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

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

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

Host-Derived Microvesicles Carrying Bacterial Pore-Forming Toxins Deliver Signals to Macrophages: A Novel Mechanism of Shaping Immune Responses

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

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

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 clearanc

Synthetic Peptides to Target Stringent Response-Controlled Virulence in a Pseudomonas aeruginosa Murine Cutaneous Infection Model

Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In Pseudomonas aeruginosa this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of P. aeruginosa PAO1 and the Liverpool epidemic strain LESB58. A ΔrelA/ΔspoT double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease aprA gene in stringent response mutants grown on blood agar plates. Promoter fusions of relA or spoT to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated in vivo by the ΔrelA/ΔspoT double mutant, but not the relA mutant nor the ΔrelA/ΔspoT complemented with either gene. Treatment of a cutaneous P. aeruginosa PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the ΔrelA/ΔspoT infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that spoT promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated relA complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the P. aeruginosa stringent response in vivo and thus offer a promising novel therapeutic approach

Ca2+-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 Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ 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 Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ 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

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

Microvesicle Shedding and Lysosomal Repair Fulfill Divergent Cellular Needs during the Repair of Streptolysin O-Induced Plasmalemmal Damage

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

Liposomal Therapy Attenuates Dermonecrosis Induced by Community-Associated Methicillin-Resistant Staphylococcus aureus by Targeting α-Type Phenol-Soluble Modulins and α-Hemolysin

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. Keywords: Liposomes, Anti-toxin therapy, CA-MRSA, USA300, Skin and soft tissue infections, Phenol-soluble modulins, α-Hemolysin, Dermonecrosi

Ca²⁺-dependent repair of pneumolysin pores:A new paradigm for host cellular defense against bacterial pore-forming toxins

AbstractPneumolysin (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 Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ 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 Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ 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