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30 août 19221922/08/30 (A51).Appartient à l’ensemble documentaire : PoitouCh

Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.

<p><i>E. coli</i> (dark grey), <i>K. pneumoniae</i> (white) and <i>S</i>. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with <i>E. coli</i>- specific Ec1000 at 3.2 µM, <i>K. pneumoniae</i>-specific Kp0001 at 3.2 µM and <i>S</i>. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of <i>E. coli</i>, <i>K. pneumoniae</i> and <i>S</i>. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of <i>S</i>. Typhimurium by Ec1000. Error bars are standard error for biological replicates (<i>n</i> = 3).</p

Bacterial strains used in this study.

a<p>American Type Culture Collection.</p>b<p><i>Salmonella</i> Genetic Stock Center.</p

<i>S</i>. Typhimurium-selective growth inhibition.

<p>Peptide-PNA Se0002 was designed to target the −5 to +5 region of the translational initiation region (TIR) of <i>ftsZ</i> in <i>S</i>. Typhimurium. Se0002 has 2 base pair mismatches in the TIR of <i>ftsZ</i> in <i>E. coli</i>. (A) Growth curve analysis of Se0002 in pure culture. <i>E. coli</i> growth in the presence of 1.25 µM Se0002 (solid line) was identical to that of untreated controls (not shown). <i>S</i>. Typhimurium growth was inhibited in the presence of 1.25 µM of Se0002 (dotted line) relative to the untreated control (dashed line). Growth in the treated samples after 10 hrs was not due to resistance (see text for details). (B) Mixed cultures of GFP-labeled <i>S</i>. Typhimurium AC02 and DsRed-labeled <i>E. coli</i> AC01 were treated with 1.25 µM Se0002; and imaged by fluorescence microscopy after 6 hrs of incubation. The filamentous growth phenotype was only observed in <i>S</i>. Typhimurium AC02 and is consistent with silencing of <i>ftsZ</i> expression.</p

Species-selective antibacterial peptide-PNAs in three-species mixed culture.

<p><i>B. subtilis</i> (dark grey), <i>K. pneumoniae</i> (white) and <i>S</i>. Typhimurium (light grey) in mixed culture were separately treated with Ec108 at 3.5 µM, Kp0001 or Se0001 at 4.5 µM or by combined treatment of Kp0001 and Se0001 both at 4.5 µM. All cultures were incubated for 16 hrs. Selective inhibition of either <i>K. pneumoniae</i> or <i>S</i>. Typhimurium individually or together, achieved with the peptide-PNAs, could not theoretically be achieved with any combination of the twenty known antimicrobial compounds tested in this study. Error bars as above.</p

Actinorhodin production in <i>S. coelicolor</i> MT1110 and effects of RNA silencing.

<p>(A) MT1110/pAS01 after 6 days of growth in R5 liquid medium (top panels, the region shown is represented by the flask on the right) and R5 agar (bottom panels) containing thiostrepton (+ induction) or DMSO (- induction). MT1110/pIJ8600 served as a control. (B) As above with MT1110/pAS02, ε-caprolactam as the inducer of expression and MT1110/pSH19 as the control.</p

Predicted secondary structures of antisense RNAs.

<p>Secondary structure of (A) the paired-termini antisense <i>act</i>I-ORF1 asRNA (−92 to +63 bp) designed in this study for silencing of actinorhodin production, and (B) <i>mic</i>X, a naturally occurring <i>trans</i>-encoded antisense RNA from <i>Streptomyces lividans</i> involved in the activation of actinorhodin biosynthesis. The <i>mic</i>X asRNA has a predicted paired-end structure which suggests that the synthetic strategy followed in this study (addition of paired termini to antisense RNA) may be analogous to natural antisense mechanisms in <i>Streptomyces</i>. <i>mic</i>X also contains non-complementary nucleotide sequences in the paired region which may serve to protect antisense RNAs from RNase III cleavage.</p

RNA silencing using peptide-PNA.

<p>(A) Schematic diagram representing the binding site of antisense peptide-PNAs designed to prevent transcription of <i>act</i>I-ORF1. The start codon of <i>act</i>I-ORF1 is shown in bold, the putative Shine-Dalgarno site in red. Sc001 and Sc002 differ in the degree of coverage of the start codon. (B) Demonstration of the ability of peptide-PNAs to silence production of actinorhodin in <i>S. coelicolor</i>. Peptide-PNA solutions were applied to a lawn of <i>S. coelicolor</i> (after 24 hrs) on MPCA agar and incubation continued for a further 72 hrs. Repression of actinorhodin production is clearly visible with 50 µM treatment using either Sc001 or Sc002; no reduction in actinorhodin production was evident when a scramble-PNA with limited complementarity to the <i>S. coelicolor</i> genome was used at the same concentration. (C) Peptide-PNA solutions were applied directly to a lawn of <i>S. coelicolor</i> MT1110 on ISP-4 agar and were photographed from on top (left) and from bottom (right) after 96 hrs incubation at 28°C.</p

Bacterial Strains and plasmids used in this study.

<p>Bacterial Strains and plasmids used in this study.</p

Effect of expressed antisense RNA on <i>act</i>I-ORF1 mRNA levels.

<p>RT-PCR of <i>act</i>I-ORF1 from RNA extracted from MT1110 strains grown for 3 days in R5 liquid medium that contained thiostrepton (+) or DMSO (-) as a control. Two primers sets (top and middle panel) designed to amplify different regions of the <i>actI</i>-ORF1 mRNA were used, with SCO4742 (bottom panel) serving as an internal control. M = 100 bp DNA ladder. (B) qRT-PCR of <i>act</i>I-ORF1; SCO4742 served as the internal reference and expression was calculated relative to uninduced MT1110. Error bars represent standard error for biological replicates (<i>n</i> = 3 ** highly significant difference, one way ANOVA, <i>F</i> = 11.96, p = <0.01).</p