Design and construction of synthetic adhesins driving the specific attachment of Escherichia coli to target surfaces, cells, and tumors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular: Fecha de lectura: 07-11-2014One of the aims of genetic engineering and synthetic biology is the design of microorganisms with novel capabilities that could be beneficial for humans, including their use of vaccines, diagnostic sensors, and therapeutic applications for major diseases such as cancer. In this regard, the availability of genetic elements able to program the adhesion of the engineered bacterium to different targets would be extremely useful. This work reports the development of synthetic adhesins (SAs) that enable to precisely program the adhesion properties of Escherichia coli bacteria to different target surfaces, including tumor cells. The structural organization of these novel SAs is defined by a domain that anchors the SA to the bacterial outer membrane, which is derived from the N-terminal fragment of Intimin comprising residues 1-659, and an adhesive domain based on the smallest antibody fragments known to date, termed VHHs. This modular organization allows the modification of the binding specificity of the SA by the exchange of the VHH sequence. We demonstrate that SAs are efficiently displayed on the surface of E. coli and are able to drive bacterial adhesion to antigen-coated abiotic surfaces and to target tumor cells expressing on their surface the antigen recognized by SAs. SAs are constitutively and stably expressing from the chromosome of an engineered E. coli strain lacking a conserved set of natural adhesins (i.e. type 1 fimbriae, Antigen 43 and mat fimbriae) and constitutively expressing the lux operon as bioluminescent reporter. Using tumor xenograft mouse models we have demonstrated that engineered E. coli strains carrying SAs colonize efficiently solid tumors expressing the cognate antigen recognized by the SA using two order of magnitude lower doses of systemically administered bacteria compared to control strains with SAs binding an unrelated antigen or the wild type E. coli strain. In addition, we observed that the engineered strains were cleared faster from non-target organs (e.g. liver and spleen) probably due to the deletion of natural adhesins. The fast and specific adhesion mediated by SAs was also employed to characterize the influence of both, flagella and YcgR protein in the adhesion process, as well as to investigate the short-term transcriptional response of E. coli upon adhesion to tumor cells. Our results indicate that, whereas active bacterial motility mediated by flagella is important for an efficient adhesion to target cells, the lack of YcgR protein does not affect the ability of bacteria to adhere to target cells, suggesting that the arrest of bacterial motility upon adhesion is independent of YcgR protein. In addition, we analyzed by RNAseq the global transcriptional response of E. coli bacteria upon adhesion (15 min) to target tumor cells. Our results indicate a common transcriptional response upon adhesion regardless of the cellular receptor recognized. Genes involved in sulfur uptake and its metabolism were upregulated upon adhesion, whereas genes involved in the transport of intermediates of the tricarboxylic acid cycle and tryptophan synthesis were downregulated. We found that the activity of gene fusions between the chromosomal yeeE promoter region and the mCherry reporter gene were upregulated in response to bacterial adhesion. Lastly, we have also demonstrated that SAs can drive the specific adhesion of non-live bacterial derived nanoparticles toward target tumor cells expressing a therapeutically relevant cell surface receptor (i.e. EGFR)