Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci.

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.

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.

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.

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.