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Bactériophages et dysbioses intestinales

Bactériophages et microbiote intestinal

Many attempts to detect phages (bacterial viruses) in various microbial ecosystems have led to similar conclusions: (i) phages are present in all ecosystems where bacteria are present; (ii) but it remains hard to assess their contribution to ecosystem functioning. This is caused by the difficulty to quantify (except in waters), classify (due to their diversity), and to assemble phage genomes out of metagenomics reads (again due to their huge genome plasticity). Last but not least, it is difficult to predict which phage infects which bacterial species. In first attempts at sequencing the viral fraction of bacterial ecosystems, it was concluded that more than 80% of the reads could not be interpreted. Recent improvements have opened the door for more promising assembly analyses: (i) the use of co-variance analysis to selectively assemble reads having the same abundance across samples(Duthil BE et al., Nat Com 2014); (ii) data mining of draft bacterial genomes for the presence of putative prophages. The later approach allows the demonstration that tetramer composition of phage genomes could be used to predict their target host(Roux S et al., eLife 2015). Based on such a comprehensive database of phage proteins, a tool allowing annotation of assembled viral sequences out of metagenomics datasets (virSorterRoux S et al., PeerJ 2014) has also been produced.VIROME ACCESS is a project that brings together molecular ecologists and statisticians from INRA to take on the challenges of virome analyses. Food, intestinal and anaerobic digester samples are used as starting materials. The project has four main objectives.

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Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity., Author Summary Dormant bacterial viruses, or prophages, are found in the genomes of almost all bacteria, but their impact on bacterial host fitness is largely unknown. Through experiments in mice, supported by a mathematical model, we quantified the activity of Escherichia coli prophage λ in monoxenic mouse gut, as well as its impact on its carrier bacteria. λ carriage negatively impacted its hosts due to frequent reactivation, but indirectly benefited its host by killing susceptible bacterial competitors. The high prophage activity unraveled in this study reflects a constant rate of SOS response, resulting from DNA damage in monoxenic mouse intestine. Our results should motivate researchers to take the presence of prophages into account when studying the action of specific bacteria in the gastrointestinal tract of mammals.

Metagenomic approaches applied to viruses have highlighted their prevalence in almost all microbial ecosystems investigated. In all ecosystems, notably those associated with humans or animals, the viral fraction is dominated by bacteriophages. Whether they contribute to dysbiosis, i.e., the departure from microbiota composition in symbiosis at equilibrium and entry into a state favoring human or animal disease is unknown at present. This review summarizes what has been learnt on phages associated with human and animal microbiota, and focuses on examples illustrating the several ways by which phages may contribute to a shift to pathogenesis, either by modifying population equilibrium, by horizontal transfer, or by modulating immunity.

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself., Author Summary Temperate bacteriophages (or phages) are bacterial viruses that, unlike virulent phages, have the ability to enter a prophage dormant state upon infection, in which they stably replicate with the bacterial genome. A majority of bacterial genomes contain multiple active or defective prophages, and numerous bacterial phenotypes are modified by these prophages, such as increased virulence. These mobile genetic elements are subject to high levels of genetic exchanges, through which new genes are constantly imported into bacterial genomes. Phage-encoded homologous recombination enzymes, or recombinases, are potentially key actors in phage genome shuffling. In this work, we show that gene acquisition in temperate phages is strongly dependent on the presence of sequence homology, but is highly tolerant to divergence. We report that gene exchanges are mainly catalyzed by recombinases found on temperate phages, and show that four different Rad52-like recombinases have a relaxed fidelity in vivo, compared to RecA. This high capacity of exchange speeds up evolution of phages, and indirectly also the evolution of bacteria.

International audience; The spread of epidemics not only depends on the average number of parasites produced per host, but also on the existence of highly infectious individuals. It is widely accepted that infectiousness depends on genetic and environmental determinants. However, even in clonal populations of host and viruses growing in homogeneous conditions, high variability can exist. Here we show that Escherichia coli cells commonly display high differentials in viral burst size, and address the kinetics of emergence of such variability with the non-lytic filamentous virus M13. By single-cell imaging of a virally-encoded fluorescent reporter, we monitor the viral charge distribution in infected bacterial populations at different time following infection. A mathematical model assuming autocatalytic virus replication and inheritance of bacterial growth rates quantitatively reproduces the experimental distributions, demonstrating that deterministic amplification of small host inhomogeneities is a mechanism sufficient to explain large and highly skewed distributions. This mechanism of amplification is general and may occur whenever a parasite has an initial phase of exponential growth within its host. Moreover, it naturally reproduces the shift towards higher virulence when the host is experimenting poor conditions, as observed commonly in host-parasite systems.

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments., Author Summary The mammalian intestine is a privileged physiological site to study how coevolution between hosts and the trillions of bacteria present in the microbiota has shaped the genome of each partner and promoted the development of mutualistic interactions. Herein we have used germ-free mice, a simplified albeit ecologically relevant system, to analyse intestinal adaptation of a model bacterial strain, Escherichia coli MG1655. Our results show that single point mutations in the ompB master regulator confer a striking selective adaptive advantage. OmpB comprises EnvZ, a transmembrane sensor with a dual kinase/phosphatase activity, and OmpR, a transcription factor controlling more than 100 target genes. In response to environmental changes, EnvZ modulates the phosphorylation and thereby the transcriptional activity of OmpR. We further show that the selective advantage conferred by OmpB mutations is related to their additive and independent effects on genes regulating permeability and flagellin expression, two major set of genes controlled by OmpR. These results suggest that global regulators may have evolved to coordinate physiological activities necessary for adaptation to complex environments and that mutations offer a complementary genetic mechanism to adjust the scale of the physiological regulation controlled by these regulators in distinct environments.