The Crohn’s disease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages

Abstract

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections.

The Crohn’s disease-related AIEC strain LF82 assembles a biofilm-like matrix to protect intracellular microcolonies from phagolysosomal attack

“…This analysis gave an image of the growth conditions encountered by AIEC LF82 within macrophages. It confirmed the presence of acid, oxidative and genotoxic stresses, and showed: i) the alteration of the bacterial envelope (membrane, cell wall and periplasm), ii) the reduction of protein biosynthesis and cell cycle activity, presumably linked to stringent response induction (Demarre et al, 2019) , iii) the switch from motile to biofilm behavior and, iv) the global reduction of catabolic and energetic metabolism. We used data available for E. coli K12 in the Regulon Database (Gama-Castro et al, 2016) to identify the key responses.…”

“…In the minutes following phagocytosis, the induction of a stringent response blocks bacterial cell division and curbs the expansion of the LF82 population. This 6 to 10 h step can lead to the formation of LF82 persisters (Demarre et al, 2019). When LF82 resumes growth, the expansion of the population is dependent on the ability of the bacteria to repair lesions (e.g.…”

“…Antibiotic challenge and viable bacterial count was performed as described before (Demarre et al, 2019). For imaging, cells were cultured on a glass microscope coverslip at the bottom of the microplate wells.…”

“…Control experiments were performed with macrophages alone and bacteria alone cultivated for 1 h and 6 h in DMEM medium at 37°C and 5% CO2 without agitation. RNA extraction was performed as described before (Demarre et al, 2019). Experiments were performed from biological duplicate.…”

“French Phage Network” Annual Conference—Fifth Meeting Report

“…This strain can survive within macrophages and produce persisters (i.e. phenotypic variants with low metabolic activity and transient resistance to antibiotics) [32]. LF82 harbours five prophages.…”