Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases

Abstract

The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.

Keywords: (p)ppGpp; HipA; bacterial persistence; single-cell analysis; toxin–antitoxin.

Fig. 1.

TA loci and molecular model integrating HipA into (p)ppGpp-mediated persistence. (A) Chromosomal locations of 11 type II TA loci of E. coli K-12. Ribosome-dependent and -independent mRNase-encoding TA modules are shown in green and blue, respectively. The serine/threonine kinase-encoding hipBA locus is shown in red. In the model (B), free HipA phosphorylates GltX and the resulting inhibition of Glu-tRNA^Glu increases the rate of uncharged tRNA^Glu loading at the A site of the ribosome and triggers RelA-dependent (p)ppGpp production. Then (p)ppGpp competitively inhibits PPX, the cellular enzyme that degrades Poly(P). In turn, Poly(P) is synthesized by polyphosphate kinase (PPK) and stimulates Lon to degrade the 11 type II antitoxins (including HipB), thereby activating the mRNases that inhibit translation and cell growth and induce persistence. Thus, activation of HipA generates a feed-forward loop that theoretically should lock the cells in a state with a high level of (p)ppGpp. However, such a locked state has never been observed experimentally and may be overcome by the ability of HipA to inactivate itself by intermolecular phosphorylation (40), as indicated in B by a line that points back to HipA. The arrow emerging from RelA and pointing back to RelA indicates a positive feedback loop in which (p)ppGpp activates further (p)ppGpp synthesis (47). This positive feedback may contribute to the observed stochasticity in the variation of (p)ppGpp in single cells (8) Broken arrows indicate the possible signaling pathway that by positive feed forward would lock the cells in a state of a permanent high level of (p)ppGpp and therefore constantly activate HipA and TA-encoded mRNases, whereas unbroken arrows indicate the signaling pathway leading to persistence as described previously.

Fig. 2.

HipA-induced persistence depends specifically on RelA. (A) Accumulation of (p)ppGpp following ectopic expression of toxins. MG1655 carrying either hipA, relE, mazF, or yafO on pBAD33 was grown exponentially in low phosphate Mops minimal medium (Materials and Methods). Samples were collected before and after toxin gene induction (0.2% arabinose) and separated by TLC. Here, we present the quantification of (p)ppGpp level after toxin induction. A representative autoradiograph of the TLC plates is shown in Fig. S1A, and growth arrest after toxin induction was monitored by growth curve (Fig. S1C). (B) Exponentially growing cells of MG1655 (gray bars) and MG1655 ∆relA (black bars) carrying either hipA, relE, mazF, or yafO on pBAD33 induced for 30 min were exposed to 2 µg/mL of ciprofloxacin (for details, see Materials and Methods). Percentage of survival after 4 h of antibiotic treatment was compared with that of the control strains carrying the pBAD33 vector plasmid (log scale). Error bars indicate the SDs of averages of at least three independent experiments.

Fig. 3.

HipA-mediated persistence depends on Poly(P), Lon, and the other type II TA loci. Exponentially growing cells of MG1655 (gray bars) and isogenic deletion strains ∆10TA (black bars), ∆(ppk ppx) (white bars), and ∆lon (dashed bars), overexpressing hipA, relE, mazF, or yafO from plasmid pBAD33, were exposed to 2 µg/mL of ciprofloxacin (for details, see Materials and Methods). Percentage of survival after 4 h of antibiotic treatment was compared with that of the control strains carrying the pBAD33 vector plasmid (log scale). Error bars indicate the SDs of averages of at least three independent experiments.

Fig. 4.

The hipA7 allele increases the frequency of (p)ppGpp ON cells independently of the 10 mRNase-encoding TA loci. (A) Snapshot of exponentially growing cells of MG1655 (A), MG1655 hipA7 (B), ∆10TA (C), or ∆10TA hipA7 (D), carrying an rpoS::mcherry translational fusion. (i) Phase contrast. (ii) RpoS-mCherry fluorescence. (iii) Overlay of i and ii. Statistical analysis of these rare fluorescent cells is provided in Fig. S3 A–D. (Scale bar, 4 μm.)

Fig. 5.

HipA7-mediated high levels of (p)ppGpp do not induce persistence in the absence of mRNase-encoding TA modules. Exponentially growing cells of MG1655 rpoS::mcherry were introduced into a microfluidic device and subjected to growth medium. (A) Time-lapse images showing growth upon rich medium injection of MG1655 hipA7 cells carrying rpoS::mcherry; the arrow indicates a growth-arrested cell with a high level of RpoS-mCherry (from Movie S1). (B) Time-lapse images showing persistence upon ampicillin injection of cells of MG1655 hipA7 rpoS::mcherry expressing a high level of RpoS-mCherry (from Movie S2). (C) Time-lapse images showing growth upon rich medium injection of ∆10TA hipA7 cells carrying rpoS::mcherry; the arrow indicates a growth-arrested cell with a high level of RpoS-mCherry (from Movie S4). (D) Time-lapse images showing the behavior of ∆10TA hipA7 expressing a high level of RpoS-mCherry upon ampicillin treatment (from Movie S5).The first images of each panel were generated by overlay of phase contrast and fluorescence images (separate phase contrast and fluorescence images are shown in Fig. S4 A–D, respectively). The arrow indicates a cell with a high level of RpoS-mCherry. (Scale bar, 4 μm.) t, time in minutes.