Protein complex from human milk enhances the activity of antibiotics and drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an α-lactalbumin-oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured α-lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here.Microbiology and Molecular Genetic

HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death

HAMLET Binding to α-Actinin Facilitates Tumor Cell Detachment

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed

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

HPV16 E5 is produced from an HPV16 early mRNA spliced from SD226 to SA3358

The HPV16 E5 open reading frame (ORF) is present on the majority of all alternatively spliced HPV16 mRNAs, but it is currently unknown how well it is translated into E5 protein. To identify HPV16 mRNAs that are efficiently translated into E5, we have generated cDNA plasmids expressing individual, alternatively spliced HPV16 mRNAs with the potential to produce E5. By replacing the E5 ORF with sLuc, we could quantitate sLuc and determine how well each cDNA was translated. Our results showed that the upstream E1 and E7 AUGs inhibited translation of the E5 ORF and revealed that only one HPV16 mRNA produced high levels of E5. This was an HPV16 early mRNA spliced from SD226 to SA3358. These results were confirmed in the context of the entire HPV16 genome. Taken together, our results indicate that E5 is expressed early in the HPV16 replication cycle since it is translated efficiently only by one early mRNA

A protein complex from human milk enhances the activity of antibiotics and drugs against Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an -lactalbumin–oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis. HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured -lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis. For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis. Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here

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