Shining a Light on Colibactin Biology

Abstract

Colibactin is a secondary metabolite encoded by the pks gene island identified in several Enterobacteriaceae, including some pathogenic Escherichia coli (E. coli) commonly enriched in mucosal tissue collected from patients with inflammatory bowel disease and colorectal cancer. E. coli harboring this biosynthetic gene cluster cause DNA damage and tumorigenesis in cell lines and pre-clinical models, yet fundamental knowledge regarding colibactin function is lacking. To accurately assess the role of pks⁺ E. coli in cancer etiology, the biological mechanisms governing production and delivery of colibactin by these bacteria must be elucidated. In this review, we will focus on recent advances in our understanding of colibactin's structural mode-of-action and mutagenic potential with consideration for how this activity may be regulated by physiologic conditions within the intestine.

Keywords: APC; DNA damage; Escherichia coli; colibactin; colorectal cancer; genotoxin; inflammation; microbiome; mutation; pks.

Figure 1

Colibactin is a product of the 54-kb pks gene island. The pks gene island consists of 19 clb genes transcribed in four polycistronic and three cistronic elements, encoding various non-ribosomal peptide synthetase (NRPS), polyketide synthetase (PKS), hybrid NRPS-PKS or accessory proteins. Colibactin production is regulated by the LuxR-type transcriptional activator ClbR and the pantetheinyltransferase (PPTase) ClbA. Following transcriptional activation, a biosynthetic scaffold coordinates production of a linear intermediate (precolibactin) harboring a N-myristoyl-D-Asn motif. The transmembrane peptidase ClbP removes these prodrug motifs, inducing spontaneous dual two-fold cyclizing events resulting in production of the bioactive colibactin molecule, characterized by two electrophilic cyclopropane warheads with high binding-affinity for adenine residues within AAWWTT nucleotide motifs.

Figure 2

Pks⁺ E. coli promote tumor formation in the colonic epithelium. During infection with pks⁺ E. coli, colibactin molecules translocate to the host nucleus via an undetermined mechanism, where the compound generates inter-strand crosslinks (ICLs) in adenine-rich nucleotide motifs and double-strand DNA breaks (DSBs). Errors during DNA repair following pks-induced damage result in the accumulation of a specific mutational signature characterized by T > N single base substitutions (SBS_pks) or insertion/deletions of varying length (ID_pks). Exposure to pks⁺ E. coli induces oncogenic phenotypes characterized by enhanced proliferation and Wnt independence. Unrepaired lesions cause cell-cycle arrest. Arrested cells adopt a senescence-associated secretory phenotype (SASP) resulting in enhanced growth factor production (hepatocyte growth factor [HGF], fibroblast growth factor [FGF]) which promote proliferation of nearby cells.

Figure 3

Transcriptional regulation of clb genes. (A) Transcription of the regulatory clb genes (clbA and clbR) is increased in low-iron or oligosaccharide rich environments. The endo- or exogenously derived polyamine spermidine is necessary for pks transcription. (B) Inflammation promotes the transcription of several individual clb components. Administration of anti-inflammatory drugs such as anti-TNF attenuate these effects and limit pks-associated tumorigenicity in vivo. Anti-polyphosphate kinase (PPK) inhibitors downregulate clbB expression and the genotoxicity of pks⁺ E. coli.

Figure 4

The oncogenic capacity of pks⁺ Escherichia coli is enhanced by increased mucosal invasion and biofilm association. In healthy tissue, E. coli typically aggregate at the mucosal surface. In biofilm-covered CRCs, pks⁺ E. coli form biofilms in cooperation with enterotoxigenic Bacteroides fragilis (ETBF), increasing depth of mucosal invasion. During inflammation, a microaerobic niche formed within the mucosa (derived from host nitric oxides or peroxides) facilitates the expansion of mucosal E. coli populations.

Figure 5

Future areas of research involving pks⁺ E. coli.