Microbiota-derived metabolites in intestinal disease: Moving from association to molecular mechanism

The human gastrointestinal tract is inhabited by trillions microbial cells that mostly form a symbiotic relationship with the host. Bacteria have been studied best with regard to the function and effects in the intestinal tract. Bacteria can promote development of colorectal cancer (CRC), but also inflammatory bowel disease (IBD). The focus of recent studies has shifted to the contribution of bacteria-derived metabolites, the intermediate or end products of metabolic reactions, on the intestinal tract. In this work, we aimed to further delineate to contribution of microbiota-derived metabolites on the intestinal diseases IBD and CRC.

First, in chapter 2, the role of microbiota-derived metabolites in colorectal cancer is comprehensively assessed. Here, the focus is on the impact of established contributors, including secondary bile acids, short-chain fatty acids, trimethylamine-N-oxide, indoles and colibactin.

A loss of epithelial barrier integrity is a hallmark of IBD. Therefore, we utilized a screen in chapter 3 using the released factors by bacteria after a two-day culture, which were added to intestinal organoids. Here, we identified the bacterial species Fusobacterium nucleatum and Fusobacterium varium to release a compound that is toxic to intestinal organoids. Butyrate is a short-chain fatty acid that has been reported to be secreted by Fusobacterium species, which was confirmed to be released by our strains. Lastly, both Fusobacterium-released metabolites and butyrate induced autophagy in intestinal epithelial cells, indicating that this prolonged autophagy could result in toxicity to the organoids. All in all, this chapter identified Fusobacterium species to secrete butyrate which promoted autophagy and organoid cytotoxicity.

Fusobacterium nucleatum is not only associated with IBD, but also with CRC. Thus, in chapter 4 we wondered whether Fusobacterium species secrete metabolites that could promote development of colorectal cancer. A pro-inflammatory compound activating ALPK1 signaling was identified to be released from Fusobacterium species, likely being the metabolite ADP-heptose. Lastly, RNA-sequencing suggested that ADP-heptose released by Fusobacterium could promote immune evasion of tumor cells by upregulating the inhibitory receptor PD-L1. Thus, Fusobacterium species secrete ADP-heptose that could promote tumor development by evading the immune system via PD-L1 upregulation.

In chapter 5 we identified another ADP-heptose secreting bacterium, Campylobacter jejuni, which can cause severe gastroenteritis in individuals. The pro-inflammatory effect of C. jejuni on epithelial cells was dependent on a functional ADP-heptose synthesis pathway in the bacterium, indicating that indeed specifically ADP-heptose or another upstream metabolite was responsible. Additionally, the ADP-heptose derived from C. jejuni activated a wide set of pro-inflammatory genes, indicating that this metabolite could contribute to the severe inflammation induced by C. jejuni infection.

The question remained what the broad transcriptional consequences of ADP-heptose on the intestinal epithelium and immune cells were. Therefore, chapter 6 assessed RNA-sequencing after ADP-heptose stimulation of intestinal epithelium and immune cells compared to a classical bacterial pro-inflammatory compound. It was identified that in addition to an extensive pro-inflammatory transcriptional signature, ADP-heptose also promoted a pro-tumor signature in intestinal epithelial cells. In immune cells, ADP-heptose induced a distinct pro-inflammatory signature, indicating that pro-inflammatory consequences of ADP-heptose could have different consequences in epithelial cells compared to immune cells.

The research conducted in this thesis provides insight into the consequences of two bacterial-derived metabolites ADP-heptose and butyrate on especially the intestinal epithelium. This is accomplished using to pathogenic bacterial species F. nucleatum and C. jejuni, which additionally provides insights into pathogenic mechanisms employed by these bacterial species. The results expand our knowledge on microbiota-derived metabolites in intestinal diseases such as inflammatory bowel disease, colorectal cancer and acute gastroenteritis as result of C. jejuni infection. Additionally, the research allows for identification of novel therapeutic approaches to combat these metabolites or bacteria in these diseases.