Peptide-Based Drug Design

Proline-Rich Antimicrobial Peptides 169
3.4. Identification of Genes Responsible for Resistant Phenotypes
1. Select the clones able to grow on medium supplemented with an inhibitory concentration
of PRP and inoculate them in 5 mL of LB broth supplemented with
ampicillin.
2. Incubate overnight at 37 ◦ C.
3. Extract the recombinant plasmids (pBSARs) (17) and double digest 5 �L (about
1 �g) of each of them with 1-5 U of EcoRI and HindIII restriction enzymes (see
Note 5).
4. Separate the products on 0.8% agarose gel to verify the presence of the inserts
corresponding to the genomic fragments and determine their length.
5. Sequence the plasmid at the 5’ and 3’ ends of the inserts using the universal primers
located on the pUC18 vector.
6. Compare the sequences with a DNA sequence databank to identify the genomic
fragment responsible of the resistant phenotype. In this respect, the on-line server
Colibri (http://genolist.pasteur.fr/Colibri/), containing a database dedicated to the
analysis of the E. coli genome, may be of great help.
3.5. Characterization of Mutants
The degree of resistance of the mutant clones to the relevant PRP may be
assayed by a number of methods. The broth microdiluition susceptibility test, a
reference method for determination of the Minimum Inhibitory Concentration
(MIC), may be performed according to the guidelines of the National Committee
for Clinical Laboratory Standard (NCCLS) as described (20). An example of the
increase in the MIC values for Bac7(1-35) in mutagenized HB101 is shown in
Table 1. Another suitable and simple method, here described, is the count of
bacterial viable cells after incubation with the peptide.
1. Add 50 �L of a log-phase bacterial suspension, diluted to approximately 1.5 × 10 6
cells/mL, in a microfuge tube and then add 100-x �L of liquid medium (e.g., MH
broth), where x corresponds to the volume of peptide to be assayed (x = 0inthe
control tubes). Use the PRP at a bactericidal concentration for the parental wildtype
strain (HB101). Different PRP peptide concentrations may be used (e.g. 5X
MIC).
2. Incubate the samples at 37 ◦ C for different times (1-4 h).
3. Serially dilute aliquots of the samples in Buffer 3.
4. Plate in duplicate on agar medium 50 �L of the 10 −3 and 10 −4 dilutions of the
control or the 10 −1 to 10 −4 dilutions of the peptide-treated samples.
5. Incubate overnight at 37 ◦ C to allow colony counts.
3.6. Synthesis of Cys-Tagged and Fluorescently Labeled Peptides
For all the cell internalization assays, PRPs have to be labeled with a
fluorescent dye as a tracer. One such dye, BODIPY, functionalized with the