Exploring novel strategies to unravel the mechanisms underlying persister recovery in Escherichia coli

Apart from genetic resistance, which is a well-known, population-wide antibiotic survival strategy, bacteria also employ persistence to overcome antibiotic treatment. Persistence is a bet-hedging strategy of the population in which a small, isogenic subpopulation is transiently tolerant to antibiotic action, mostly by being in a slow- or non-growing state. Persistence is a transient phenotype, meaning that these so-called persister cells can exit the persister state and switch back to the sensitive, growing state. Indeed, when antibiotic treatment is ceased, persister cells can recover and regrow, eventually establishing a new population that again contains a small fraction of persister cells. Although long overlooked, evidence is mounting that persistence is partly responsible for the recalcitrance of chronic infections, including pulmonary infections in cystic fibrosis patients, tuberculosis infections and recurrent urinary tract infections. Moreover, persistence has been shown to contribute to resistance development. Given its clinical relevance, it is important to gather knowledge on the persister phenotype to steer the development of anti-persister therapies. Research interest in persistence strongly increased in the past two decades, mostly leading to the identification of persister formation mechanisms. However, the knowledge on persister recovery remains limited. Given the heterogenous timing of persister exit and the low abundance of persister cells in the population (typically ranging between 0.001 and 1 %), studying persister recovery requires single-cell, high-throughput techniques. Therefore, in this work, we explored the use of high-throughput droplet microfluidics to study single-cell persister recovery. Apart from that, we explored CRISPR interference (CRISPRi) as a genetic screening method to identify genes important for persister formation and persister recovery. In the first part of this work, droplet microfluidics was tested as a method to study the recovery of single persister cells. More specifically, the treated culture was washed and diluted in fresh medium so that upon encapsulation in picodroplets, 10 % of the droplets contained a single persister cell surrounded by treated dead cells. Droplets were incubated and screened for bacterial growth, indicative of persister recovery. However, running a few test experiments revealed that the washed treated culture needs to be strongly diluted in fresh medium prior to encapsulation to allow for persister recovery and regrowth, which automatically abolished the throughput that the droplet microfluidics setup could offer. By further examining this need for a strong dilution, we found that a high density of treated dead cells surrounding the persister cells hampers persister regrowth and that antibiotic present in the medium after the washing steps at a sub-MIC concentration can hinder persister recovery. Finally, we showed that picodroplets still provide a valuable alternative for existing methods to image the recovery of singe persister cells with time-lapse microscopy, although its use remains restricted to high-persister strains. In the second part of this work, we implemented CRISPRi to find genes important for persister formation and genes important for persister recovery. In CRISPRi, dCas9 in complex with a single-guide RNA (sgRNA) binds a specific DNA sequence, thereby causing a gene knockdown. Compared to other genetic screening methods, the CRISPRi system can be induced, i.e. turned on and off, and as such, offers the possibility to study persister formation and recovery independently from each other, for the first time in persistence research. Two separate pooled sgRNA CRISPRi screening experiments were performed to find genes important for persister formation and genes important for persister recovery. To this end, first, different aspects of the CRISPRi screenings were optimized. Most importantly, we compared different inducible dcas9 expression systems and found that dcas9 expression can be most tightly regulated from the chromosome. Performing both CRISPRi screenings, data analysis and subsequent validation experiments revealed that persister cells are mostly formed through dormancy and that following fluoroquinolone treatment, DNA repair and cell division delay are important for the recovery of persister cells, thereby confirming findings from previous studies. From this, we conclude that CRISPRi is a valid and promising approach to study persister formation and persister recovery. In conclusion, studying the recovery of single persister cells remains challenging, not only due to their low abundance and their heterogenous nature, but also because the washed treated culture needs to be strongly diluted in fresh medium to allow for persister recovery and regrowth. Although the droplet microfluidics setup provides a high throughput in theory, its use remained limited to high-persister strains. Therefore, studying single-cell persister recovery of any strain in droplets in the future will require a platform with an even higher throughput. Finally, CRISPRi was shown to be a valid and promising method to identify genes underlying persister formation and genes underlying persister recovery. Since the current knowledge on persister recovery is mostly limited to fluoroquinolone persistence, and since persister recovery mechanisms seem to strongly depend on the type of antibiotic that is used, we envision that performing such CRISPRi screenings in the future with other antibiotic classes will lead to important novel insights.

Jaar van publicatie:2024

Toegankelijkheid:Closed