Figure 3. c D4+ c D45RBhigh T cells elicit an aggressive wasting disease leading to exacerbated mortality in transferred sc ID mice fed with Nissle ΔclbA. c D4+ c D45RBhigh T cells were purified from spleens of BalB/c a nNc rl mice and 2 × 105 cells were injected (i.p.) into recipient sc ID mice. Groups of 5–11 (untransferred controls) mice were fed with Nissle WT, Nissle ΔclbA (2.5 × 109 cfu/mouse) or with pBs every 3 d and sacrificed 45 d after transfer. pooled body weight gain and surviving rates of mice from two independent experiments are represented. (A) Body weight gain (percentage of initial) is expressed as the mean ± seM (B) percentages of survival mice at the indicated time points are shown. the colon (Fig. 4C), and significantly decreased histology scores (Fig. 4D) and IL-1β expression (Fig. 4E). Colonic IL-10 expression levels observed in mice fed with Nissle WT were similar to the levels observed in control healthy mice. As previously observed in the DSS-induced colitis model, the probiotic effect of Nissle WT treatment was lost when SCID mice were treated with the non-genotoxic mutant, as evidenced by increased macroscopic damage (Fig. 4A and B), colon weight/ length ratio (Fig. 4C) and mucosal inflammation (Fig. 4D). The colonic cytokine expression pattern of mice fed with Nissle ΔclbA was also similar to that seen in PBS-treated mice (Fig. 4E). Complementation of clbA-mutant restores probiotic proper- ties of E. coli Nissle 1917. In order to confirm that the dele- tion of clbA was solely responsible for the differences observed in experimental colitis, we constructed a complemented strain by re-introducing the wild-type clbA gene into the chromosome of Nissle ΔclbA. We first confirmed the ability of this comple- mented mutant (Nissle ΔclbA + clbA) to induce DNA-double strand breaks in transiently infected IEC-6 cells (Fig. S2). Given the in vitro restoration of genotoxicity displayed by the comple- mented clbA mutant, we next re-assessed colitis development in CD45RBhigh T cells injected SCID mice fed with Nissle WT, Nissle ΔclbA or Nissle ΔclbA + clbA. Consistent with our pre- vious data (Fig. 3A), body weight loss kinetics were similar in the three groups (Fig. S3A). Although global colitis severity was exacerbated in this experiment, we re-observed an increased mor- tality among the group of transferred mice fed with Nissle ΔclbA as compared with the group of transferred mice fed with Nissle WT (Fig. S3B). These results were confirmed by a significantly higher clinical score associated to the treatment of transferred SCID mice with Nissle ΔclbA (Fig. 5A and B). Moreover, res- toration of clinical scores similar to those observed with Nissle WT treatment was obtained for mice fed with the complemented clbA mutant. In the same way, as indicated by the measurement of colon weight/length ratio (Fig. 5C) or by the examination of histological sections (Fig. 5D), increased intestinal inflamma- tion resulting from clbA deletion was completely reversed when transferred SCID mice were fed with the Nissle strain in which the wild-type clbA gene was re-introduced within the pks island. These data clearly demonstrate that the immunomodulatory properties of Nissle 1917 required the clbA gene of the pks island and that the probiotic activity of E. coli Nissle 1917 cannot be dissociated from its genotoxicity. Discussion Our results together with previous studies20-23 and clinical tri- als13-15 confirm the therapeutic potential of this E. coli Nissle 1917 strain in promoting gut homeostasis upon mucosal injury. This probiotic was regarded, so far, as nonpathogenic,11 but we have shown that this bacterial strain induce DNA double strand breaks and chromosomal abnormalities in eukaryotic cells. Even if in vivo genotoxicity of this probiotic strain remains to be inves- tigated, we hypothesized that construction of a non-genotoxic mutant of E. coli Nissle 1917 could improve its safety. However, we were unable to dissociate the genotoxic activity of the strain from its probiotic activity since inactivation of the biosynthetic pathway leading to the production of Colibactin abrogated also the probiotic activity in two different models of experimental colitis. Whereas Colibactin appears to be a beneficial compound in the probiotic activity of E. coli Nissle 1917, the nature of this activity remains to be clarified and raises several considerations: Colibactin was first reported to be a genotoxin but our results now suggest that the same molecule may also possess anti-inflam- matory activity and could be thus considered as an immuno- modulin. This beneficial activity could be directly or indirectly linked to the mechanism triggering DNA damage in the host cell. Alternatively, it is possible that Colibactin may consist of more than one molecule. The biosynthetic gene clusters of the pks island may encode additional compounds that could mediate the probiotic activity observed in the gut. Since the genotoxic activity of E. coli Nissle 1917 cannot be dissociated from its probiotic activity, safety considerations about large-scale use of this probiotic strain have to be re-evaluated. We have previously shown that the transient DNA damage inflicted 504 Gut Microbes Volume 3 Issue 6