Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

Abstract

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.