Elucidating a mechanistic role of colibactin induced DNA damage.

Anu,¹ Thisuri Wanniarachchi,¹ Nicholas Randazo,¹ Yougant Airan,² Seth B. Herzon,² Steven D. Bruner¹

1Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA
2Department of Chemistry, Yale University, New Haven, CT, 06520, USA

The human gut microbiota constitutes a complex, diverse community of microorganisms that influence human physiology, clinical responses to drugs, and disease progression¹. The natural product colibactin, along with its associated biosynthetic gene cluster, is an example system for the role microbially derived small molecules play in the human microbiome. This is particularly relevant in the human gut, where host microbiota is involved in various disorders, including colorectal cancer pathogenesis. Colibactins are hybrid polyketide-nonribosomal peptides that are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis². Owing to difficulty in isolating E. coli natural active colibactins we will utilize synthetic colibactin analogs³ to investigate and elucidate mechanisms by which colibactins damage and bring about genotoxic effects in human gut microbiome. In this current work we establish that our synthetic colibactin analogs show similar levels of activities as the naturally virulent colibactins. Our results indicate that colibactin interacts with DNA by forming DNA-alkylated adducts at adenine rich motifs. We verified DNA alkylation by gel electrophoresis, enzymatic reaction and mass spectrometry.  ClbS, a gene product of the pks island, confers resistance to genotoxicity of colibactin in host bacteria by functioning as a cyclopropane hydrolase¹. In this work, we confirmed the enzyme activity of ClbS against synthetic colibactin analogs. Our results aim to delineate the mechanistic role of colibactin in DNA-damage associated tumorigenesis. We hope that these results show the finer details of the complex chemistry of host-microbe and microbe-microbe interactions.

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