Inulin and GOS enhance clbA expression in EcN
To study the impact of oligosaccharides on the growth of EcN and colibactin expression, we cultured the bacteria in the presence of inulin or GOS. As shown in Fig. 1A and Supplementary Fig. 1A-C the addition of inulin from 20 mg/mL to 40 mg/mL to minimal media significantly increased bacterial growth (OD600). Similarly, GOS supplementation stimulated the growth of EcN, albeit to a lesser extent than inulin (OD600, Fig. 1B and Additional Fig. 1D-F).
To determine colibactin expression, the transcript levels of the genes clbA, clbB, clbQ, and clbR fused to a promoterless luciferase reporter construct (lux) were quantified by relative luminescence (RLU, Fig. 1A-B and Additional Fig. 1A-F). As previously reported (7), the clbA gene had the highest expression, followed by clbB, clbR and clbQ. For clbA, we found an increase in RLU over time, peaking around 7 hours. When bacteria were supplemented with 40 mg/mL of oligosaccharides, a significant increase in RLU was seen when compared to control conditions of bacteria grown in the absence of prebiotics (Fig. 1A-B and Additional Fig. 1A-F). As shown in Fig. 1C-D and Supplementary Fig. 1G-L, the area under the curve (AUC) of the RLU divided by the OD600 showed increased expression of the clbA gene at the highest inulin and GOS concentrations. We also found a dose-dependent response of colibactin expression with inulin (Fig. 1C, Additional Fig. 1G-I).
These results indicate that oligosaccharides stimulate the expression of clb genes in EcN.
Iron decreases the clbA expression of EcN stimulated by oligosaccharides
In addition to colibactin synthesis, the clbA gene from the pks island is also involved in the synthesis of siderophores, such as enterobactin and yersiniabactin, which are small molecules synthesized by bacteria that scavenge and solubilize ferric iron (Fe3+) (21). Iron was previously shown to downregulate the expression of the colibactin gene cluster including the clbA gene (21–23). Hence, we tested, using the clbA reporter construct, whether the addition of iron abrogated the increased expression of the colibactin gene cluster induced by oligosaccharides supplementation. The addition of iron sulfate to the minimal medium resulted in significantly increased growth of EcN (Fig. 2A), whereas clbA transcript levels were reduced in a concentration-dependent-manner (Fig. 2B), as expected (22, 23). Addition of inulin increased the growth of EcN as confirmed by our previous experiments (Fig. 2C-E; line with circle). However, when comparing the growth curves of bacteria exposed to increasing inulin concentrations in medium containing iron sulfate at 125 µM, the inulin-induced growth was visibly inhibited, as shown in Figs. 2C-E (line with diamond).
We then calculated the individual luciferase activity levels (AUC of RLU/OD600) when the bacteria were grown in medium supplemented with inulin and iron. The addition of 5 to 125 µM of iron sulfate to the medium with inulin led to a decrease in clbA expression in a concentration-dependent manner (Fig. 2F-H). Similar results using GOS supplementation were obtained regarding bacterial growth (Fig. 2I-K) and clbA expression (Fig. 2L-N).
These results indicate that iron supplementation inhibits the effects of oligosaccharides on bacterial growth and clbA induction as an indicator for the expression of the colibactin gene cluster.
Inulin and GOS enhance clbA expression in tumor-promoting E. coli strain NC101
We tested the effects of inulin and GOS on tumor- and inflammation-promoting E. coli strain NC101, which also harbors the pks genomic island (4, 24). We incubated E. coli NC101 in the presence of the highest concentrations of both oligosaccharides (40 mg/ml) and 100 µM of iron sulfate. As shown in Fig. 3A-B, inulin and GOS supplementation did not influence the growth of E. coli NC101. However, oligosaccharide supplementation increased the transcript levels of clbA compared to control (Fig. 3C). Similar to our luciferase results with EcN, when iron was added to the medium with oligosaccharides, the expression of the clbA gene was reduced.
These results indicate that inulin and GOS increase the expression of the clbA gene in the E. coli strain NC101 and that this increase can be inhibited by iron supplementation.
Oligosaccharides increase colibactin-induced cytotoxicity and double-strand DNA breaks in Caco-2 cells
Our results indicate that inulin and GOS upregulate clbA expression and thus expression of the colibactin operon. We investigated whether this increased expression of the colibactin gene cluster at the transcriptional level could also result in greater genotoxicity. We used the E. coli K-12 strain, which is psk-, and the pks + strain NC101 to infect the adenocarcinoma cell line Caco-2 and assessed cytotoxicity through the megalocytosis assay. As expected (25), Fig. 4A shows that cells infected with E. coli NC101 displayed enhanced megalocytosis compared to cells infected with the control E. coli K-12 strain. The addition of 40 mg/mL of oligosaccharides to the medium resulted in a significant increase in abnormal cell enlargement (Fig. 4A) as determined by the lower absorbance (660 nm), as shown in Fig. 4B (inulin, 1.6-fold decrease) and C (GOS, 2.3-fold decrease compared to cells infected with E. coli NC101 in the absence of oligosaccharides).
Taking into account the well-described effects of colibactin causing DNA DSBs (9) we used an in-cell Western assay to quantify DNA DSBs in Caco-2 cells infected with E. coli strains K-12 and NC101. DSBs in Caco-2 cells infected with E. coli NC101 were evident compared to the K-12 strain (Fig. 4D). Most importantly, the addition of 40 mg/mL of inulin led to an increase of the DSBs in Caco-2 cells as indicated by levels of γ-H2AX, a marker of DNA damage (Fig. 4E, 2-fold increase). Similar results were obtained when using GOS treatment (Fig. 4E, 1.9-fold increase).
Taken together, these data show that oligosaccharides not only increase the expression of colibactin genes, but also lead to an increase in cytotoxicity and DNA DSBs in Caco2 cells.