Abstract
Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.
Highlights
Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are a major global health concern that affects over 3 million adults in the United States alone (Kaplan, 2015; Dahlhamer et al, 2016)
We hypothesized that NC101 was an auxotroph, unable to synthesize a key nutrient found in the murine gut
In addition to evaluating model E. coli strains (K12 and 25922), we evaluated clinical specimens isolated from the intestinal mucosa of IBD or non-IBD patients (E. coli LF82, 42ET-1, 568-3, HM670, 37RT-2, 5329, and 39ES-1) (Darfeuille-Michaud et al, 1998; Martin et al, 2004; Baumgart et al, 2007) (Table 1)
Summary
Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are a major global health concern that affects over 3 million adults in the United States alone (Kaplan, 2015; Dahlhamer et al, 2016). Sequencing of a selected spontaneous prototrophic revertant, termed NADerivative or NAD NC101, revealed that NAD NC101 had a single nucleotide substitution in nadA (G263T, compared to WT) that matched the prototrophic E. coli strains LF82 and K12 (Figure 4A).
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