The enemy from within: a prophage of Roseburia intestinalis systematically turns lytic in the mouse gut, driving bacterial adaptation by CRISPR spacer acquisition

Abstract

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal’s microbiota has been largely unexplored. Here we studied temperate phage–bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counter-evolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

New insights into intestinal phages

“…colonized with a limited number of bacterial strains), in which the majority of the susceptible bacteria were killed by phage, suggest that virion loss is highly dependent on individual phages considered. In some reports, free phage to bacteria ratios over 100 were observed in the feces of mice, 37,57 whereas in others, ratios close to 1 were observed, 58,59 suggesting either virion loss or very low viral amplification per infected bacteria, i.e., about one new virion produced per lytic cycle.…”

“…However, the presence of active prophages, which produce virions, has been demonstrated experimentally in almost all gut bacterial strain tested, suggesting that a significant proportion of prophages detected by genomic analyses are active. 33,34,37,38 High prevalence of temperate phages in human microbiota could participate in the stability of the phageome over time, as populations of lysogenic bacteria constantly produce virions. (4) The identity of the hosts targeted by the phages is a crucial but largely unanswered question.…”

“…However, and in contrast with results obtained in vitro, these mutants never entirely replaced the susceptible bacteria. 37,57,89 In other cases, phage-mediated bacterial mortality was relatively limited and neither depletion of the bacterial population, nor selection of phage-resistant mutants were observed. 57,59,87,88 Overall, these data suggest that the intestinal environment provides bacteria with spatial refuges or alternative resistance mechanisms, such as phenotypic resistance (Fig.…”

Probing the “Dark Matter” of the Human Gut Phageome: Culture Assisted Metagenomics Enables Rapid Discovery and Host-Linking for Novel Bacteriophages

“…Unlike symbiotic gut bacteria, the majority of which can be propagated in the lab with varied level of effort ( Rajilić-Stojanović and de Vos, 2014 ), isolation and propagation of a bacteriophage requires a suitable bacterial host. Given the narrow host range of many phages ( de Jonge et al., 2019 ), prevalence of temperate phages in the gut ( Reyes et al., 2010 ; Cornuault et al., 2018 ; Fitzgerald et al., 2018 ; Cornuault et al., 2020 ), rapid acquisition of reversible resistance through phase variation of surface receptors and other mechanisms ( De Sordi et al, 2019 ; Hryckowian et al., 2020 ; Porter et al., 2020 ), isolation of metagenomically detected, uncultured phages proves to be a daunting task ( Dutilh et al., 2014 ). Traditional approaches of phage isolation, characterisation and linking to specific bacterial hosts rely on detection of plaques formed by phage-mediated lysis of bacterial cells embedded in semi-solid agar.…”

How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

“…Since these pioneering studies, spacer uptake from phages and other mobile genetic elements in bacteria and archaea from natural and human-associated environments has been inferred from variation in spacer sequences within and between populations of the same species and from their homology to mobile genetic element (MGE) genomes (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Experimental observations of spacer uptake in the lab in response to plasmid and phage infection have been made amongst others in engineered E. coli strains (41)(42)(43) and Staphylococcus aureus (44)(45)(46)(47) and in wild type Pectobacterium atrosepticum (48), Pseudomonas aeruginosa (49,50), Roseburia intestinalis (51), Sulfolobus solfataricus (52), Streptococcus mutans (37) and other species (reviewed in (53)). Consistent with the hypothesis that CRISPR-Cas protects bacteria from infections, some mobile genetic elements encode so-called anti-CRISPR genes (reviewed in (54)).…”