RESEARCH SUMMARY

Our group conducts both basic and biotech-oriented research aimed to understand and exploit protein secretion in pathogenic, commensal and laboratory E. coli strains. Our basic research focuses on the molecular mechanisms that these bacteria employ for the secretion of proteins (e.g. cytotoxins, proteases, adhesins) and for the assembly of cell surface organelles (e.g. fimbriae) that participate in bacterial virulence. We focus especially on those proteins and surface organelles secreted and assembled by enteropathogenic (EPEC), enterohemorragic (EHEC) and uropathogenic (UPEC) E. coli strains. The biotechnological projects exploit these systems for the development of novel expression and selection technologies for recombinant antibodies in non-pathogenic commensal and laboratory E. coli strains. Among the recombinant antibody formats available (e.g. single-chain Fv, Fab, Fc-fusions, etc.), we focus on single-domain antibodies (sdAbs) or nanobodies, the smallest antibody fragment known-to-date (~15 kDa) with full antigen-binding capacity. nanobodies are based on a single VH domain obtained by recombinant DNA technology from heavy-chain-only antibodies from camelids (e.g. dromedaries, llamas). Despite the lack of a paired V_L domain, nanobodies show high-affinity and specificity for their cognate antigens. In addition they are highly similar to human V_H3 sequences, making them excellent candidates for multiple applications, including human therapy.

Some of our current projects are:

1) The secretion mechanism of the bacterial type V secretion system (T5SS) and its application for the bacterial display of single-domain antibodies in E. coli.

The T5SS include proteins with "self-translocation" capacity across the outer membrane like the Intimin-Invasin family and the so-called autotransporters (ATs), which are the major family of proteins secreted by Gram-negative bacteria. ATs have distinct biological functions important during the pathogenesis of the producer microorganism (e.g. proteolysis of host proteins and antibodies, cytotoxicity of host cells, adhesion to host tissue, etc). In addition to investigate the secretion mechanism of ATs, we are exploiting the translocator domains of T5SSs to display nanobodies on the surface of E. coli for selection of binders against specific antigens (bacterial display).

2) Assembly of type 1 fimbriae.

Type 1 fimbriae are thin proteinaceous filaments assembled on the surface of E. coli cells by the ordered polymerization of a major protein subunit (FimA) and several minor protein subunits (FimF, FimG, FimH) assembled by the chaperone-usher pathway. Type 1 fimbriae and FimH adhesin are essential for effective colonization and invasion of the epithelial cells of the urinary bladder by UPEC strains. We found that the N-terminal lectin domain of FimH is recognized by the fimbrial usher FimD in order to initiate the assembly the adhesin and the polymerization of the fimbrial filament. The mechanism of activation of FimD by the N-lectin domain of FimH is currently under investigation.

3) Injection of nanobodies from E. coli into human cells.

We are employing the type III protein secretion system (T3SS) from EPEC and EHEC E. coli strains, to directly deliver single-domain recombinant antibodies from E. coli cells into the cytosol of human cells. During infection T3SSs act as molecular syringes for the translocation of proteins from bacteria into eukaryotic cells. E. coli injection of nanobodies does not require bacterial invasion of the eukaryotic cell or the transfer of any genetic material.