The human gut mobile metagenome: a metazoan perspective

Abstract

Using the culture independent TRACA system in conjunction with a comparative metagenomic approach, we have recently explored the pool of plasmids associated with the human gut mobile metagenome. This revealed that some plasmids or plasmid families are present in the gut microbiomes of geographically isolated human hosts with a broad global distribution (America, Japan and Europe), and are potentially unique to the human gut microbiome. Functions encoded by the most widely distributed plasmid (pTRACA22) were found to be enriched in the human gut microbiome when compared to microbial communities from other environments, and of particular interest was the increased prevalence of a putative RelBE toxin-antitoxin (TA) addiction module. Subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes, but homologues of the RelE toxin were associated with all major bacterial divisions comprising the human gut microbiota. In this addendum, functions of the gut mobile metagenome are considered from the perspective of the human host, and within the context of the hologenome theory of human evolution. In doing so, our original analysis is also extended to include the gut metagenomes of a further 124 individuals comprising the METAHIT dataset. Differences in the incidence and relative abundance of pTRACA22 and associated TA modules between healthy individuals and those with inflammatory bowel diseases are explored, and potential functions of pTRACA22 type RelBE modules in the human gut microbiome are discussed.

Keywords: gut microbiota; holobiont; hologenome; horizontal gene transfer; metagenomics; mobile genetic elements; mobile metagenome; toxin-antitoxin addiction module.

Figure 1

Incidence and relative abundance of plasmids pTRACA10, pTRACA17, pTRACA18, pTRACA20, pTRACA22 and pTRACA30 in the METAHIT gut metagenome data set. The complete nucleotide sequences of each plasmid were used to search the METAHIT dataset using Blastn, and calculate incidence and relative abundance according to methods described previously in reference and . For this analysis, only hits matching the following criteria were considered significant: An identity of 90% or greater over 100 nucleotides or more, and an e-value of 1 e⁻¹⁰ or lower. In addition to the complete METAHIT data set, incidence and relative abundance of plasmids in sub sets of individuals represented in the METAHIT dataset was also explored based on nationality (Danish or Spanish), gender, and disease status (healthy, Crohn disease or ulcerative colitis). For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative colitis (UC) or Crohn disease (CD), regardless of age or body mass index. The statistical significance of observed incidence and relative abundance data was explored using the χ² distribution. Where significant differences in relative abundance of plasmids between metagenomic datasets were identified, the statistical power afforded by the available sample sizes in each group was calculated using the Piface program. This indicates the probability that the observed differences are truly reflective of the wider population, with a probability of 0.8 considered to indicate adequate power. Symbols above bars indicate the level of significance in χ² analysis, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been computed to p = 0.05 using variation (standard deviation) observed within the total METAHIT dataset. Danish HEALTHY, Spanish HEALTHY = Combined gut metagenomes from individuals of each nationality in the METAHIT data set, excluding those diagnosed with IBD; Male HEALTHY, Female HEALTHY = Combined gut metagenomes from individuals of each gender in the METAHIT data set, excluding those diagnosed with IBD; Healthy TOTAL = Combined gut metagenomes from all healthy individuals in the METAHIT dataset; CD, UC = Combined gut metagenomes from individuals diagnosed with respective IBDs; TOTAL METAHIT = Combined gut metagenomes of all 124 individuals regardless of nationality, gender or disease status. (A) Shows the incidence of each plasmid (and closely related elements) among the gut metagenomes comprising the METAHIT data set, when grouped by nationality, gender and disease statues, as well in the METAHIT cohort as a whole. Incidence is expressed as the percentage of metagenomes in which at least one significant hit to each plasmid was detected, (according to criteria outlined above). In this analysis, only differences found to be supported by adequate statistical power (0.8 at p = 0.05) were considered significant overall. (B) Shows the relative abundance of each plasmid, expressed as hits/Mb, among the gut metagenomes comprising the METAHIT data set, as grouped in (A)., For this analysis relative abundance was calculated based on the total number of significant hits (according to criteria outlined above) in each group (gender, nationality, disease status), and the average relative abundance in each cohort displayed. INSET: Shows the overall inter-individual variation for each plasmid among all 124 individuals in the METHIT dataset, with yellow bars representing lowest observed abundance, red bars highest observed abundance, and green bars showing the average abundance over all 124 individuals. (C) Shows the percentage of each plasmid nucleotide sequence that could be recovered from the total METHIT dataset. Colors of segments represent % identity of recovered METAHIT sequences corresponding to respective regions of each plasmid, and numbers below pie charts provide the coordinates in the complete plasmid sequence for each segment. Yellow segments = 92–94% identity; orange segments = 94–96% identity; red segments = 96–98% identity; black segments = 98–100% identity.

Figure 2

Relative abundance of pTRACA22 type RelBE modules in the METAHIT dataset and variation in inflammatory bowel disease. The relative abundance of pTRACA22 type RelBE modules in the gut microbiomes of individuals represented in the METHIT dataset was calculated exactly as described previously in reference . METHIT data was searched with pTRACA22 RelB and RelE amino acid sequences using tBlastn to identify homologous sequences, and only hits generating e-values of 1 e⁻⁵ or lower with a length of 30 aa or over were considered significant, and used to calculate hits/Mb. For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative colitis (UC) or Crohn disease (CD), regardless of age or body mass index. Statistically significant differences in relative abundance or incidence of RelBE modules within and between metagenomic datasets were explored using the χ² distribution. Where significant differences in relative abundance of RelBE homologues were identified within or between groups comprised of metagenomes from the METAHIT dataset, the statistical power afforded by the available sample sizes in each group was calculated using the Piface program. This provides the probability that the observed differences reflect the wider population, with a probability of 0.8 generally considered to indicate adequate power. Symbols above bars indicate the level of significance in χ² analysis, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been computed to p = 0.05 using variation (standard deviation) observed within the total METAHIT dataset. (A) Shows the relative abundance of pTRACA22 type RelBE homologues in the individual gut microbiomes of all 124 individuals represented in the METAHIT dataset. Each bar represents the relative abundance data (Hits/Mb) for RelB (blue) overlaid with that of RelE (red). Purple regions show the least abundant ORF in each individual metagenome, while the color of upper regions indicates the abundance of the most prevalent ORF in each individual metagenome. Individuals in which the RelB anti-toxin is most abundant are indicated by blue upper regions of bars, while those in which the RelE toxin is most abundant are indicated by red upper regions of bars. Individuals in which the level of RelB and RelE are equal are indicated by exclusively purple bars. (B) Shows the percentage of individuals represented in the entire METAHIT dataset with varying ratios of RelB:RelE abundance. Blue = % of individuals in which RelB dominates, red = % of individuals in which RelE dominates, purple = % of individuals with equal relative abundance of both RelB and RelE; white = % of individuals in which neither RelB nor RelE could be detected within the criteria employed in this study. (C) Comparison of overall relative abundance in combined human gut metagenomes of Japanese and American individuals (utilized in our original analysis), and the METAHIT dataset.,, Red bars show relative abundance of RelE homologues and Blue bars show relative abundance of RelB homologues. Am&Jp = American and Japanese gut metagenomes utilized in our original study;, METAHIT Healthy = Combined gut metagenomes of individuals from the METAHIT dataset without IBD; METAHIT CD = Combined gut metagenomes of individuals from the METAHIT dataset diagnosed with Crohn disease; METAHIT UC = Combined gut metagenomes of individuals from the METAHIT dataset diagnosed with ulcerative colitis; METAHIT TOTAL = Combined gut metagenomes of all 124 individuals represented in the METAHIT dataset.