Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

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.

Tailored liposomal nanotraps for the treatment of Streptococcal infections.

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.

Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.

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.

The Contribution of the Left Phrenic Nerve to Innervation of the Esophagogastric Junction.

The contribution of the left phrenic nerve to innervation of the esophagogastric junction. The esophagogastric junction is part of the barrier preventing gastroesophageal reflux. We have investigated the contribution of the phrenic nerves to innervation of the esophagogastric junction in humans and piglets by dissecting 30 embalmed human specimens and 14 piglets. Samples were microdissected and nerves were stained and examined by light and electron microscopy. In 76.6% of the human specimens, the left phrenic nerve participated in the innervation of the esophagogastric junction by forming a neural network together with the celiac plexus (46.6%) or by sending off a distinct phrenic branch, which joined the anterior vagal trunk (20%). Distinct left phrenic branches were always accompanied by small branches of the left inferior phrenic artery. In 10% there were indirect connections with a distinct phrenic nerve branch joining the celiac ganglion, from which celiac plexus branches to the esophagogastric junction emerged. Morphological examination of phrenic branches revealed strong similarities to autonomic celiac plexus branches. There was no contribution of the left phrenic nerve or accompanying arteries from the caudal phrenic artery in any of the piglets. The right phrenic nerve made no contribution in any of the human or piglet samples. We conclude that the left phrenic nerve in humans contributes to the innervation of the esophagogastric junction by providing ancillary autonomic nerve fibers. Experimental studies of the innervation in pigs should consider that neither of the phrenic nerves was found to contribute. Clin. Anat. 33:265-274, 2020. © 2019 Wiley Periodicals, Inc.

Pneumolysin-damaged cells benefit from non-homogeneous toxin binding to cholesterol-rich membrane domains.

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.

Pathological manifestations of Farber disease in a new mouse model.

Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1tmEx1 mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.

Defying death: Cellular survival strategies following plasmalemmal injury by bacterial toxins.

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.

Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack.

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.

Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit Remarkable Properties Forming Lamellar Phases and Giant Vesicles.

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (Cn-IMe·HI, n = 7, 11, 15) and 1,3-dibenzyl-4,5-dialkylimidazolium (Cn-IBn·HBr, n = 7, 11, 15) salts. Diverse alkyl chain lengths and headgroups differing in their steric demand were employed for the membrane interface interaction with bilayer membranes imitating the cellular plasma membrane. Membrane hydration properties and domain fluidization were analyzed by fluorescent bilayer probes in direct comparison to established model membranes in a buffered aqueous environment, which resembles the salt content and pH of the cytosol of living cells. Membrane binding and insertion was analyzed via a quartz crystal microbalance and confocal laser scanning microscopy. We show that short-chain 4,5-dialkylimidazolium salts with a bulky headgroup were able to disintegrate membranes. Long-chain imidazolium salts form bilayer membrane vesicles spontaneously and autonomously without the addition of other lipids. These 4,5-dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.

Ca²âº-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins.

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.

Clinical Streptococcus pneumoniae isolates induce differing CXCL8 responses from human nasopharyngeal epithelial cells which are reduced by liposomes.

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.

P2X7 receptors mediate resistance to toxin-induced cell lysis.

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.

miR-199a-5p regulates urothelial permeability and may play a role in bladder pain syndrome.

Defects in urothelial integrity resulting in leakage and activation of underlying sensory nerves are potential causative factors of bladder pain syndrome, a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. Herein, we identified the microRNA miR-199a-5p as an important regulator of intercellular junctions. On overexpression in urothelial cells, it impairs correct tight junction formation and leads to increased permeability. miR-199a-5p directly targets mRNAs encoding LIN7C, ARHGAP12, PALS1, RND1, and PVRL1 and attenuates their expression levels to a similar extent. Using laser microdissection, we showed that miR-199a-5p is predominantly expressed in bladder smooth muscle but that it is also detected in mature bladder urothelium and primary urothelial cultures. In the urothelium, its expression can be up-regulated after activation of cAMP signaling pathways. While validating miR-199a-5p targets, we delineated novel functions of LIN7C and ARHGAP12 in urothelial integrity and confirmed the essential role of PALS1 in establishing and maintaining urothelial polarity and junction assembly. The present results point to a possible link between miR-199a-5p expression and the control of urothelial permeability in bladder pain syndrome. Up-regulation of miR-199a-5p and concomitant down-regulation of its multiple targets might be detrimental to the establishment of a tight urothelial barrier, leading to chronic pain.

Annexin A1 is a biomarker of T-tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy.

Skeletal muscle complaints are a common consequence of cholesterol-lowering therapy. Transverse tubular (T-tubular) vacuolations occur in patients having statin-associated myopathy and, to a lesser extent, in statin-treated patients without myopathy. We have investigated quantitative changes in T-tubular morphology and looked for early indicators of T-tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol-lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin-naive subjects. In addition, morphometry of the T-tubular system was performed. Only the gene expression for annexin A1 was up-regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin-treated patients, the volume fraction of the T-tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca(2+)-dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T-tubular sites. The up-regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T-tubular repair.

Down-regulation of annexin A1 in the urothelium decreases cell survival after bacterial toxin exposure.

PURPOSE: We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. MATERIALS AND METHODS: Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. RESULTS: Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. CONCLUSIONS: Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function.

Ceramide in plasma membrane repair.

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca(2+)-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca(2+) sensors and as membrane fusion agents.

Tailored protection against plasmalemmal injury by annexins with different Ca2+ sensitivities.

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.

Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes.

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.

Inhibition of suicidal erythrocyte death by blebbistatin.

Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 µM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 µM, annexin V binding ≥ 10 µM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 µM, annexin V binding ≥ 25 µM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.

MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.