Single cell observations show persister cells wake based on ribosome content

Abstract

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising after antibiotic treatment at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through eight sets of experiments), we used microscopy of single cells to determine that the resuscitation of dormant persisters is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found during the observation of persister cells when fresh nutrients were added: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD

“…The GFP reporter indicates transcription of rrsB (16S rRNA), gltT (tRNAglu), rrlB (23S rRNA) and rrfB (5S rRNA); hence, it indicates production of the three major rRNA building blocks. Although this is not a direct observation of 70S ribosomes, this method is a suitable proxy for the number of active ribosomes based on the measurement of rRNA concentrations and has been used frequently (Lu et al, 2009;Piques et al, 2009;Burger et al, 2010;Kim et al, 2018b), and we have verified its use by isolating ribosomes and comparing GFP fluorescence (Kim et al, 2018b). Hence, the increased persister cell resuscitation with RluD is directly due to the increase in active (70S) ribosomes of persister cells since persisters wake via ribosome activation (Kim et al, 2018b).…”

“…Using a GFP reporter that indicates the number of 70S ribosomes in individual persister cells (Kim et al, 2018b), we found that overproducing RluD from pCA24N before making persister cells (i.e. before rifampicin treatment) makes 85% AE 6% of the cells have high ribosome fractions compared with 22% AE 2% of the cells that do not overproduce RluD (Fig.…”

“…These proteins are related to contacting with BPOET because addition of the diluent DMSO alone and sequencing larger colonies did not identify these five proteins but instead identified TmcA, a tRNA Met cytidine acetyltransferase, which is a general factor required for translation that likely led to larger colony sizes with the diluent. Of the proteins related to BPOET, only RluD (23S rRNA pseudouridine synthase) and SrlR (represses the gut operon for glucitol metabolism) have been characterized; we focused on RluD because it is related to ribosomes, and we have shown inactivating ribosomes causes persistence (Kwan et al, 2013) and activating ribosomes resuscitates persister cells (Kim et al, 2018b;; Table 1. Compounds that resuscitate persister cells and their structures that were identified in the initial screen.…”

“…It has been suggested that persister cells resuscitate by inactivating toxins such as TacT acetyltransferase via peptidyl-tRNA hydrolase Pth (Cheverton et al, 2016), but this has not been demonstrated. It is established that persister cells revive in response primarily to environmental signals, such as fresh nutrients (rather than stochastically) (Kim et al, 2018b). In addition, persisters revive in a heterogeneous manner, by activating ribosomes; cells increase their ribosome content until a threshold is reached, then they begin to elongate or divide (Kim et al, 2018b).…”

“…It is established that persister cells revive in response primarily to environmental signals, such as fresh nutrients (rather than stochastically) (Kim et al, 2018b). In addition, persisters revive in a heterogeneous manner, by activating ribosomes; cells increase their ribosome content until a threshold is reached, then they begin to elongate or divide (Kim et al, 2018b). For resuscitation, the persisters sense nutrients by chemotaxis and phosphotransferase membrane proteins, reduce cAMP levels to rescue stalled ribosomes, unhybridize 100S ribosomes via HflX and undergo chemotaxis towards fresh nutrients .…”