Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Molecular assembly of the pneumococcal pilus . From a natural to a biotechnological toolbox – PILIPATH

New anti-bacterial targets

Antibiotics inhibit essential physiological processes in bacteria, which consequently develop mecanisms to escape to antibiotics action. To reduce and/or delay the emergence of resistant strains, strategies to design new anti-bacterial drugs aim to target important steps in the disease physiopathology.

Assembly inhibition of the adhesive pneumococcal pilus

Streptococcus pneumoniae causes most cases of pneumonia, otitis, sinusitis, sepsis and meningitis in humans leading to about 1.6 million people yearly. Combating pneumococcal diseases is hampered by the replacement of vaccine serotypes within populations and the dissemination of antibiotic resistant strains. Therefore, innovative therapeutic targets and/or vaccine candidates must be identified and characterized such as the pili, a virulence factor on which the present research project focuses.

The pneumococcal pilus is an extended fiber composed by three proteins, the pilins, exposed at the bacterial surface through covalent link catalyzed by the sortases. We propose to characterize at the molecular level how the pilins are assembled by the sortases and thus to question the pilus architecture and the enzymatic properties of the sortases. These studies will be performed by in vitro and in vivo approaches.

This proposal is based on the current collaborative work performed by two groups of the Institut de Biologie Structurale, Grenoble. We aim at discovering the complete blue print for the construction of the pilus and to describe its full structure. We have already developed the tools required to progress into this project.

This goal will entail a detailed description of all the steps required for the assembly of the pilus in parallel with the determination of the high-resolution structure of complexes of the pilus proteins. These data will be helpful to develop innovative ways to inhibit the formation of this important virulence factor and thus investigate new strategies to combat pneumococcal infections.

Partners of this project have already co-publisehd 7 papers on the pneumococcal pilus.

Bacterial infections still represent a considerable threat for human life in developed and developing countries. Among the Gram-positive bacteria, Streptococci and Enterococci are the most important human pathogens, causing either community-acquired or nosocomial infections. Streptococcus pneumoniae causes most cases of pneumonia, otitis, sinusitis and many cases of invasive diseases such as sepsis and meningitis in humans. The WHO estimates that this pathogen is responsible for the death of about 1.6 million people yearly, mostly young children, elderly and immunocompromised individuals. Despite important advances in pneumococcal vaccines and antibiotic treatments, combating pneumococcal diseases is hampered by the replacement of vaccine serotypes within populations and the dissemination of antibiotic resistant strains. Therefore, innovative therapeutic targets and/or vaccine candidates must be identified and characterized such as the pili, a virulence factor on which the present research project focuses. The pneumococcal pilus is an extended fiber composed by three protein, the pilins, exposed at the bacterial surface through covalent link to the peptidoglycan.
This proposal is based on the current collaborative work performed by two groups of the IBS leading to the following assets central to our proposal: (1); a sortase-based in-vitro technique for the synthesis of the fiber using recombinant proteins, (2); an efficient pilins-sortases co-expression platform for the identification and characterization of complexes between pilin building blocks. (3), the high resolution structures of five of the six proteins involved in pneumococal pilus biogenesis. We have assessed the specific role of each of the three sortases of the pilus operon and the importance of distinctive isopeptide bonds in the pilus structure. These technological developments, associated know-how and basic knowledge should combine in ensuring the success of our proposal. Structural characterization of pilins revealed an unusual intramolecular covalent bond between the side chains of an Asn and a Lys, a feature reminiscent to disulphide bonds increasing protein fold stabilization. Two small domains identified from the high-resolution structure of one of the pilins were engineered. They are being used in an innovative method called “Bio Molecular Welding” based on the fact that these two domains form a spontaneous covalent complex either in vitro or in vivo.

We aim at discovering the complete blue print for the construction of the pilus and to describe its full structure. This goal will entail a detailed description of the all the steps required for the assembly of the pilus in parallel with the determination of the high-resolution structure of complexes of the pilus proteins. Ultimately, these efforts should render the first complete structural and biochemical characterization of a Gram-positive pilus. Applications related to pneumococcal biomedical uses that are derived from our pending patent for the “Bio Molecular Welding” method are included in this proposal.

Project coordinator

Madame Anne Marie DI GUILMI (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES) – Anne-marie.Di-guilmi@ibs.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CEA, CNRS, Univ. Joseph Fournier CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
CEA, CNRS, Univ. Joseph Fournier CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

Help of the ANR 348,479 euros
Beginning and duration of the scientific project: - 36 Months

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