Présentation sous forme d’affiche au congrès CBDN (Canadian Bacterial Diseases Network) à Calgary le 25 avril 2003.

Strategy for Screening and Identification of Inhibitor Peptides of the Bacterial Divisosome and Peptidoglycan Synthesis. Paradis-Bleau Catherine, El Zoeiby Ahmed, Beaumont Mélanie, Boudreault Lydia, Sanschagrin François, Loyd Adrian and Levesque Roger C. Pavillon Charles-Eugène-Marchand, Dept. Biologie Médicale, Faculté de Medicine, Université Laval, Ste-Foy, Québec, G1K 7P4.

With the objective of developing new classes of antimicrobial agents and inhibitors, we exploited the phage display technology against essential bacterial divisosome proteins. The FtsZ-FtsA protein complex is responsible for septation of daughter cells; while the MurD, E and F enzymes are essential for the synthesis of the peptidoglycan precursor. Pseudomonas aeruginosa genes encoding FtsZ, FtsA, MurD, MurE and MurF were cloned into pET expression vectors with His-tag fusions and proteins were expressed in E. coli BL21 (λDE3) and purified. Enzyme purity was estimated at >99% homogeneity and their identities were confirmed by N-terminal sequencing. The ATPase activity of FtsZ and MurD along with the GTPase activity of FtsZ was confirmed by thin layer chromatography and by development of an HTS microtiter plate coupled enzyme assay. We used FtsZ, FtsA, MurD, MurE and MurF purified proteins to screen C-7-C and 12-mers libraries containing 109 peptides fused to the phage M13 pIII minor coat protein. Screening was done using 3 rounds of biopanning with stringent washes; competitive elution was done using specific substrates synthesized in vitro , analogues and with FtsA known to bind FtsZ. Phages were selected from each biopanning step and DNA encoding peptides sequenced. Bioinformatics analysis identified consensus peptide sequences for the 5 enzymes studied. A collection of peptides were synthesized, purified and used to determine specific inhibition of ATPase and GTPase activity. Peptide/protein interactions were analysed by ELISA. The peptides FtsZp1 and FtsZp2 showed inhibition of FtsZ GTPase activity with an IC50 value of 450 μM and of 1 mM for FtsZp3. Studies done with MurD identified a peptide with an IC50 of <10 μM. Peptides identified will be used for HTS screening of a combinatorial library of peptidomimetic molecules.