Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents

Characterization of the Uptake and Trafficking of AvB3-targeted and Non-targeted Nanoemulsions in Human Endothelial Cells in vitro

RGD-functionalized and non-functionalized oil-in-water nanoemulsions of approximately 100 nm containing DSPC, PEGylated DSPE, cholesterol and Gd-DTPA-DSA at a molar ratio of 1.1/0.15/1/0.75 were prepared. In vitro uptake and trafficking in HUVECs of the nanoemulsions was characterized using confocal laser scanning microscopy and flow cytometry. The RGD peptide recognizes αvβ3 and αvβ5 integrin receptors, which play central roles in angiogenesis. Moreover, the αvβ3 integrin receptor is overexpressed in the endothelium of angiogenic tumor vasculature. It was found that the RGD-emulsion showed a remarkably high uptake in HUVECs expressing αvβ3 integrins compared to its non-conjugated control version. Furthermore, the RGD-emulsion was able to evade the lysosomes at least within the first 3 hours of incubation, while the control-emulsion was not. The uptake of both emulsions was mainly facilitated by caveolae-mediated endocytosis, but also to a lesser extent by clathrin-mediated endocytosis and other un- known mechanisms. It was shown that RGD and control-emulsions were internalized or sorted into distinct vesicles. Both emulsions bypassed the early endosomes, and it was hypothesized that they were mainly trafficked to caveosomes before subsequent traffick- ing of control-emulsion to late endosomes/lysosomes and of RGD-emulsion to cis-Golgi or endoplasmatic reticulum. The results suggest that the RGD-emulsion has promising feasibility as a site-specific targetable delivery system

Characterization of the Uptake and Trafficking of AvB3-targeted and Non-targeted Nanoemulsions in Human Endothelial Cells in vitro

RGD-functionalized and non-functionalized oil-in-water nanoemulsions of approximately 100 nm containing DSPC, PEGylated DSPE, cholesterol and Gd-DTPA-DSA at a molar ratio of 1.1/0.15/1/0.75 were prepared. In vitro uptake and trafficking in HUVECs of the nanoemulsions was characterized using confocal laser scanning microscopy and flow cytometry. The RGD peptide recognizes αvβ3 and αvβ5 integrin receptors, which play central roles in angiogenesis. Moreover, the αvβ3 integrin receptor is overexpressed in the endothelium of angiogenic tumor vasculature. It was found that the RGD-emulsion showed a remarkably high uptake in HUVECs expressing αvβ3 integrins compared to its non-conjugated control version. Furthermore, the RGD-emulsion was able to evade the lysosomes at least within the first 3 hours of incubation, while the control-emulsion was not. The uptake of both emulsions was mainly facilitated by caveolae-mediated endocytosis, but also to a lesser extent by clathrin-mediated endocytosis and other un- known mechanisms. It was shown that RGD and control-emulsions were internalized or sorted into distinct vesicles. Both emulsions bypassed the early endosomes, and it was hypothesized that they were mainly trafficked to caveosomes before subsequent traffick- ing of control-emulsion to late endosomes/lysosomes and of RGD-emulsion to cis-Golgi or endoplasmatic reticulum. The results suggest that the RGD-emulsion has promising feasibility as a site-specific targetable delivery system

Topoisomerase IV tracks behind the replication fork and the SeqA complex during DNA replication in Escherichia coli

Abstract Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication. There exists a short time lag of about 10–20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate. We show here that TopoIV localizes to the origin proximal side of the fork trailing protein SeqA and follows the movement pattern of the replication machinery in the cell

Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity

In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies

<i>Escherichia coli</i> SeqA Structures Relocalize Abruptly upon Termination of Origin Sequestration during Multifork DNA Replication

<div><p>The <i>Escherichia coli</i> SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, <i>E. coli</i> cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.</p></div

Model of SeqA relocalization events.

<p>A schematic drawing to illustrate how new and unreplicated DNA may change places during the SeqA relocalization events reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1</a>. (<b>A</b>) Images in red square (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1C</a>) showing SeqA at the end of the period at midcell (t min), the first relocalization to one cell quarter (at t+1 min), and the second relocalization to the other cell quarter (at t+2 min). (<b>B</b>) Illustration of the position of SeqA and newly replicated DNA of two chromosomes during the period at midcell (cell number one), after the movement of first new and unreplicated DNA of the lower chromosome (cell number two), and then the upper chromosome (cell number three). It is here suggested that origins in sequestration are part of the midcell SeqA structures. The SeqA foci are illustrated as green dots, the origin foci as red squares, unreplicated DNA as a black line and newly replicated DNA as a grey line. The position of the unreplicated DNA is simplified and the Ter region localization at midcell is not included.</p

Live-cell fluorescence imaging of SeqA-YFP during rapid growth.

<p>Cells with the <i>YFP</i> gene inserted at the C-terminal end of the chromosomal <i>seqA</i> gene (SF128) were grown at 28°C on an agarose pad containing 1% glucose-CAA. The agarose pad was attached to a microscopy slide, and images were recorded every 1 minute over a 40 minutes period. (<b>A</b>) Cell cycle diagram with parameters obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s006" target="_blank">Table S1</a>). The replication period (C-period) spanned about one doubling time (τ = 66 min). The origin sequestration period is shown in purple as part of the black line that indicates the C-period. Initiation of replication occurred at age a_i = 2 minutes (average for the population). The D-period is shown as a stippled line. Numbers presented in the diagram are average of four independent experiments. Schematic drawings of cells are shown above the diagram to illustrate DNA content, numbers of origins, numbers of replication forks and replication fork progression at different stages of the cell cycle (the drawings are not indicative of chromosome positioning or organization patterns). Chromosomes are shown as black lines and origins as black dots. (<b>B</b>) Histogram showing numbers of SeqA foci per cell in categories I–IV representing the progression along the cell cycle (see text for description of categories). Representative illustrations of the cells with SeqA foci (as green dots) within each category are shown above the histogram. (<b>C</b>) Time-lapse series of a representative cell from category I with one SeqA focus at midcell. Scale bar is 1 µm. Numbers on pictures indicate time after start of imaging. (<b>D</b>) Analysis of SeqA dynamics during live-cell imaging. The positions of the SeqA foci along the cell length were plotted as a function of time for the cell from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1C</a>. The average positions of SeqA foci relative to the cell pole (obtained from six cells from category I) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s003" target="_blank">Figure S3</a>. The red box in (<b>C</b>) and (<b>D</b>) indicates relocalization of SeqA from midcell towards the quarter positions at 14–16 minutes after start of imaging.</p

Simultaneous imaging of SeqA and the Ter region.

<p>(<b>A</b>) Cell cycle diagram of cells (SF163), with tagged SeqA protein (SeqA-YFP) and Ter region (FROS), obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s006" target="_blank">Table S1</a>). Cells were grown in glucose-CAA medium with a doubling time (τ) of 65 minutes. Initiation of replication at four origins occurred at age a_i = 39 minutes (average for the population), and the replication period (C-period) lasted for about 83 minutes. The C-period is indicated as a black line, and the D-period is shown as a stippled line (<b>B</b>) Representative images from snapshot fluorescence imaging shows formation of SeqA(pseudo-colored red) and Ter (pseudo-colored green) foci. N = 232 cells. (<b>C</b>) Schematic drawing of DNA and foci below the images, SeqA as red dot, Ter region as green dot, origin as black dot, DNA replicated by old forks in grey, DNA to be replicated by old forks in black and DNA replicated by new forks in blue. The placement of origins and DNA is in this illustration hypothetical and simplified, based on observed positioning of SeqA and Ter region in fluorescence images in addition to cell cycle parameters from 3A.</p

Categories of cells (SF128) with characteristic features from snapshot imaging (Figure 1B).

<p>Categories of cells (SF128) with characteristic features from snapshot imaging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1B</a>).</p