibition by UNAG-3P.

(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 (λ_exc = 300 nm). (C) Determination of the K _i of UNAG-3P for MurA under steady state conditions. The UNAG-3P concentrations for each saturation curve were 0 µM (black circles), 15 µM (dark red circles), and 30 µM (red triangles). A Lineweaver-Burk plot is shown as inset. The saturation curves were fitted globally by nonlinear regression assuming competitive inhibition (black line), yielding a K _m of 15 µM for UNAG and a K _i of 7 µM for UNAG-3P.

Figure 6. Growth rate determines toxicity of PezT expression.

(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.

Figure 7. Model for the function of the pneumococcal PezAT systems during 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 S. pneumoniae . Moreover, partial autolysis and the general inhibition of capsular polysaccharide synthesis by UNAG-3P will favor biofilm formation. Cells surviving PezT release will eventually recover by production of PezA in absence of the stress conditions.