CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Abstract

Background: Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets.

Results: We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.

Conclusions: We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.

Figure 1

The distribution of MGEs across HMP samples. A MGE is considered to be present in a sample if 70% of its length is covered by at least one read. The X-axis represents the number of samples that contain each MGE, Y-axis represents MGEs. (A) Distribution of the 959 MGEs each with at least 40 protospacers. (B) Prevalence of 155 MGEs selected from stool microbiomes, each with at least 10 protospacers. The inserts are the histograms of the MGE sizes. MGE, mobile genetic element.

Figure 2

MGE contig SRS016200_WUGC_scaffold_12207 represents a mosaic genomic island. The contig is 101,291 bp long, and is shown as an open box in the middle, with protospacers shown as vertical lines within the box (gray lines each show a protospacer that is similar to spacers from individual HMP datasets, while lines of the same color show sets of protospacers matching spacers all from the same HMP dataset). Similarities between this contig and two bacterial genomes, H. parainfluenzae T3T1 (with sequence identity of 93%) and H. parainfluenzae ATCC 33392 (with sequence identity of 97%), and one ICE (ICEhin1056 found in H. influenzae), are represented as green, blue and yellow shades, respectively, between the contig and the corresponding genome lines. The location of the tRNA-Leu gene found in the contig is highlighted by a red triangle. The prophage regions found in this contig by Prophage finder and PHAST are shown as red lines, below the contig box. The regions corresponding to the genomic island regions in the H. parainfluenzae T3T1 genome as predicted by various methods (IslandViewer, IslandPick, SIGI-HMM and IslandPath-DIMOB) are represented as blue lines above the corresponding genome line.

Figure 3

SRS053630_LANL_scaffold_2818 contains three MGEs with differential abundance patterns across HMP samples. (A) Annotation of the contig: the bottom box represents the contig (with protospacers shown as vertical lines), and the top line represents the genome of H. parainfluenzae T3T1, with which the contig shares similarity. Alignment between the genome and the contig is shown in green. This contig contains three MGEs, two of which are similar to phages based on PHAST predictions (the regions predicted to be prophages by PHAST are shown in red lines below the contig box), and one contains six protospacers (highlighted in the red box). The MGE similar to phage Entero SfV contains most of the protospacers. (B) Relative abundance of each MGE (Y-axis; MGE_UNK in open circles, PHAGE_Entero_SfV in red triangles, and PHAGE_Entero_philP27 in green diamonds) versus the abundance of bacterial host (X-axis) across the HMP samples. MGE, mobile genetic element; RPKM, reads per kbp per million reads.

Figure 4

Abundance analysis of the MGE found in SRS044662_LANL_scaffold_46036. The MGE is predicted to be a prophage with genes encoding phage-related proteins (phage structural proteins, and so on). (A) Correlation between the abundance of the MGE and the abundance of its host, with the data points for 'outliers' in green, and the data point for the source sample (SRS044662) of this contig in red. (B) Detailed reads recruitment to the contig in sample SRS044662, from which the contig was assembled (green lines; the identity bar is shown on the left) and the coverage curve (in blue) along the contig. The genes predicted in this contig are shown as open arrows at the top (for the genes on the forward strand) and the bottom (for the genes on the reverse strand) of the line representing the contig. (C) Coverage and reads recruitment to the contig in another sample (SRS024015), demonstrating a case where MGE significantly outnumbers the host bacterium. MGE, mobile genetic element.

Figure 5

A network of MGEs and CRISPR-Cas defense systems mediated through PAMs. Only selected MGEs and CRISPR types are shown, for demonstration purposes. Blue rectangles represent CRISPRs; red rectangles represent MGE contigs. The WebLogo of PAM for the protospacers is plotted on the edges between the corresponding nodes of the MGE and CRISPR. The logos include 10 positions: -4 to 0 for the protospacers and 1 to 5 for the adjoining regions containing the PAM. PAM, protospacer-adjacent motifs.