Bacterial membrane activity of α-peptide/β-peptoid chimeras: Influence of amino acid composition and chain length on the activity against different bacterial strains

<p>Abstract</p> <p>Background</p> <p>Characterization and use of antimicrobial peptides (AMPs) requires that their mode of action is determined. The interaction of membrane-active peptides with their target is often established using model membranes, however, the actual permeabilization of live bacterial cells and subsequent killing is usually not tested. In this report, six α-peptide/β-peptoid chimeras were examined for the effect of amino acid/peptoid substitutions and chain length on the membrane perturbation and subsequent killing of food-borne and clinical bacterial isolates.</p> <p>Results</p> <p>All six AMP analogues inhibited growth of twelve food-borne and clinical bacterial strains including Extended Spectrum Beta-Lactamase-producing <it>Escherichia coli</it>. In general, the Minimum Inhibitory Concentrations (MIC) against Gram-positive and -negative bacteria were similar, ranging from 1 to 5 μM. The type of cationic amino acid only had a minor effect on MIC values, whereas chain length had a profound influence on activity. All chimeras were less active against <it>Serratia marcescens </it>(MICs above 46 μM). The chimeras were bactericidal and induced leakage of ATP from <it>Staphylococcus aureus </it>and <it>S. marcescens </it>with similar time of onset and reduction in the number of viable cells. EDTA pre-treatment of <it>S. marcescens </it>and <it>E. coli </it>followed by treatment with chimeras resulted in pronounced killing indicating that disintegration of the Gram-negative outer membrane eliminated innate differences in susceptibility. Chimera chain length did not influence the degree of ATP leakage, but the amount of intracellular ATP remaining in the cell after treatment was influenced by chimera length with the longest analogue causing complete depletion of intracellular ATP. Hence some chimeras caused a complete disruption of the membrane, and this was parallel by the largest reduction in number of viable bacteria.</p> <p>Conclusion</p> <p>We found that chain length but not type of cationic amino acid influenced the antibacterial activity of a series of synthetic α-peptide/β-peptoid chimeras. The synthetic chimeras exert their killing effect by permeabilization of the bacterial cell envelope, and the outer membrane may act as a barrier in Gram-negative bacteria. The tolerance of <it>S. marcescens </it>to chimeras may be due to differences in the composition of the lipopolysaccharide layer also responsible for its resistance to polymyxin B.</p

Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32 × MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32 × MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16-32 ×) to the selection agent. Five transfers (≈ 35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32 × MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ≈ 30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics

PALB2, CHEK2 and ATM rare variants and cancer risk : data from COGS

AbstractBackground: The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study.Methods: We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant.Results: For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p = 7.1 × 10⁻⁵), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p = 6.9 × 10⁻⁸) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p = 0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p ≤ 0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p = 0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p = 0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants.Conclusions: This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important.Abstract Background: The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. Methods: We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. Results: For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p = 7.1 × 10⁻⁵), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p = 6.9 × 10⁻⁸) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p = 0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p ≤ 0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p = 0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p = 0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants. Conclusions: This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important

Continuous selection of <i>Escherichia coli</i>.

<p>Continuous selection exerted by peptidomimetic 1 (A), or by polymyxin B (B) leads to tolerance in <i>E. coli</i>. Solid lines represent individual lineages. All lineages exposed to the peptidomimetic were cultured up to 32×wt MIC (A); for lineages selected against polymyxin B this was eight out of ten (the last two were not able to grow after two doublings in concentration i.e. at 0.25 µg/mL). Punctuated lines indicate wt MIC of the selective agents (8 µg/mL and 1 µg/mL, respectively).</p

MIC (µg/mL) for wild-type <i>E. coli</i> and four peptidomimetic-adapted isolates against peptidomimetic 1 in standard MHB media and in MHB media supplemented with 25% human blood plasma.

a<p>Ability of plasma to interact synergistically with peptidomimetic 1.</p

Grouping of genome-sequenced peptidomimetic-adapted isolates based on comparison to wild type (wt) MIC value (8 µg/mL).

<p>Grouping of genome-sequenced peptidomimetic-adapted isolates based on comparison to wild type (wt) MIC value (8 µg/mL).</p