Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut

Abstract

Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/hosts. To characterize their role as agents shaping gut microbial community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage-bacterial host dynamics in the gut.

Keywords: artificial gut communities; microbiome; prophage function; viral diversity; viral metagenomics.

Fig. 1.

Sequential changes in the relative abundance of two members of the 15-member artificial human gut microbiota and correlation with the appearance of two previously undescribed phages. (A) Relative abundance plot for each bacterial species as a function of time for either the live p-VLP or the heat-killed p-VLP treatment groups. Mean values ± SEM are shown (n = 5 mice). The color key next to the plot indicates the identity of each bacterial species. (B and C) Plots of the relative abundance (fraction of the total community; mean ± SEM; n = 5 animals per treatment group) of B. caccae and B. ovatus in the fecal microbiota of gnotobiotic mice as a function of time before and after gavage with live purified VLPs pooled from the fecal microbiota of five human donors or a control heat-killed version of the same p-VLP preparation (time of gavage indicated by the upward pointing arrow; t = 0 on the x axis refers to the time of introduction of the 15-member artificial community into germ-free animals). The change in abundance of these Bacteroides spp. occurs in a reproducible sequence among individually caged mice that received live but not heat-killed p-VLPs. (D and E) Changes in the abundance of two phages, derived from the p-VLP sample, in the fecal microbiota of recipient gnotobiotic mice. Differences in the time course of change in bacterial and viral abundances are highlighted by the gray shading (lighter for B. caccae and ϕHSC01). Insets in D and E are assembled genome sequences for ϕHSC01 and ϕHSC02. The location of genes on the positive strand (green) and negative strand (red) are shown; those that have significant sequence similarity to known viral genes are colored blue (blastp E-value <10⁻⁵; Dataset S1). The inner plot represents GC skew based on 200-bp windows (yellow, G/C ratio is greater than the average for the genome; purple, ratio is lower than the average).

Fig. 2.

Prophage induction in B. cellulosilyticus WH2. (A) VLP-derived sequencing reads from mouse fecal samples mapped to a 150-kbp fragment of the B. cellulosilyticus WH2 genome containing prophage 1. The y axis corresponds to the log (10) of the read coverage (blue) for a given position in the prophage genome. Mapping VLP reads to the bacterial genome identified the prophage insertion site at an arg-tRNA gene, with the corresponding duplicated region generating the attachment (att) sites. No reads were obtained from potential cos sites (red arrow, zoomed-in fragment). (B–D) Relative abundance (mean ± SEM) of the bacterial host and its prophage in the fecal microbiota of individually caged mice. Relative abundance was measured based on the ratio of COPRO-Seq reads mapping to each prophage and elsewhere in the bacterial genome. Bacterial relative abundance was scaled to its community relative abundance (Fig. S3B); prophage abundance was also scaled accordingly. Equivalent abundances correspond to phage in an uninduced state, whereas relative increases in prophage abundance indicate induction.