The remarkable biomechanical properties of the Type 1 Chaperone-Usher Pilus: a structural and molecular perspective

Chaperone-usher (CU) pili are long, supramolecular protein fibers tethered to the surface of numerous bacterial pathogens. These virulence factors function primarily in bacterial adhesion to host tissues, but they also mediate biofilm formation. Type 1 and P pili of uropathogenic Escherichia coli (UPEC) are the two best-studied CU pilus examples, and here we primarily focus on the former. UPEC can be transmitted to the urinary tract by fecal shedding. It can then ascend up the urinary tract and cause disease by invading and colonizing host tissues of the bladder, causing cystitis, and the kidneys, causing pyelonephritis. FimH is the subunit displayed at the tip of type 1 pili and mediates adhesion to mannosylated host cells via a unique catch-bond mechanism. In response to shear forces caused by urine flow, FimH can transition from a low-affinity to high-affinity binding mode. This clever allosteric mechanism allows UPEC cells to remain tightly attached during periods of urine flow, while loosening their grip to allow dissemination through the urinary tract during urine stasis. Moreover, the bulk of a CU pilus is made up of the rod, which can reversibly uncoil in response to urine flow to evenly spread the tensile forces over the entire pilus length. We here explore the novel structural and mechanistic findings relating to the type 1 pilus FimH catch-bond and rod uncoiling and explain how they function together to enable successful attachment, spread, and persistence in the hostile urinary tract

Combattre la résistance aux glycopeptides... un défi d'impact clinique

Doctorat en sciences chimiques -- UCL, 199

Elaboration d'une méthode de sélection in vitro pour activité catalytique : application à l'évolution d'une β-lactamase

Doctorat en sciences chimiques -- UCL, 199

Correlation between serum lactate, lactate clearance and survival after cardiac arrest

status: publishe

Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop.

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue

In vitro selection for catalytic turnover from a library of beta-lactamase mutants and penicillin-binding proteins.

A library of mutants of the RTEM beta-lactamase displayed on phage was created; it contained penicillin binding proteins (PBPs) as well as a small fraction of active beta-lactamases. The library was submitted to a selection process to extract the beta-lactamases i.e. the enzymes that turnover efficiently. This was achieved by a two steps procedure. In the first step, the beta-lactamases were labelled by reaction with a biotinylated suicide inhibitor while the PBPs were blocked by incubation in the presence of benzylpenicillin. In the second step, the labelled active phage-enzymes were separated by affinity chromatography on streptavidin coated beads. (C) 1997, Elsevier Science Ltd