Luis Ángel Fernández
Group Leader
Research summary
Our work focuses on engineering E. coli bacteria for biomedical applications, including the selection and production of small recombinant antibodies in bacteria and the design of bacteria for use in diagnosis and therapy in vivo. We study protein secretion systems found in pathogenic E. coli strains and modified them to become nanomachines that may be useful in the selection and expression of proteins of therapeutic interest in nonpathogenic strains of E. coli.
Publications
Cepeda-Molero M, Berger CN, Walsham ADS, Ellis SJ, Wemyss-Holden S, Schüller S, Frankel G, Fernández LÁ (2017). Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors. PLoS Pathog. 13(10):e1006706. PMID: 29084270 DOI: 10.1371/journal.ppat.100670
Salema V., Mañas C., Cerdán L., Piñero-Lambea C., Marín E., Roovers R., van Bergen en Henegouwen P.M., and L.A. Fernández (2016) High affinity nanobodies against human Epidermal Growth Factor Receptor selected on cells by E. coli display. MAbs 8:1286-1301. PMID: 27472381 DOI: 10.1080/19420862.2016.1216742
Ruano-Gallego D., Álvarez B., and L.A. Fernández (2015) Engineering the controlled assembly of filamentous injectisomes in E. coli K-12 for protein translocation into mammalian cells. ACS Synthetic Biology 4:1030-1041. PMID: 26017572 DOI: 10.1021/acssynbio.5b00080
Piñero-Lambea C., Ruano-Gallego D., and L.A. Fernández. (2015) Engineered bacteria as therapeutic agents Current Opinion in Biotechnology 35: 94-102.
PMID: 26070111 DOI: 10.1016/j.copbio.2015.05.004
Piñero-Lambea C, Bodelón G, Fernández-Periáñez R, Cuesta AM, Álvarez-Vallina L, Fernández LA. (2015) Programming controlled adhesion of E. coli to target surfaces, cells and tumors with synthetic adhesins. ACS Synth Biol; 4:463-473 PMID: 25045780 DOI: 10.1021/sb500252a
Our research is aimed to engineer E. coli bacteria for biomedical applications, including the selection of small recombinant antibodies and the design of bacteria for diagnostic and therapeutic use in vivo. We study protein secretion systems found in pathogenic E. coli strains and engineer them to develop protein nanomachines that can be applied for selection of recombinant antibodies and the delivery of therapeutic proteins by non-pathogenic E. coli strains. Among the recombinant antibodies, we employ single-domain antibodies (sdAbs) or nanobodies, the smallest antibody fragments known-to-date with full antigen-binding capacity. Nanobodies are based on VHH domains obtained from heavy-chain-only antibodies found in camelids (e.g. dromedaries, llamas). We use synthetic biology approaches and genome engineering to combine the expression of these modular parts in the designed bacteria.
Current projects:
1) E. coli display technology for selection of nanobodies from libraries. Members of the type V secretion system (T5SS) are proteins with "self-translocation" capacity across the bacterial outer membrane like those belonging to the Intimin-Invasin and autotransporter families. We have engineered the translocator domains of T5SS-proteins to display nanobodies on the surface of E. coli and we are using this technology to select high-affinity binders against antigens relevant in human disease and infection.
2) Re-programming E. coli adhesion to tumors with synthetic adhesins. The display of nanobodies on the surface of E. coli has allowed us to generate "synthetic adhesins" that can drive the attachment of bacteria to target antigenic surfaces, including tumor cells expressing cell surface antigens. We have demonstrated that specific tumors in vivo can be targeted and colonized efficiently by low doses of engineered E. coli strains expressing synthetic adhesins binding antigens expressed on the surface of the tumor cells.
3) Injection of therapeutic proteins from E. coli into human cells. We are exploiting the type III protein secretion system (T3SS) from enteropathogenic E. coli (EPEC) to directly deliver therapeutic proteins and nanobodies from E. coli into the cytosol of human cells. During infection these filamentous T3SSs act as molecular syringes (injectisomes) for the translocation of proteins from bacteria into mammalian cells. We have engineered the expression of EPEC injectisomes in non-pathogenic E. coli K-12 strain allowing us to specifically deliver a protein of interest in the cytosol of mammalian cells. E. coli injection of proteins does not require bacterial invasion of the eukaryotic cell or the transfer of any genetic material.
