Lysis-Hi-C as a method to study polymicrobial communities and eDNA

Abstract

Microbes interact in natural communities in a spatially structured manner, particularly in biofilms and polymicrobial infections. While next generation sequencing approaches provide powerful insights into diversity, metabolic capacity, and mutational profiles of these communities, they generally fail to recover in situ spatial proximity between distinct genotypes in the interactome. Hi-C is a promising method that has assisted in analysing complex microbiomes, by creating chromatin cross-links in cells, that aid in identifying adjacent DNA, to improve de novo assembly. This study explored a modified Hi-C approach involving an initial lysis phase prior to DNA cross-linking, to test whether adjacent cell chromatin can be cross-linked, anticipating that this could provide a new avenue for study of spatial-mutational dynamics in structured microbial communities. An artificial polymicrobial mixture of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli was lysed for 1-18 h, then prepared for Hi-C. A murine biofilm infection model was treated with sonication, mechanical lysis, or chemical lysis before Hi-C. Bioinformatic analyses of resulting Hi-C interspecies chromatin links showed that while microbial species differed from one another, generally lysis significantly increased links between species and increased the distance of Hi-C links within species, while also increasing novel plasmid-chromosome links. The success of this modified lysis-Hi-C protocol in creating extracellular DNA links is a promising first step toward a new lysis-Hi-C based method to recover genotypic microgeography in polymicrobial communities, with potential future applications in diseases with localized resistance, such as cystic fibrosis lung infections and chronic diabetic ulcers.

Keywords: DNA cross-linking; Hi-C; biofilm structure; genomics; polymicrobial infections.

FIGURE 1

Sample treatments and predicted DNA interactions during formaldehyde crosslinking with predicted Hi‐C link outcomes expected. EC, Escherichia coli (DNA in red); PA, Pseudomonas aeruginosa (DNA in yellow); SA, Staphylococcus aureus (DNA in blue). Plasmid DNA is shown as small circles and as thinner lines in Hi‐C links

FIGURE 2

Distance of Hi‐C links within species (i.e., self‐links) measured from origin of replication, normalized between species (i.e., link distance from origin was scaled to a value of 1). CLL, cells long lysis; CNL, cells no lysis; CSL, cells short lysis; EC, Escherichia coli; MLL, mouse long lysis; MML, mouse mechanical lysis; MS, mouse sonicated; PA, Pseudomonas aeruginosa; SA, Staphylococcus aureus. Note: in the SA–SA plot, all six treatments are depicted, but points are difficult to distinguish because they fall on the same line

FIGURE 3

Proportion of Hi‐C links and reads occurring between species plotted for cell polymicrobial “communities” and mouse wound biofilms. Asterisks show statistically significant differences (two‐proportion z‐test) and error bars show 95% confidence intervals calculated by the Wilson score method without continuity correction (Newcombe, 1998). CLL, cells long lysis; CNL, cells no lysis; CSL, cells short lysis; EC, Escherichia coli; MLL, mouse long lysis; MML, mouse mechanical lysis; MS, mouse sonicated; PA, Pseudomonas aeruginosa; SA, Staphylococcus aureus

FIGURE 4

Proportion of Hi‐C links and reads occurring between cells and plasmids (pG527 and pUPC18) plotted for cell polymicrobial “communities” and mouse wound biofilms. Asterisks show statistically significant differences (two‐proportion z‐test) and error bars show 95% confidence intervals calculated by the Wilson score method without continuity correction (Newcombe, 1998). CLL, cells long lysis; CNL, cells no lysis; CSL, cells short lysis; EC, Escherichia coli; MLL, mouse long lysis; MML, mouse mechanical lysis; MS, mouse sonicated; PA, Pseudomonas aeruginosa; SA, Staphylococcus aureus

FIGURE 5

Hi‐C link positions within and between cells and plasmids for cell mixtures (3‐species artificial polymicrobial “communities”), drawn in Circos. Upper row shows plots for all links (not normalized); lower row shows only interspecies or chromosome‐to‐plasmid links, normalized to the same number of links between samples. CLL, cells long lysis; CNL, cells no lysis; CSL, cells short lysis; EC, Escherichia coli; PA, Pseudomonas aeruginosa; pU, pUCP18 plasmid; pG, pG527 plasmid; SA, Staphylococcus aureus