Étude de la biogenèse de l'autotransporteur AIDA-I d'Escherichia coli

Monomeric autotransporters, a family of proteins that use the type V secretion pathway, are important mediators of virulence for many bacterial pathogens. Many functions important for host colonization and survival have been described for these proteins. Despite the recognized importance of this family of proteins, the mechanisms that are required for the biogenesis and functionality of monomeric autotransporters still remain poorly understood.   The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a classical multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. Extracellular domains of autotransporters are responsible for the protein function and fold into a characteristic β-helical structure. We performed a random mutagenesis of the AIDA-I passenger domain in order to identify regions involved in the various phenotypes associated with the expression of this protein. Our study suggests that the passenger domain of AIDA-I possesses a modular organization, which means that AIDA-I is built with individual functional modules.  Autotransporter passenger domains can be cleaved from the β-domain and released into the extracellular milieu. However, despite the fact that diverse cleavage mechanisms have been previously described, many autotransporters, like AIDA-I, are cleaved by an unknown mechanism. By monitoring the in vitro refolding and cleavage following by site-directed mutagenesis, we showed that AIDA-I processing is an autocatalytic event that involves two acidic residues. Our results unveil a new mechanism of auto-processing in the autotransporter family.  AIDA-I is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. Our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif. Furthermore, we showed that glycosylation occurs in the cytoplasm by a cotranslational mechanism. These observations are unique in bacteria and represent a significant advance in our comprehension of prokaryotic glycosylation. We also showed that glycosylation is required to ensure a normal conformation of AIDA-I and, as a consequence, is necessary for its cell-binding function. The finding that other autotransporters or large adhesin-encoding genes are linked to Aah homologue-encoding genes suggests that glycosylation may be important, if not essential, for the function of these proteins, as for AIDA-I.  In conclusion, the results presented in this thesis bring new information about the autotransporter family and also give new insight into the mechanisms that are important for different aspects of the biogenesis of monomeric autotransporters.

Keyphrases: Autotransporter, Secretion, Glycosylation, Heptose, Autocatalytic cleavage, AIDA-I, Aah, Adhesion, Biofilm, Escherichia coli