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Caractérisation structurale et fonctionnelle des composants du pilus de Streptococcus pneumoniae : vers une meilleure compréhension de la biogenèse des pili.

Abstract : Streptococcus pneumoniae, a piliated pathogen, is the causative agent of otitis, pneumonia, bacteremia, and meningitis, and is a major cause of community-acquired illnesses, especially in the very young and the elderly. Pili have been shown to play key roles in pneumococcal infection, and although the pilus biogenesis mechanism has been well studied in Gram-negative bacteria, it remains poorly understood in Gram-positive species. Pilus assembly in S. pneumoniae involves a membrane-associated macromolecular machinery encoded on a single pathogenicity islet, and includes the backbone fiber protein (RrgB), two minor pilins (RrgA and RrgC), and three dedicated sortases (SrtC-1, SrtC-2, SrtC-3). These pilin subunits contain the recognition LPXTG motif of sortases. Here, we provide structural, biochemical, and microbiological insight into this novel pilus formation mechanism by (1) reconstituting the main backbone fiber of the pneumococcal pilus in vitro upon identification that SrtC-1 acts as the main pilus-polymerase and can form RrgB fibers; (2) employing electron microscopy in order to visualize fibers produced in vitro and showing that they structurally mimic the pneumococcal pilus backbone; (3) confirming this result in vivo; (4) solving the high resolution crystal structures of SrtC-1 and SrtC-3 which reveal active sites whose access is controlled by flexible lids, unlike in nonpilus sortases, suggesting that substrate specificity is dictated by surface recognition coupled to lid opening. In addition, we characterize the catalytic triad present in the sortase active site as well as lid residues, and identify key amino acids which are relevant for sortase activity as well as structural stabilization.
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https://tel.archives-ouvertes.fr/tel-00537697
Contributor : Clothilde Manzano <>
Submitted on : Friday, November 19, 2010 - 9:05:50 AM
Last modification on : Wednesday, November 29, 2017 - 3:02:25 PM
Long-term archiving on: : Friday, December 2, 2016 - 11:33:10 AM

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Clothilde Manzano. Caractérisation structurale et fonctionnelle des composants du pilus de Streptococcus pneumoniae : vers une meilleure compréhension de la biogenèse des pili.. Biochimie [q-bio.BM]. Université Joseph-Fourier - Grenoble I, 2009. Français. ⟨tel-00537697⟩

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