Group Leader: LUIS A. FERNÁNDEZ HERRERO

The research of our group concentrates on the mechanisms of protein secretion in Gram negative bacteria and on various biotechnological applications derived from the use of these protein secretion systems in the expression, secretion, and surface display of recombinant antibodies, especially of domain antibodies (dAbs) derived from camelids (Nanobodies). Our model microorganisms are E. coli strains, both non-pathogenic and pathogenic isolates, like enteropathogenic (EPEC),  enterohemorragic (EHEC) and uropathogenic (UPEC) E. coli. Our research is focused on the following systems:

Figure 1. Comparison of the 3D structures of the inmunoglobulin (lg) domains from a human VL, a Nanobody VHH, and the lectin N-terminal domain from FimH. Structures shown from the PDB files 1F6L, 1KXV and 1QUN. Numbering is according to conventions established for antibody domains.

 

Figure 2. Protein secretion system of Gram negative bacteria.

 

1.- Bacterial autotransport proteins. We have compared the translocator domains obtained from autotransporters isolated from different bacterial pathogenic species of the a, b, g and e proteobacteria. Similarities in functionality, structure, and dependence on cellular chaperones have been investigated.

2.- Type 1 fimbriae. These filamentous organelles required for colonization of the bladder epithilium by UPEC strains. A fundamental role for the N-terminal lectin domain of the adhesin FimH during fimbriae assembly has been found. FimH is located at the tip of the fimbrial filament and its N-terminal domain must be recognized by the outer membrane protein FimD for assembly of FimH in fimbriae.

3.- a-hemolysin secretion system. a-hemolysin (HlyA) is secreted by UPEC strains during infection.  We have used its type I secretion system, composed by the proteins TolC/HlyB/HlyD, for the secretion of dAbs to the extracellular medium. The system has been improved and used for cloning of dAb libraries from immunized camels.  We performed direct selection of secreted dAbs binding antigens using this system.

4.- Intrabodies. Recombinant antibodies were produced in a functional and oxidized form, with disulfide bonds, in the cytoplasm of E. coli cells carrying mutations in the trxB and gor genes. Recombinant antibodies were fused to the thioredoxin 1 (TrxA) acting as a chaperone during protein folding. Intrabodies were selected against cytoplasmic proteins in E. coli (a bacterial transcription factor and a relaxase involved in plasmid conjugation) and we demonstrated that these intrabodies inhibited the activity of these proteins in vivo.