Identification of Pseudomonas aeruginosa FtsZ peptide inhibitors as a tool for development of novel antimicrobials

Sir, The alarming increase and spread of antibiotic resistance among bacterial pathogens is one of the most serious public health problems of the last decade.1 This critical situation necessitates the design of novel classes of antibacterial agents having new mechanisms of action against novel targets. We selected the essential and highly conserved FtsZ protein from the cell division machinery of Pseudomonas aeruginosa as the most attractive target. FtsZ is at the top of hierarchic recruitment in the divisosome, its polymerization into the Z-ring coupled with GTPase activity and interactions with FtsA allow the physical separation of daughter cells.2 To identify specific inhibitors, we used the phage-display technique for the selection of short peptide ligands having high binding affinities to FtsZ among a large pool of random peptide permutations.3 The P. aeruginosa FtsZ and FtsA proteins fused with a His-tag at their C terminus were expressed in Escherichia coli under the control of the T7 promoter and purified to homogeneity on an affinity nickel column (Novagen) and by the renaturation of purified inclusion bodies for FtsA (Protein Refolding Kit, Novagen, Madison, USA). N-terminal sequencing confirmed the identity of both proteins. The GTPase activity of FtsZ and the ATPase activity of FtsA were measured using a thin layer chromatography (TLC) assay with 32P-labelled nucleotides (Perkin-Elmer) as substrates. A UV cross-link specific nucleotide binding assay confirmed that FtsZ binds preferentially GTP and that FtsA binds preferentially ATP. Purified FtsZ was used to screen for GTPase peptide inhibitors by phage-display using the PH.D.-12 and PH.D.-C-7-C phage libraries (New England Biolabs) containing ∼2.7 × 109 12-mer and ∼3.7 × 109 C-7-C-mer random peptide sequences, respectively. The specificity of the three rounds of biopanning was raised by increasing the stringency of the washes and by decreasing the time of contact between the phage encoding peptide fusions and FtsZ. Phages encoding peptides having specific affinity with FtsZ were eluted by non-specific disruption using glycine and by competitive elutions using nucleotide substrate GTP, non-hydrolysable substrate analogue 5′-guanylylimidodiphosphate and FtsA. This unique biopanning identified two C-7-C mers and one 12-mer consensus peptide sequences against FtsZ, reported here as FtsZp1 (CSYEKRPMC), FtsZp2 (CLTKSYTSC) and FtsZp3 (GAVTYSRISGQY). These three peptides were synthesized and their inhibitory capacities of the GTPase activity of FtsZ were evaluated by calculating the percentage of residual enzyme activity. Various concentrations of peptide buffered solutions were pre-incubated with 12 μM of FtsZ in reaction buffer (50 mM Bis-Tris propane pH 7.4, 10 mM MgCl2) for 20 min at room temperature. [32P]GTP was added and mixtures were immediately incubated for 1 h at 37°C. The enzyme reaction was visualized using 2 μL of each reaction sample separated by TLC along with positive (FtsZ alone) and negative (without FtsZ) controls. The percentage of hydrolysis of radioactive substrate was measured by autoradiography. The values of 50% inhibitory concentration (IC50s) for peptides were obtained by plotting the percentage of residual enzymic activity as a function of increasing peptide concentrations (Figure 1). When compared to BSA protein in a competitive assay and to random peptides, the three consensus peptides were able to specifically inhibit the FtsZ GTPase activity. The C-7-C-mers FtsZp1 and FtsZp2 gave an IC50 of 0.45 mM and 1.2 mM whereas the 12-mer gave an IC50 of 5 mM (Figure 1). The three-dimensional crystal structure of FtsZ showed a compact and globular protein.4 We hypothesized that the C-7-C-mer peptides would have more affinity for FtsZ than the 12-mers. The reducing agent dithiothreitol (DTT) had no effect on the inhibitory capacity of the 12-mer peptide FtsZp3. In contrast, DTT reduced significantly the inhibitory potential of the two C-7-C peptides. These results indicated that the disulphide bond formed by the two cysteines of the C-7-C peptides and the subsequent loop conformation were important for their inhibitory potential. Cell division proteins have rarely been used as target proteins and the sole small molecule viriditoxin that inhibits FtsZ was discovered during the course of this work.5 This study allowed the identification of three peptides inhibiting the GTPase activity of the essential cell division protein FtsZ. In perspective, it will be useful to determine their inhibitory constant (Ki) values and to analyse their inhibitory capacities on other GTPases so as to define their specificity. In order to obtain promising lead compounds, inhibitory peptides will undergo chemical modifications and their sequences will constitute the core for the synthesis of libraries of peptidomimetic molecules.6 IC50 determinations for the phage-display-derived peptide inhibitors of FtsZ GTPase. The residual enzymic activity was measured as a function of the concentration of the C-7-C peptides (a) FtsZp1, (b) FtsZp2 and (c) the 12-mer FtsZp3. The peptide sequences are given in the text. We thank Le Service de séquence de peptides de l'Est du Québec and Le Service d'analyse et de synthèse d'acides nucléiques de l'Université Laval. This work was funded by The Canadian Bacterial Diseases Network via the Canadian Centers of Excellence and a FCAR infrastructure team grant to R. C. Levesque and a CRSNG studentship to C. Paradis-Bleau.

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