Sequencing of E. coli Nissle 1917: Functional Genome Analysis in vivo and in vitro and Molecular Analysis of probiotic effects

Our project is focussing on the molecular and functional analysis of the host-microbe interactions responsible for the probiotic effect of E. coli Nissle 1917. The entire genome sequence of E. coli Nissle 1917 is sequenced concomitantly in the department of Genome Analysis at the GBF. In the previous funding period, we have identified coding sequences and also reconstructed and visualized the metabolic network of E. coli Nissle 1917 from low-coverage, unannotated genome data, using a new biomathematical method. The metabolic network of E. coli Nissle 1917 is largely identical to that of the sequenced uropathogenic E. coli isolate CFT073. Furthermore, we could identify 108 genes, specific for E. coli Nissle 1917. While in the initial phase of our project, we were focused on establishing the genome sequence of the probiotic as well as to set up in vitro and in vivo model systems for the analysis of probiotic effects, in the forthcoming period we will investigate the molecular basics of the probiotic phenotype of E. coli Nissle 1917. So far we could identify in vitro surrogate markers of E. coli Nissle´s probiotic effect, using a coculture system with human intestinal epithelial cells. Based on these data, we will look for genes of E. coli Nissle 1917, which do influence gene expression of these surrogate markers, thus having a potential impact on the probiotic effect of the strain. Therefore, we will at first delete the 108 genes specific for E. coli Nissle 1917, using a single step mutagenesis procedure with PCR products, a method recently described by Datsenko and Wanner. Alongside, using gene expression analysis arrays and, in cooperation with project A09, using a promotor trap library, we will search for in vivo expressed genes of E. coli Nissle 1917. They will be functionally characterized and their impact for the probiotic phenotype of E. coli Nissle will be dissected. Based on previous experiments, where we have established protocols to stably colonize the intestine of conventional and gnotobiotic mice with E. coli Nissle 1917, we will investigate the in vivo gene expression of intestinal epithelial cells in mice, using AFFYMETRIX MOE430A arrays and real-time RT-PCR. This part of the project is aimed at identifying candidate genes for the probiotic effect in the intestinal mucosa of mice, as well as characterizing the in vivo impact of the surrogate markers detected in vitro . Furthermore, we have developed concepts to employ E. coli Nissle 1917 as an intestinal therapeutic for in situ synthesis of recombinant immune modulating molecules. Based on the E. coli alpha hemolysin, we have constructed a plasmid, which allows for synthesis and expression of heterologous proteins using the E. coli hemolysin transport system. Currently we are establishing this system in making E. coli Nissle 1917 isolates that produce murine IL-10 or IFN g or human IL-2. In cooperation with project A04 we will then investigate the impact of recombinant E. coli Nissle 1917 isolates for treatment of chronic infectious bowel diseases as well as for induction or breaking of mucosal and peripheral tolerance.