The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae

HAMLET is a complex of human a-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bis-phosphate aldolase (FBPA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

Background: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae

HAMLET is a complex of human a-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bis-phosphate aldolase (FBPA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis

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

Bacteroides fragilis Synthesizes a DNA Invertase Affecting both a Local and a Distant Region

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of Bacteroides fragilis was characterized. Its gene, tsr19, is adjacent to mpi, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of B. fragilis, Tsr19 brings about inversion of two DNA regions, one local and one located distantly

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

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

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

Expression of a Uniquely Regulated Extracellular Polysaccharide Confers a Large-Capsule Phenotype to Bacteroides fragilis▿

Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, more than any described bacterium outside the order Bacteroidales. Here, we show that this organism also produces a high-molecular-weight extracellular polysaccharide (EPS). Expression of the EPS results in the formation of a large polysaccharide layer around the bacteria which prevents them from forming a tight pellet upon centrifugation and from entering a Percoll density gradient. Like expression of the capsular polysaccharides, expression of the EPS is phase variable and dictated by DNA inversion of its promoter. EPS expression is regulated at one level by the DNA invertase Tsr19, which is encoded by a gene immediately upstream of the EPS locus and inverts the EPS promoter, causing an on or off phenotype. Expression of the EPS is also regulated at another level, which dictates the amount of EPS produced. By analyzing a panel of tsr19 deletion mutants, we found that the number of inverted repeats (IRs) flanking the promoter is variable. Transcription into the EPS genes is greater in mutants with a single IR between the promoter and the downstream EPS genes than in mutants with more than one IR in this region, correlating with the synthesis of more EPS. By analyzing the relative orientations of the EPS promoter of bacteria obtained from human fecal samples, we showed that both DNA inversion and variation in the number of IRs are active processes of B. fragilis in the endogenous human intestinal ecosystem