New insights into intestinal phages

Abstract

The intestinal microbiota plays important roles in human health. This last decade, the viral fraction of the intestinal microbiota, composed essentially of phages that infect bacteria, received increasing attention. Numerous novel phage families have been discovered in parallel with the development of viral metagenomics. However, since the discovery of intestinal phages by d'Hérelle in 1917, our understanding of the impact of phages on gut microbiota structure remains scarce. Changes in viral community composition have been observed in several diseases. However, whether these changes reflect a direct involvement of phages in diseases etiology or simply result from modifications in bacterial composition is currently unknown. Here we present an overview of the current knowledge in intestinal phages, their identity, lifestyles, and their possible effects on the gut microbiota. We also gather the main data on phage interactions with the immune system, with a particular emphasis on recent findings.

Fig. 1. Phage life cycles.

The production of new virions is realized either through lytic cycles for Caudovirales and Microviridae phages (left side of the figure, brown arrows) or through chronic infection in the case of filamentous phages, or Inoviridae (blue arrows). Both start with the recognition and infection of the targeted bacteria (1), followed by phage DNA replication and synthesis of new virions (2). In lytic cycles, new virions are released through bacterial lysis (3), while new virions of filamentous phages exit bacteria through a dedicated secretion apparatus, without bacterial lysis (4). Phages that reproduce only through lytic cycles are called virulent. By opposition,  some phages, called temperate phages, in addition to performing either lytic or chronic cycles, are able to perform lysogenic cycles (pink arrows), whereby they enter a dormant state in the infected bacteria, the prophage state (5). The prophage, either integrated within the bacterial genome or in an episomal state, is replicated with the bacterial chromosome as long as bacteria divide (6). In some bacteria, generally when submitted to a stress, the prophage is induced and the phage resumes a lytic or a chronic cycle.

Fig. 2. Main intestinal phage types.

a Epifluorescence microscopy image of a human fecal filtrate. Virus-like particles (VLPs) appear as bright dots following Sybr-gold staining of DNA (M. De Paepe, unpublished results). b Transmission electronic microscopy images of major intestinal phage types. Scale bars are 100 nm except for the Microviridae virion, for which it is 50 nm. 1: CrAssphage PhiCrAss001 (Podoviridae), reproduced from ref. 2: Microviridae virion isolated from surface water near a coastal aquaculture site, reproduced from ref. 3 and 4: Myoviridae and Siphoviridae virions respectively, extracted from human intestinal contents. Several Myoviridae virions are bound to a membrane vesicle. Reproduced from ref. Copyright © 2014 Elsevier Masson SAS. c Inter-individual virome specificity and conservation in four human subjects over 12 months. Each phage cluster, which roughly corresponds to a phage genus, is outlined in black. Reproduced from ref.

Fig. 3. Phage-bacterium interactions in the GIT.

a Illustration of the variability in the populations of phages and targeted bacteria in feces of gnotobiotic mice, reproduced from ref. Germ-free mice were colonized with ten bacterial strains representing the major phyla of the human gut microbiota (Clostridium sporogenes, E. faecalis, Bacteroides fragilis, B. ovatus, B. vulgatus, Parabacteroides distasonis, Klebsiella oxytoca, Proteus mirabilis, E. coli Nissle 1917, and Akkermansia muciniphila). Dotted lines indicate the time of addition of 2 × 10⁶ PFU of a virulent phage targeting one bacterial strain exclusively. Bacteria and phages were quantified by quantitative PCR (blue and red lines, respectively). In the case of E. coli, after a rapid drop in bacterial population after T4 phage administration, phage and bacteria coexist without selection of phage-resistant mutants. In the cases of C. sporogenes and B. fragilis, bacterial populations are transiently affected by phage administration, and phages reach very high concentrations. Finally, in the case of E. faecalis, the bacterial population drops and does not recover its initial level over time, due to a defect of colonization by phage-resistant mutants. b Hypotheses for the co-existence of phage and susceptible bacteria in the GIT. (1) Important bacterial phenotypic variability, e.g., in phage receptor expression or growth rate, would render some bacteria phage-susceptible, whereas other genetically identical bacteria would be resistant. (2) Spatial heterogeneity of the environment (such as intestinal crypts, biofilm-like structures on food debris, or the inner part of the mucus layer) would create a refuge for bacteria and prevent access to phages. (3) Important loss of virions due to specific and nonspecific adsorption would sufficiently lower free-phage concentration to protect bacteria. (4) The presence of numerous long chain carbohydrates fibers from food and mucus would hinder phage diffusion, preventing them to adsorb on bacteria. None of these mechanisms has been formally demonstrated in the GIT.

Fig. 4. Potential interactions between phages, epithelial cells, and host immune cells in the intestine.

Phage tropism for the mucus may promote penetration of phages within the body through endocytosis and transcytosis in intestinal epithelial cells (1), or through sampling by dendritic cells (2). Dendritic cells endocytose phages, may be captured in the intestinal lumen via extended dendrites, or exocytosed in the subepithelial compartment. Once endocytosed, phage nucleic acids can trigger TLR pathways, notably TLR9-dependent pathways (3), and stimulate adaptive immune responses (4). Although mechanisms of B and T-cell activation by phages are not fully elucidated, recent studies showed that activation of B cells leads to the secretion of phage-specific antibodies, both in the intestine and in the systemic compartment. In addition, activation of T cells in the Peyer’s patches and mesenteric lymph nodes results in production of cytokines, such as IFN-γ.