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1.
Front Microbiol ; 13: 867449, 2022.
Article in English | MEDLINE | ID: mdl-35369481

ABSTRACT

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

2.
Cells ; 11(1)2022 01 05.
Article in English | MEDLINE | ID: mdl-35011729

ABSTRACT

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis-important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens.


Subject(s)
Cytotoxins/toxicity , Leukocytes/metabolism , Liposomes/chemistry , Streptococcus pyogenes/metabolism , Streptococcus/metabolism , Cell Death/drug effects , Cell Line , Cell Line, Transformed , Cholesterol/metabolism , Humans , Leukocytes/drug effects , Neutralization Tests
3.
Front Cell Infect Microbiol ; 12: 1106063, 2022.
Article in English | MEDLINE | ID: mdl-36683678

ABSTRACT

Introduction: Streptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries. Methods: Here we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD. Results and Discussion: Only the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin.


Subject(s)
Meningitis , Pneumococcal Infections , Mice , Rats , Animals , Serogroup , Liposomes , Streptococcus pneumoniae , Pneumococcal Infections/microbiology , Cytokines , Inflammation , Pneumococcal Vaccines
4.
Toxins (Basel) ; 13(2)2021 02 09.
Article in English | MEDLINE | ID: mdl-33572185

ABSTRACT

Pore-forming toxins (PFTs) form multimeric trans-membrane pores in cell membranes that differ in pore channel diameter (PCD). Cellular resistance to large PFTs (>20 nm PCD) was shown to rely on Ca2+ influx activated membrane repair mechanisms. Small PFTs (<2 nm PCD) were shown to exhibit a high cytotoxic activity, but host cell response and membrane repair mechanisms are less well studied. We used monocytic immune cell lines to investigate the cellular resistance and host membrane repair mechanisms to small PFTs lysenin (Eisenia fetida) and aerolysin (Aeromonas hydrophila). Lysenin, but not aerolysin, is shown to induce Ca2+ influx from the extracellular space and to activate Ca2+ dependent membrane repair mechanisms. Moreover, lysenin binds to U937 cells with higher efficiency as compared to THP-1 cells, which is in line with a high sensitivity of U937 cells to lysenin. In contrast, aerolysin equally binds to U937 or THP-1 cells, but in different plasma membrane areas. Increased aerolysin induced cell death of U937 cells, as compared to THP-1 cells, is suggested to be a consequence of cap-like aerolysin binding. We conclude that host cell resistance to small PFTs attack comprises binding efficiency, pore localization, and capability to induce Ca2+ dependent membrane repair mechanisms.


Subject(s)
Bacterial Toxins/toxicity , Calcium Signaling/drug effects , Calcium/metabolism , Cell Membrane Permeability/drug effects , Cell Membrane/drug effects , Monocytes/drug effects , Pore Forming Cytotoxic Proteins/toxicity , Toxins, Biological/toxicity , Cell Death/drug effects , Cell Membrane/metabolism , Cell Membrane/pathology , Drug Resistance , Genes, Reporter , Humans , Monocytes/metabolism , Monocytes/pathology , THP-1 Cells , U937 Cells
5.
J Nanobiotechnology ; 19(1): 46, 2021 Feb 15.
Article in English | MEDLINE | ID: mdl-33588835

ABSTRACT

BACKGROUND: Streptococcal infections are associated with life-threatening pneumonia and sepsis. The rise in antibiotic resistance calls for novel approaches to treat bacterial diseases. Anti-virulence strategies promote a natural way of pathogen clearance by eliminating the advantage provided to bacteria by their virulence factors. In contrast to antibiotics, anti-virulence agents are less likely to exert selective evolutionary pressure, which is a prerequisite for the development of drug resistance. As part of their virulence mechanism, many bacterial pathogens secrete cytolytic exotoxins (hemolysins) that destroy the host cell by destabilizing their plasma membrane. Liposomal nanotraps, mimicking plasmalemmal structures of host cells that are specifically targeted by bacterial toxins are being developed in order to neutralize-by competitive sequestration-numerous exotoxins. RESULTS: In this study, the liposomal nanotrap technology is further developed to simultaneously neutralize the whole palette of cytolysins produced by Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis-pathogens that can cause life-threatening streptococcal toxic shock syndrome. We show that the mixture of liposomes containing high amounts of cholesterol and liposomes composed exclusively of choline-containing phospholipids is fully protective against the combined action of exotoxins secreted by these pathogens. CONCLUSIONS: Unravelling the universal mechanisms that define targeting of host cells by streptococcal cytolysins paves the way for a broad-spectrum anti-toxin therapy that can be applied without a diagnostic delay for the treatment of bacterial infections including those caused by antibiotic-resistant pathogens.


Subject(s)
Liposomes/pharmacology , Liposomes/therapeutic use , Streptococcal Infections/drug therapy , Anti-Bacterial Agents/therapeutic use , Bacterial Toxins , Delayed Diagnosis , Hemolysin Proteins , Humans , Streptococcus , Streptococcus pyogenes
6.
Nat Commun ; 11(1): 1338, 2020 03 12.
Article in English | MEDLINE | ID: mdl-32165633

ABSTRACT

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.


Subject(s)
Acid Ceramidase/metabolism , Herpes Simplex/enzymology , Herpes Simplex/prevention & control , Herpesvirus 1, Human/physiology , Macrophages/enzymology , Multivesicular Bodies/virology , Acid Ceramidase/genetics , Animals , Female , Herpes Simplex/virology , Humans , Macrophages/virology , Mice , Mice, Inbred C57BL , Mice, Knockout , Virus Replication
7.
FASEB J ; 34(1): 1665-1678, 2020 01.
Article in English | MEDLINE | ID: mdl-31914676

ABSTRACT

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.


Subject(s)
Bacterial Toxins/metabolism , Cell Membrane Structures/metabolism , Cell Membrane Structures/pathology , Cell Membrane/metabolism , Cell Membrane/pathology , Cell-Derived Microparticles/metabolism , Cell-Derived Microparticles/pathology , Calcium/metabolism , Cell Death/physiology , Cell Line, Tumor , Cell Survival/physiology , Humans , Jurkat Cells , Monocytes/metabolism , Monocytes/pathology , Myeloid Cells/metabolism , Myeloid Cells/pathology , Streptococcus pneumoniae/pathogenicity , THP-1 Cells , U937 Cells
8.
Biomater Sci ; 7(9): 3693-3705, 2019 Aug 20.
Article in English | MEDLINE | ID: mdl-31187801

ABSTRACT

Protein-membrane interactions that modify the shape of membranes are important for generating curvature, membrane deformation by protein-protein crowding or trafficking of vesicles. Giant vesicles represent a simplified but versatile model for biological membranes and are commonly employed for the study of lipid domains and permeation across compartments. In this study, we investigated the interaction of pneumolysin (PLY), a pore-forming toxin secreted by Streptococcus pneumoniae, with multilamellar and unilamellar membranes. It reveals an enlargement of membrane area due to the insertion of pores into the bilayer and protein-membrane aggregations that induce membrane deformation and wrinkling. Moreover, we demonstrate that PLY peel-off layers from multilamellar giant vesicles in a hitherto unknown layer-by-layer peeling mechanism, which reveals the structure and number of membrane lamellae. We employed microfluidic methods to capture giant vesicles and confocal laser scanning microscopy, transmission microscopy, dynamic light scattering and cryo-electron microscopy to disclose the structure of multilamellar vesicles. Based on our findings we suggest how back-to-back pore arrangements stabilize large PLY-membrane entities and that pore-displaced lipids possibly remain in the membrane.


Subject(s)
Cell Membrane/chemistry , Streptococcus pneumoniae/chemistry , Streptolysins/chemistry , Unilamellar Liposomes/chemistry , Bacterial Proteins/chemistry
9.
FASEB J ; 33(1): 275-285, 2019 01.
Article in English | MEDLINE | ID: mdl-29979630

ABSTRACT

Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.


Subject(s)
Bacterial Toxins/pharmacology , Cell Membrane/metabolism , Heat-Shock Proteins/pharmacology , Hemolysin Proteins/pharmacology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Streptolysins/pharmacology , Bacterial Proteins/pharmacology , Biological Transport , CRISPR-Cas Systems , Calcium/metabolism , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Survival , Cell-Derived Microparticles/chemistry , Cell-Derived Microparticles/drug effects , Cell-Derived Microparticles/metabolism , Humans , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
10.
Front Immunol ; 9: 1688, 2018.
Article in English | MEDLINE | ID: mdl-30100903

ABSTRACT

Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14+MHCIIlowCD86low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance.


Subject(s)
Bacterial Toxins/immunology , Cell-Derived Microparticles/immunology , Cell-Derived Microparticles/metabolism , Macrophages/immunology , Macrophages/metabolism , Pore Forming Cytotoxic Proteins/immunology , Signal Transduction , Cytokines/metabolism , Host-Pathogen Interactions , Humans , Immunity , Immunomodulation , Immunophenotyping , Models, Biological , Monocytes/immunology , Monocytes/metabolism , Phenotype
11.
EBioMedicine ; 33: 211-217, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29936135

ABSTRACT

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Methicillin-Resistant Staphylococcus aureus/metabolism , Sphingomyelins/administration & dosage , Staphylococcal Skin Infections/therapy , Animals , Cell Line , Community-Acquired Infections , Disease Models, Animal , Hemolysin Proteins/antagonists & inhibitors , Humans , Liposomes , Methicillin-Resistant Staphylococcus aureus/drug effects , Mice , Necrosis , Sphingomyelins/pharmacology , Treatment Outcome
12.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(8): 795-805, 2018 08.
Article in English | MEDLINE | ID: mdl-29679741

ABSTRACT

Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca2+-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.


Subject(s)
Host-Pathogen Interactions/immunology , Lipid Bilayers/metabolism , Pneumococcal Infections/immunology , Streptococcus pneumoniae/physiology , Bacterial Proteins/metabolism , Bacterial Shedding/immunology , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/microbiology , Cholesterol/metabolism , HEK293 Cells , Humans , Intravital Microscopy , Lipid Bilayers/immunology , Microfluidics , Pneumococcal Infections/microbiology , Streptolysins/metabolism
13.
Biochim Biophys Acta ; 1860(11 Pt A): 2498-2509, 2016 11.
Article in English | MEDLINE | ID: mdl-27481675

ABSTRACT

BACKGROUND: Streptococcus pneumoniae is a potent human pathogen. Its pore-forming exotoxin pneumolysin is instrumental for breaching the host's epithelial barrier and for the incapacitation of the immune system. METHODS AND RESULTS: Using a combination of life imaging and cryo-electron microscopy we show that pneumolysin, released by cultured bacteria, is capable of permeabilizing the plasmalemma of host cells. However, such permeabilization does not lead to cell lysis since pneumolysin is actively removed by the host cells. The process of pore elimination starts with the formation of pore-bearing plasmalemmal nanotubes and proceeds by the shedding of pores that are embedded in the membrane of released microvesicles. Pneumolysin prepores are likewise removed. The protein composition of the toxin-induced microvesicles, assessed by mass spectrometry, is suggestive of a Ca(2+)-triggered mechanism encompassing the proteins of the annexin family and members of the endosomal sorting complex required for transport (ESCRT) complex. CONCLUSIONS: S. pneumoniae releases sufficient amounts of pneumolysin to perforate the plasmalemma of host cells, however, the immediate cell lysis, which is frequently reported as a result of treatment with purified and artificially concentrated toxin, appears to be an unlikely event in vivo since the toxin pores are efficiently eliminated by microvesicle shedding. Therefore the dysregulation of cellular homeostasis occurring as a result of transient pore formation/elimination should be held responsible for the damaging toxin action. GENERAL SIGNIFICANCE: We have achieved a comprehensive view of a general plasma membrane repair mechanism after injury by a major bacterial toxin.


Subject(s)
Cell Membrane/ultrastructure , Streptococcus pneumoniae/pathogenicity , Streptolysins/pharmacology , Bacterial Proteins/pharmacology , Bacterial Proteins/toxicity , Cell Membrane/drug effects , Cell Membrane/microbiology , Cell Membrane Permeability , HEK293 Cells , HeLa Cells , Humans , Streptolysins/toxicity
14.
BMC Microbiol ; 16(1): 154, 2016 07 19.
Article in English | MEDLINE | ID: mdl-27430279

ABSTRACT

BACKGROUND: Streptococcus pneumoniae causes several human diseases, including pneumonia and meningitis, in which pathology is associated with an excessive inflammatory response. A major inducer of this response is the cholesterol dependent pneumococcal toxin, pneumolysin. Here, we measured the amount of inflammatory cytokine CXCL8 (interleukin (IL)-8) by ELISA released by human nasopharyngeal epithelial (Detroit 562) cells as inflammatory response to a 24 h exposure to different pneumococcal strains. RESULTS: We found pneumolysin to be the major factor influencing the CXCL8 response. Cholesterol and sphingomyelin-containing liposomes designed to sequester pneumolysin were highly effective at reducing CXCL8 levels from epithelial cells exposed to different clinical pneumococcal isolates. These liposomes also reduced CXCL8 response from epithelial cells exposed to pneumolysin knock-out mutants of S. pneumoniae indicating that they also reduce the CXCL8-inducing effect of an unidentified pneumococcal virulence factor, in addition to pneumolysin. CONCLUSION: The results indicate the potential of liposomes in attenuating excessive inflammation as a future adjunctive treatment of pneumococcal diseases.


Subject(s)
Epithelial Cells/metabolism , Interleukin-8/metabolism , Liposomes/pharmacology , Nasopharynx/metabolism , Streptococcus pneumoniae/metabolism , Bacterial Capsules , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Cell Line , Cells, Cultured , Cholesterol/pharmacology , Cytokines/metabolism , Epithelial Cells/drug effects , Humans , Mutation , Nasopharynx/drug effects , Sphingomyelins/pharmacology , Streptococcus pneumoniae/isolation & purification , Streptococcus pneumoniae/pathogenicity , Streptolysins/genetics , Streptolysins/pharmacology
15.
Semin Cell Dev Biol ; 45: 39-47, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26481974

ABSTRACT

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic constituents. In order to ensure survival, the cell needs to identify, plug and remove lesions from its membrane. Quarantined by membrane folds and isolated by membrane fusion, the pores are removed from the plasmalemma and expelled into the extracellular space. Outward vesiculation and microparticle shedding seem to be the strategies of choice to eliminate toxin-perforated membrane regions from the plasmalemma of host cells. Depending on the cell type and the nature of injury, the membrane lesion can also be taken up by endocytosis and degraded internally. Host cells make excellent use of an initial, moderate rise in intracellular [Ca(2+)], which triggers containment of the toxin-inflicted damage and resealing of the damaged plasmalemma. Additional Ca(2+)-dependent defensive cellular actions range from the release of effector molecules in order to warn neighbouring cells, to the activation of caspases for the initiation of apoptosis in order to eliminate heavily damaged, dysregulated cells. Injury to the plasmalemma by bacterial toxins can be prevented by the early sequestration of bacterial toxins. Artificial liposomes can act as a decoy system preferentially binding and neutralizing bacterial toxins.


Subject(s)
Bacterial Toxins/pharmacology , Cell Membrane/physiology , Animals , Annexins/physiology , Calcium Signaling , Cell Survival/drug effects , Cell-Derived Microparticles/physiology , Endocytosis , Humans
16.
Biochim Biophys Acta ; 1853(9): 2045-54, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25219550

ABSTRACT

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca²âº entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca²âº sequestration that prevents excessive Ca²âº elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca²âº signals in cells that were able to survive after PLY attack. Intracellular Ca²âº dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca²âº signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.


Subject(s)
Calcium/metabolism , Streptococcus pneumoniae/chemistry , Streptolysins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Membrane , HEK293 Cells , Humans , Streptolysins/chemistry
17.
Nat Biotechnol ; 33(1): 81-8, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25362245

ABSTRACT

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.


Subject(s)
Bacterial Infections/prevention & control , Bacterial Toxins/chemistry , Exotoxins/chemistry , Genetic Engineering , Liposomes/chemistry , Animals , Mice
18.
Biochimie ; 107 Pt A: 66-72, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25183513

ABSTRACT

Eukaryotic cells have developed repair mechanisms, which allow them to reseal their membrane in order to prevent the efflux of cytoplasmic constituents and the uncontrolled influx of calcium. After injury, the Ca(2+)-concentration gradient fulfils a dual function: it provides guidance cues for the repair machinery and directly activates the molecules, which have a repair function. Depending on the nature of injury, the morphology of the cell and the severity of injury, the membrane resealing can be effected by lysosomal exocytosis, microvesicle shedding or a combination of both. Likewise, exocytosis is often followed by the endocytic uptake of lesions. Additionally, since plasmalemmal resealing must be attempted, even after extensive injury in order to prevent cell lysis, the restoration of membrane integrity can be achieved by ceramide-driven invagination of the lipid bilayer, during which the cell is prepared for apoptotic disposal. Plasmalemmal injury can be contained by a surfeit of plasma membrane, which serves as a trap for toxic substances: either passively by an abundance of cellular protrusions, or actively by membrane blebbing.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Exocytosis , Lysosomes/metabolism , Animals , Cell-Derived Microparticles/metabolism , Endocytosis , Humans , Lipid Bilayers/metabolism , Models, Biological
19.
PLoS One ; 9(2): e89743, 2014.
Article in English | MEDLINE | ID: mdl-24587004

ABSTRACT

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.


Subject(s)
Cell Membrane/physiology , Cell-Derived Microparticles/physiology , Lysosomes/physiology , Streptolysins/pharmacology , Actin Cytoskeleton/metabolism , Bacterial Proteins/pharmacology , Cell Line, Tumor , Cell Membrane/drug effects , Cell Survival , HEK293 Cells , Humans , Immunity, Innate , Membrane Fusion , Myosins/metabolism
20.
Biochim Biophys Acta ; 1843(5): 915-22, 2014 May.
Article in English | MEDLINE | ID: mdl-24487066

ABSTRACT

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.


Subject(s)
Receptors, Purinergic P2X7/physiology , Streptolysins/toxicity , Bacterial Proteins/toxicity , Base Sequence , Blotting, Western , Cell Line , DNA Primers , Humans , Polymerase Chain Reaction
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