Genome maintenance of induced bacterial persisters – Persist3Rs

Bacteria can enter into a non-growing but surviving state called persistence. It allows them to tolerate antibiotic treatment, and restart growth once the treatment is stopped. This is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Various environmental and stressing signals induce entry into persistence, through poorly characterized pathways, and almost nothing is known about the chromosome maintenance during persistence. And whether prophages, the bacterial viruses integrated in their host in a dormant stage, contribute positively or negatively to persistence is also far from being understood.

Our studies on the Escherichia coli strain LF82, carrying 5 prophages, show that during macrophage infection, 10-20% of bacteria enter into persistence. Moreover, bacterial DNA is likely damaged, because several genes of the SOS response are induced. Finally, prophage encoded genes for DNA repair are also induced in macrophages, which may contribute to a non-homologous end-joining pathway (NHEJ).

The Persist3Rs project will investigate how genome integrity is maintained during Escherichia coli persistence. To access to this knowledge, we will first invest efforts into the method of separation of persisters sub-populations. Indeed, we suspect that several pathways leading to persistence coexist in the bacterial population, so that if they are studied globally, signals are lost. To facilitate the recovery of persisters, clinically relevant treatments known to induce persistence will be used: growth with non-lethal doses of antibiotics and phagocytosis inside macrophages. Also, as the E. coli species is diverse, two strains will be taken as starting points: the commensal strain MG1655, devoid of functional prophage, and the pathogen LF82, an adhesion invasive strain (AIEC) hosting 5 prophages.

The project revolves around three axes. (1) To sort out the distinct persister populations, strains containing transcriptional fluorescent fusions to promoters induced at different growth stages, or by different stresses, will be constructed. The proportion of persisters, among these sub-populations marked for a specific growth stage or stress response, will be measured by FACS. (2) Then the transcriptome of each sub-population will be studied. We also want to detect messenger RNA specifically transcribed in these bacteria (by Nascent-RNAseq), and those specifically translated (by Ribo-seq). This will give access to the key cellular functions requested for the survival of persisters. (3) Finally, single cell epifluoresence and microfluidics, genetics and biochemistry approaches will be used to detect DNA lesions, mutations, as well as repair pathways put into action in persisters.

Persist3R will therefore give unprecedented access to the pathway(s) leading to persistence in E. coli, and to quantifications of persister proportions as a function of the stress applied. Strain comparisons will allow distinguishing common behaviors and strain particularities. We will identify how the genome is impacted by persistence, and what are the genetic pathways involved in genome maintenance. Finally, we expect to uncover prophage bacteria interactions beneficial for persistence, whereby prophage genes may contribute novel functions for the repair of DNA in cells devoid of sister chromatids. In the long run, these results might lead to develop new ways to combat persisters in the clinic.