Towards profiles of resistance development and toxicity for the small cationic hexapeptide RWRWRW-NH 2

RWRWRW-NH 2 (MP196) is an amphipathic hexapeptide that targets the bacterial cytoplasmic membrane and inhibits cellular respiration and cell wall synthesis. In previous studies it showed promising activity against Gram-positive bacteria and no significant cytotoxicity or hemolysis. MP196 is therefore used as lead structure for developing more potent antibiotic derivatives. Here we present a more comprehensive study on the parent peptide MP196 with regard to clinically relevant parameters. We found that MP196 acts rapidly bactericidal killing 97% of initial CFU within 10 min at two times MIC. We were unable to detect resistance in standard 24 and 48 h resistance frequency assays. However, MP196 was effective against some but not all MRSA and VISA strains. Serum binding of MP196 was intermediate and we confirmed its low toxicity against mammalian cell lines. MP196 did neither induce NFκB activation nor cause an increase in IL8 levels at 250 μg/mL, and no IgE-dependent activation of basophil granulocytes was detected at 500 μg/mL. Yet, MP196 demonstrated acute toxicity in mice upon injection into the blood stream. Phase contrast microscopy of mouse blood treated with MP196 revealed a shrinking of erythrocytes at 250 μg/mL and severe morphological changes and lysis of erythrocytes at 500 μg/mL. These data suggest that MP196 derivatization directed at further lowering hemolysis could be instrumental in overcoming acute toxicity. The assessment of hemolysis is a critical step in the evaluation of the clinical potential of promising antimicrobial peptides and should be accompanied by microscopy-based morphological analysis of blood cells

Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA

<div><p>Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion <em>in vivo</em> using degron tagging in budding yeast. The previously described DHFR–based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 “core” subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.</p> </div

Depletion of Scc3 and Pds5 with a “conventional” temperature-sensitive degron.

<p>(A–C) Strains 2395 (<i>SCC1-HA6</i>), 2452 (<i>SCC1-HA6</i>, degron-<i>MYC18-PDS5</i>), 2455 (<i>SCC1-HA6</i>, degron-<i>MYC18</i>-<i>SCC3</i>) and 2456 (<i>SCC1-HA6</i>, degron-<i>MYC18- PDS5, degron-MYC18-SCC3</i>) were arrested with nocodazole in YEP raffinose at 30°C for 2 hours, resuspended in YEP galactose containing nocodazole and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were shifted to 37°C in YEP galactose containing nocodazole and doxycycline to deplete Pds5 and/or Scc3. (A) Chromosomal spreads were prepared at the indicated time points and stained with DAPI for DNA, anti-HA (mouse, 16B12) and anti-MYC (rabbit, 71D10) antibodies. The secondary antibodies were Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit. Protein fluorescence was quantified using Metamorph software. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. (B) Western blot of TCA protein extracts probed with anti-HA (16B12), anti-MYC (71D10) and anti-Cdc28 (sc-28550, Santa Cruz). (C) FACS analysis of cellular DNA content. (D–F) Strains were staged in G1 with <i>α</i>-factor in YEP raffinose at 30°C, resuspended in YEP galactose containing <i>α</i>-factor and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were then shifted to 37°C in YEP galactose containing doxycycline and <i>α</i>-factor, incubated for 90 minutes to deplete Pds5 and/or Scc3 and subsequently released in YEP galactose containing nocodazole and doxycycline at 37°C. Chromosomal spreads (D), Western blot (E), and FACS analysis of cellular DNA content (F) are shown.</p

Interaction of Pds5, Scc3 and Wpl1 with cohesin ring.

<p>Lysates of nocodazole/benomyl arrested yeast cultures were incubated with IgG sepharose to precipitate Scc1-TAP or Smc3-TAP. The presence of different proteins on the IgG beads was analysed by Western blot probed with anti-HA (12CA5), anti-MYC (71D10) and PAP (P1291, Sigma). The strains were in (A): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 1829 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>), 1958 (<i>SCC3-MYC18, PDS5-HA6, SCC1-TAP</i>); in (B): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 1834 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>), 1956 (<i>PDS5-MYC18, SCC3-HA6, SCC1-TAP</i>); in (C): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2014 (<i>WPL1-MYC18, PDS5-HA6, SCC1-TAP</i>), 2016 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>); in (D): 1880 (<i>WPL1-MYC18, SCC3-HA6</i>), 2012 (<i>WPL1-MYC18, SCC3-HA6, SCC1-TAP</i>), 2018 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>); in (E): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 2251 (<i>SCC3-MYC18, PDS5-HA6, SMC3-TAP</i>), 2290 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (F): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 2249 (<i>PDS5-MYC18, SCC3-HA6, SMC3-TAP</i>), 2264 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>); in (G): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2253 (<i>WPL1-MYC18, PDS5-HA6, SMC3-TAP</i>), 2265 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (H): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2261 (<i>WPL1-MYC18, SCC3-HA6, SMC3-TAP</i>), 2271 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>).</p

Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.

<p>Strains 1021 (untagged), 1813 (<i>SCC3-HA6</i>, <i>SCC1-Myc18</i>), 1625 (<i>SCC3-HA6-</i>degron, <i>SCC1-Myc18</i>), 2525 (<i>PDS5-HA6</i>, <i>SCC1-Myc18</i>) and 1818 (<i>PDS5-HA6</i>-degron, <i>SCC1-Myc18</i>) carried the centromeric minichromosomes. (A) Yeast lysates were incubated with BglII restriction enzyme as indicated. Minichromosomes were co-immunoprecipitated with Scc1-Myc18. DNA was prepared by phenol/chloroform extraction and separated on a 1% agarose gel with ethidium bromide. Southern blot probed with a <i>TRP1</i>-specific probe is shown. Nicked (N), linear (L), and closed circular (C) forms of the minichromosome are indicated. (B) Minichromosomes were immunoprecipitated with anti-HA antibody. Minichromosomes from <i>SCC3-HA6</i> but not <i>SCC3-HA6</i>-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the <i>SCC3-HA6</i>-degron strain. Since Pds5 association with minichromosomes is very salt-sensitive, they could not be co-immunoprecipitated with Pds5-HA6 in either the wild type or <i>PDS5-HA6</i>-degron strains under our experimental conditions.</p

Depletion of Scc3 and Pds5 does not affect cohesin association with chromatin.

<p>Yeast strains were staged in G1 with <i>α</i>-factor and released into media with nocodazole. Chromosomal spreads were prepared as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen-1002856-g002" target="_blank">Figure 2</a>. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. FACS analysis of cellular DNA content is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856.s013" target="_blank">Figure S13</a>. The strains were in (A): 1813 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>), 1625 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>-degron), 1815 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>), 1818 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>-degron), in (B): 1771 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>), 1796 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>-degron), 1734 (<i>PDS5-MYC18</i>, <i>SCC3-HA6</i>) and 1744 (<i>PDS5-Myc18</i>, <i>SCC3-HA6</i>-degron) in (C): 1479 (<i>SCC3-HA6</i>), 1864 (<i>SCC3-HA6, Δwpl1</i>), 1677 (<i>PDS5-HA6</i>), 1866 (<i>PDS5-HA6, Δwpl1</i>), 10589 (<i>SCC1-Myc18</i>) and 1906 (<i>SCC1-Myc18, Δwpl1</i>).</p

A model of how Scc3 and Pds5 play a role in the establishment of sister chromatid cohesion but are not required to stabilize cohesin rings on the DNA.

<p>In the normal cell cycle of budding yeast Scc1 subunit is synthesized in the late G1 or early S phase. Scc1 binds to Smc1/Smc3 heterodimer and completes the cohesin ring. Scc3 and Pds5 stably associate with cohesin via Scc1 subunit. Cohesin complexes are loaded on the chromosomes. During DNA replication two newly generated sister chromatids are captured inside a single cohesin ring in a process which remains poorly understood. Scc3 and Pds5 function to ensure that two sister chromatids are captured inside a cohesin ring. In their absence, cohesin complexes are stably loaded on the DNA but fail to embrace both of the sister chromatids resulting in defective sister chromatid cohesion (A). We can speculate that Pds5 and Scc3 could stabilize cohesin rings specifically during the replication fork passage (B) or transiently bind sister chromatids during the establishment of cohesion (C). Alternatively they could mediate a transient interaction between two cohesin rings as proposed by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856-Zhang2" target="_blank">[61]</a> (D).</p

Scc3, Pds5, and Wpl1 form a complex and associate with Scc1 “core” subunit of cohesin.

<p>(A) Purified recombinant Scc3, Pds5 and Wpl1 proteins were mixed as indicated and separated by ultracentrifugation on a 10–30% glycerol gradient. A total of 44 gradient fractions were collected and analyzed on a Coomassie-stained 6% SDS-PAGE. Only fractions containing the proteins are shown. Positions of molecular size markers are indicated. (B) Recombinant His6-tagged Scc3, Pds5, and Wpl1 were mixed together and incubated with glutathione-agarose beads charged with GST or GST-fused to the N-terminal (aa 1–168), middle (aa 169–337) or C-terminal (aa 338–566) regions of Scc1. Beads eluates were analyzed by Western blotting with Penta-His antibody (QIAGEN) (upper panel). Coomassie-stained GST-beads are shown in the lower panel.</p

Eco1 contains a degron.

<p>(A) 1222 strain with <i>CDC20</i> expressed from a methionine-repressible promoter (<i>ECO1-TAP</i>) was staged in G1 with <i>α</i>-factor and then released into three different media. Release into the medium without methionine allows cell cycling (top) while release into full medium with methionine or nocodazole (middle and bottom) results in progression through replication and eventual G2 arrest. Expression of S phase cyclin Clb5 is induced during DNA replication while cyclin Clb2 accumulates in G2 and is destroyed in mitosis. TCA protein extracts were prepared and analyzed by Western blot. Eco1 was detected with peroxidase-anti-peroxidase, Clb5 with sc-6704, Clb2 with sc-9071, loading control with anti-Cdc28 (sc-28550, Santa Cruz). (B) Depletion of Scc3 and Pds5 with an Eco1-derived degron. G1-staged strains 12544 (<i>SCC3-HA6</i>), 1323 (<i>SCC3-HA6</i>-degron), 1677 (<i>PDS5-HA6</i>) and 1675 (<i>SCC3-HA6</i>-degron) were released into full media with nocodazole. Western blot was probed with anti-HA antibody. (C) Scc1 protein level is unchanged in the strains with Eco1-derived degron. Strains 1815 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>), 1818 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>-degron), 1813 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>), 1625 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>-degron), 10589 (<i>SCC1-MYC18</i>) and 1906 (<i>SCC1-MYC18, Δwpl1</i>) were staged in G1 with <i>α</i>-factor and released into media with nocodazole. Western blot was probed with anti-HA, anti-Myc and anti-Cdc28 antibodies. The same yeast cultures were used for chromosomal spreads (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen-1002856-g005" target="_blank">Figure 5</a>) and for FACS analysis of cellular DNA content (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856.s013" target="_blank">Figure S13A</a>). (D and E) Determination of Scc3 and Pds5 copy number per yeast cell. (D) Coomassie-stained gel with serial dilutions of purified recombinant Scc3-HA6, Pds5-HA6 and BSA (NEB #B9001) as a standard. (E) Protein extracts were prepared from nocodazole-arrested strains 1323 (<i>SCC3-HA6</i>-degron), 1479 (<i>SCC3-HA6</i>), 1675 (<i>PDS5-HA6</i>-degron) and 1677 (<i>PDS5-HA6</i>). Extracts from the indicated number of cells were analyzed by Western blot with anti-HA antibody. Known quantities of recombinant Scc3-HA6 and Pds5-HA6 were used as standards. Bands were quantified with MetaMorph.</p