title: Investigation of colibactin regulation by exogenous amino acids using molecular analyses and colorectal cancer models
Creators: name: Sandalli, Sofia
Publisher: name: University of Glasgow
Year: 2024

description: Colorectal cancer (CRC) is the third most common malignant disease and the second deadliest form of cancer worldwide. Risks of CRC development can arise from genetic predispositions, intestinal disorders, environmental factors like lifestyle and diet, and from oncogenic microorganisms, which are increasingly being investigated for their role in cancer aetiology. In 2006, a potent genotoxic metabolite produced by strains of Escherichia coli was identified, and subsequently named colibactin. The biosynthetic machinery for production of this genotoxin is encoded on the 54 kb polyketide-synthase (pks) pathogenicity island, which is comprised of 19 genes (clbA-S). The pks island has been identified in Klebsiella pneumoniae strains, in a Citrobacter koseri isolate, and in Escherichia coli strains of the B1 phylogroup, however the vast majority of colibactinproducing bacteria are E. coli strains belonging to the B2 phylogroup. Colibactin behaves as both a genotoxin and a cyclomodulin and is capable of inducing DNA damage, such as interstrand cross links (ICLs), double strand breaks (DSBs), single base pair substitutions (SBSs), and insertion-deletion (indel) mutations. These genomic aberrations lead to cell cycle arrest in the G2/M phase, chromosomal instability, senescence, and over time have the potential of triggering CRC. Although several pks+ E. coli strains are commensals of the human gut, they are heavily overrepresented in patients suffering from CRC, with studies indicating that, on average, 50-60% of CRC patients harbour colibactin producing E. coli, as opposed to 20% of healthy individuals. Interestingly, the pks island is also carried by the widely administered probiotic strain, E. coli Nissle 1917 (EcN), which is sold under the name Mutaflor® and used to manage several inflammatory intestinal disorders. Indeed, the probiotic activity of EcN is tightly linked to its genotoxic one, with studies showing that deletion of clb genes results in a lack of antagonistic activity. 

As research has demonstrated a strong interplay between CRC, diet, and the gut microbiome, this project aimed to investigate the impact of dietary components on colibactin expression and subsequent downstream effects on CRC. Previously, D-Serine has been shown to downregulate transcription of colibactin, but it was unclear whether this effect is unique to D-Serine or could be driven by different isomers of multiple AAs. The impact of inclusion of L- and D- isomers of all proteinogenic AAs on colibactin expression was screened in four different types of growth media using a transcriptional GFP reporter assay. RT-qPCR was then used to confirm L-Histidine, L-Isoleucine, L-Serine, D-Serine, and D-Tyrosine as the most significantly inhibitory AAs. The functional impact of colibactin transcriptional repression was further validated through in cellulo assays and DNA crosslinking activity. Results showed that L-Serine and D-Serine reduced the megalocytotic, senescent phenotype induced by EcN, and that D-Serine and DTyrosine decreased DNA crosslinking caused by EcN. Moreover, the combination of D-Serine and D-Tyrosine greatly repressed the transcription of colibactin biosynthesis genes, indicating additive activity between the two AAs. The activity of D-Serine on genotoxin activity was further validated in several colibactinproducing clinical isolates and two different EcN stocks. In vivo work conducted in C57BL/6 mice indicated that EcN elicited probiotic activity, driving down colonic inflammation, while EcN-gavaged mice who were fed D-Serine did not seem to display the probiotic effects of EcN. This was likely because, by acting on colibactin, D-Serine counteracted EcN’s probiotic activity. D-Serine was also found to have ameliorative effects on the intestinal mucosa of AOM/DSS mice by reducing inflammation. Stool sampling performed to analyse the impact of DSS, D-Serine, and EcN administration highlighted that DSS treatment was responsible for the biggest changes to microbiota composition. Finally, data from this PhD project also showed that the nitrogen regulatory protein glnG may play a role in colibactin transcription. 

Overall, this thesis advances what is currently understood about colibactin’s endogenous and exogenous regulation and contributes to elucidating how D-Serine may reduce colibactin production. Furthermore, it lays the groundwork for future research that could focus on further probing D-Serine’s effect on pks+ strains in vivo, on understanding the mechanism by which D-Serine reduces colibactin expression, and on investigating how D-Tyrosine downregulates colibactin transcription.