Peptide-Based Drug Design

Proline-Rich Antimicrobial Peptides 173
which is within the most conserved region of the protein in different Gramnegative
species. This corresponds to the short hydrophilic EAA sequence
motif that is highly conserved in ABC import systems, and is localized in a
cytoplasmic loop that constitutes an important interaction site with the cognate
ABC-ATPase subunit (24). Taking everything into account, the hypothesis that
SbmA may be an uptake system carrying compounds into the bacterial cells
is quite solid, and PRPs appear to use it to be internalized into bacterial cells
(21,25).
In conclusion, our genetic approach has allowed us to demonstrate that the
entrance of PRPs into Gram-negative bacterial cells is not simply mediated by
the intrinsic capacity of these peptides to cross the membrane lipid bilayers,
but that a specific protein–mediated transport mechanism is necessary. Our data
clearly indicate that SbmA is involved in the transport of peptides and that it
likely represents an important part of this process, as its inactivation leads to
an increased resistance to PRPs. This type of bacterial transporters may thus
represent a Trojan horse for translocation of specific peptides into bacterial cells.
This also suggests possible novel strategies for using PRPs or their analogs as
shuttle systems for the internalization of non-permeant drugs with anti-infective
potential into bacteria.
4. Notes
1. By this approach, positive clones are expected to arise from overexpression of
wild-type genes conferring resistance or from trans-dominant mutations giving a
mutant (resistant) phenotype (14). To identify loss-of-function recessive mutations
the method should be reversed: P-RES resistant clones should be transformed with
a genomic library constructed with the DNA of the wild-type HB101, and clones
with restored peptide susceptibility analyzed.
2. Isogenicity between the wild type strain and the mutated colonies may be checked
by ERIC-PCR based on the enterobacterial repetitive intergenic consensus
(ERIC) sequences (26). Genomic DNA is extracted and amplified by PCR with
the specific primer 5 ′ -AAGTAAGTGACTGGGGTGAGCG-3 ′ for the conserved
ERIC sequence. Amplified PCR products are separated by electrophoresis on 2%
agarose gel and the band patterns compared with those of the parental HB101
strain. A nonisogenic E. coli strain may be used as a control.
3. To obtain highly purified DNA samples, an additional extraction with
phenol/chloroform followed by one with chloroform may be performed. The
DNA is then precipitated by adding NaCl to a final concentration of 0.3 M and
2.5 volumes of 100% ethanol. After centrifugation, the pellet is washed twice
with 70% ethanol, dried on air and resuspended in sterile water. This additional
procedure greatly increases the efficiency of transformation.
4. The plasmid library can be subjected to a round of amplification using the E. coli
XL-1 Blue strain before being introduced into the final strain. In this case, the