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