Bacterial engineering for biomedical applications

Group Leader: LUIS A. FERNÁNDEZ HERRERO

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 Β-domains of T5SS-proteins, like Intimin, 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.

 

Figura 1.(A) Scheme of conventional IgGs with heavy (H) and light (L) chains, indicanting the crystallizable fragment (Fc) region, and variable (V) and constant (C) domains. Common antigen-binding fragments, Fab and single-chain Fvs are depicted. (B) Schematic representation of a heavy chain only antibody and derived single-domain fragments (VHH) called nanobodies.

 

Figura 2. Scheme showing the process of selection of nanobodies by E. coli display. The VHHs gene segments are amplified by the lymphocytes of an immunized dromedary and cloned in a vector with Intimin Β-domain for their expression on the surface of E. coli. These bacteria are incubated with the target antigen labelled with biotin followed by anti-biotin magnetic beads. Antigen-binding clones are captured in magnetic cell sorting (MACS) columns and plated for amplification.

 

Figura 3. Fluorescence confocal microscopy image showing E. coli bacteria (red fluorescence) with synthetic adhesins targeting an antigen (green fluorescence) expressed on the surface of human tumor cells (nuclei and bacterial DNA stained in blue).

 

Figura 4. Scheme showing protein translocation into mammalian cells with Synthetic Injector E. coli (SIEC) bacteria engineered to express the filamentous T3SS injectisomes of EPEC.