Association of HAMLET with mitochondria and pneumococci.

<p>Confocal micrographs of mitochondria (left) and <i>S. pneumoniae</i> D39 (right), incubated with a cytotoxic concentration of Alexa Fluor 488-conjugated HAMLET (100 µg/ml, green) for 1 hour at 37°C, and counterstained with DAPI (300 nM, pseudo-stained red). A light microscopy image (DIC) of each section is included (bottom panels). HAMLET associated with both bacteria and isolated mitochondria.</p

Membrane depolarization and death induced by HAMLET in mitochondria and pneumococci.

<p><i>A</i>) Visualization of the membrane potential after HAMLET-treatment of bacteria and tumor cells for 30 minutes. Confocal micrographs depict untreated (left) and HAMLET-treated (right) <i>S. pneumoniae</i> AL2 bacteria (D39 <b>Δ</b><i>lytA</i>) and A549 carcinoma cells. Bacterial membrane potential was visualized using the anionic bis-oxonol dye DiBAC₄(3) that accumulates in depolarized bacteria and the tumor cell mitochondrial potential was visualized with the cationic dye TMRE that dissipates from depolarized mitochondria. Treatment of the cells with HAMLET resulted in dissipation of both the bacterial and mitochondrial membrane potential in tumor cells seen by an increased staining with DiBAC₄(3) and a decreased staining with TMRE, respectively. <i>B</i>) Membrane potential and <i>C</i>) membrane rupture measurements in <i>S. pneumoniae</i> AL2 (D39 <b>Δ</b><i>lytA</i>), or in isolated rat liver mitochondria. Bacterial membrane potential was monitored by the DiBAC₄(3) and membrane rupture by an influx of propidium iodide, after treatment with 31 (HL31), 62 (HL62), or 125 (HL125) µg/ml HAMLET or 125 µg/ml ALA in the absence or presence of 30 µM Ruthenium Red (RuR). Each experiment was repeated six times and the data represents the mean ratio of the six experiments. Membrane potential in isolated mitochondria was measured by the distribution of TPP⁺ ions in the suspension in the presence of 40 nmoles of Ca²⁺ per mg protein after treatment with 50 µg/ml HAMLET in the presence or absence of 10 µM RuR. Arrow indicates addition of mitochondria. The experiment was repeated three times. The graph represents one of the three traces obtained. <i>D)</i> Effect of calcium transport inhibition on HAMLET-induced pneumococcal death. <i>S. pneumoniae</i> D39 was incubated with increasing concentrations of HAMLET in the presence or absence of 30 µM Ruthenium Red (hatched lines) and viability was monitored after 1 h of incubation by viable plate counts after overnight culture. Viability of bacteria is presented as colony forming units (CFUs) per ml suspension (detection limit in the assay was 10² CFU/ml). Ruthenium Red significantly reduced HAMLETs bactericidal activity. The data represent the mean of four individual experiments with standard deviation error bars.</p

Role of serine proteases in HAMLET-induced death of tumor cells and pneumococci.

<p>A) A549 carcinoma cells were preincubated for 10 minutes with diluent, 25 µM zVAD-fmk (pan-caspase inhibitor), 100 µM dichloroisocoumarin (serine protease inhibitor), or 100 µM calpeptin (calcium-dependent cysteine protease inhibitor) before being treated with 300 µg/ml of HAMLET. After 6 hours of incubation cell viability was measured using trypan blue exclusion. The graph depicts the mean death in % obtained after 3 individual experiments. The error bars represent the standard deviation. * and *** represent <i>P</i><0.05 and <i>P</i><0.001, respectively. B and C) <i>S. pneumoniae</i> AL2 (D39 <b>Δ</b><i>lytA</i>) were preincubated for 10 minutes with diluent, 25 µM Aprotinin (serine protease inhibitor), 25 µM zVAD-fmk (pan-caspase inhibitor), or 10 µM E-64 (cysteine protease inhibitor) before being treated with 50 µg/ml of HAMLET. After 2 hours viability was determined and samples were analyzed for high molecular weight DNA fragmentation. <i>B)</i> Viability. The graph depicts the mean log₁₀ death obtained from five individual experiments. The error bars represent the standard deviation. ** represents <i>P</i><0.01. C) DNA fragmentation. Untr indicates untreated bacteria. The remaining samples were treated with HAMLET in the presence of diluent (none) or proteasee inhibitors. Only the serine protease inhibitors aprotinin rescued pneumococci from death and DNA fragmentation.</p

Strains of <i>S. pneumoniae</i> tested for HAMLET-sensitivity.

<p>Strains of <i>S. pneumoniae</i> tested for HAMLET-sensitivity.</p

Patients and samples.

<p>Samples from patients participating in a clinical trial of induced <i>E. coli</i> 83972 ABU were analyzed. All collected urine samples were subjected to PMN, IL-6 and IL-8 quantification, and blood samples from eleven patients were collected for genotyping of promoter polymorphisms in TLR4 and IRF3. Blood and urine samples from these eleven patients were also selected for an extended urine protein analysis.</p

Apoptosis-like changes in HAMLET-treated <i>H. influenzae</i>.

<p>A) Association of HAMLET with bacteria. Confocal micrographs of <i>H. influenzae</i> 2019, incubated with a cytotoxic concentration of Alexa Fluor 488-conjugated HAMLET (250 µg/ml, green) for 1 hour at 37°C, and counterstained with DAPI (300 nM, pseudo-stained red). A light microscopy image (DIC) of each section is included in the bottom row. <i>B</i>) Membrane potential and <i>C</i>) membrane rupture measurements in <i>H. influenzae</i> 2019. Bacterial membrane potential was monitored by the DiBAC₄(3) and membrane rupture by an influx of propidium iodide, after treatment with 62 (HL62), 125 (HL125), or 250 (HL250) µg/ml HAMLET or 250 µg/ml ALA in the absence or presence of 30 µM Ruthenium Red (RuR). Each experiment was repeated six times and the data represents the mean ratio of the six experiments. <i>D)</i> Effect of calcium transport inhibition on HAMLET-induced pneumococcal death. <i>H. influenzae</i> 2019 was incubated with increasing concentrations of HAMLET in the presence or absence of 30 µM Ruthenium Red (hatched lines) and viability was monitored after 1 h of incubation by viable plate counts after overnight culture. Viability of bacteria is presented as colony forming units (CFUs) per ml suspension (detection limit in the assay was 10² CFU/ml, (mean of four experiments with standard deviation error bars). E) Chromatin fragmentation induced by HAMLET in <i>H. influenzae</i>. High molecular weight DNA fragments were induced by HAMLET in <i>H. influenzae</i> 2019 cells and detected after 1 h of incubation. (HAMLET concentration in µg/ml). Increasing concentrations of HAMLET resulted in accumulation of DNA fragments over time. Low molecular weight oligonucleosomal DNA fragments were not observed (lower panel).</p

Host response to <i>E. coli</i> 83972 bacteriuria.

<p><i>E. coli</i> 83972 ABU triggered an increase in PMN numbers and IL-8 concentrations (p<0.0001) but IL-6 levels were unchanged (n.s., Mann-Whitney test). Group-wise comparison of monthly urine samples collected during <i>E. coli</i> 83927 ABU or after PBS inoculations. Coded patient IDs are noted on the x-axis. A. Means + SEs of neutrophil numbers, IL-8 and IL-6 concentrations during <i>E. coli</i> 83972 bacteriuria (pink) or sterile conditions (blue). B. Intra-individual comparison of samples obtained during <i>E. coli</i> 83972 bacteriuria (pink) and sterile intervals (blue). C. Box-plot of intra-individual host response variation during <i>E. coli</i> 83972 ABU.</p

Bacterial and tumor cell death induced by HAMLET.

<p>A) Jurkat cells and <i>S. pneumoniae</i> D39 were incubated with increasing concentrations of HAMLET or human alpha-lactalbumin (ALA) and viability was monitored after 6 h or 1 h of incubation for Jurkat and bacterial cells, respectively. Viability of Jurkat cells are presented on the right Y axis in per cent viable cells in the suspension as determined by trypan blue staining and viability of bacteria are presented on the left Y axis as colony forming units (CFUs) per ml suspension (detection limit in the assay was 10¹ CFU/ml). ALA (hatched lines) did not kill any of the organisms whereas HAMLET (solid lines) killed both Jurkat and bacterial cells in a dose-dependent manner. The data represent the mean of three individual experiments with standard deviation error bars. B) Role of bacterial virulence factors in HAMLET-induced killing of <i>S. pneumoniae.</i> Pneumococcal strains lacking capsule, Pneumococcal surface proteins A and C (PspA and PspC), autolysin (LytA), pneumococcal surface adhesin A (PsaA), or dihydrolipoamide dehydrogenase (DLDH), all associated with virulence, were treated with 50, 120 and 200 µg/ml of HAMLET for 1 hour at 37°C and viable organisms were determined by plating organisms on solid agar and counting colony forming units after overnight growth. The graph depicts the mean of three experiments. There was no significant difference in sensitivity related to lack of these virulence factors.</p

Role of autolysin in HAMLET-induced lysis and killing of <i>S. pneumoniae.</i>

<p>Pneumococcal strains D39 and AL2 (D39 <b>Δ</b><i>lytA</i>) were treated with 150 µg/ml of HAMLET (HL) or 0.1% of the bile salt deoxycholate (DOC) over 90 minutes. As pneumococci are bile salt sensitive streptococci and lysis from bile salts is LytA dependent, deoxycholate was added as a LytA-dependent control. <i>A</i>) The optical density of the suspension was monitored at 600 nm every 15 minutes to assess the lysis of bacteria. <i>B</i>) After 10 minutes and 60 minutes bacteria were serially diluted and plated on blood agar overnight to determine viable colony forming units (detection limit in the assay was 10² CFU/ml). The data represent the mean of three individual experiments with standard deviation error bars. HAMLET and DOC killed both strains equally well, but lysis was only observed in the autolysin positive D39 strain.</p

Nuclease activity in HAMLET induced DNA fragmentation.

<p><i>A</i>) HAMLET was mixed with either pneumococcal chromosomal DNA or chromosomal DNA from Jurkat cells in the presence of both 1 mM each of Ca²⁺ and Mg²⁺ and the mixture was incubated for 1 hour and digestion was examined by gel electrophoresis. Bovine pancreas DNAse I was used as a positive control for DNA cleavage. HAMLET was unable to cleave either DNA preparation. (<i>B</i>) <i>S. pneumoniae</i> R6 (WT) and the isogenic strains 577 (<b>Δ</b><i>endA</i>), 641 (<b>Δ</b><i>exoA</i>) and 642 (<b>Δ</b><i>endA, </i><b>Δ</b><i>exoA</i>) were incubated with 100 or 200 µg/ml of HAMLET for 2 h and high molecular weight DNA fragmentation was examined. All strains displayed an accumulation of HMW DNA fragments.</p