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.
Highlights
The gut is a rich microbial ecosystem that contains numerous species, many of them implicated in human diseases
For the pks island genes, a fragment of 800 bp from the clbN gene was amplified directly from 11 stool samples that had been found to be positive for this gene (2 healthy, 5 adenomas, and 4 colorectal cancer (CRC))
For the tcpC gene, a fragment of 283 bp was amplified from seven stool samples
Summary
The gut is a rich microbial ecosystem that contains numerous species, many of them implicated in human diseases. It is increasingly clear that the involvement of E. coli in CRC takes place through mechanisms that are encoded by specific sets of genes (Bonnet et al, 2014; Raisch et al, 2014). These genes encoding genotoxic cyclomodulins and other pro-inflammatory molecules have been identified in certain strains of gramnegative bacteria. The pks island genes, a cluster of genes encoding enzymes for the production of the elusive natural product colibactin, has been found more frequently in mucosa samples from CRC individuals than in healthy donors (Arthur et al, 2012; Gomez-Moreno et al, 2019). The gene for the uropathogenic specific protein (usp) initially identified in E. coli from urinary tract infections encodes a DNAse with genotoxic activity that promotes cell death (Nipic et al, 2013)
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