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.

Small Pore-Forming Toxins Different Membrane Area Binding and Ca2+ Permeability of Pores Determine Cellular Resistance of Monocytic Cells.

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.

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.

Differentially regulated promoters for antigen expression in Salmonella vaccine strains.

In most attenuated Salmonella enterica vaccines, heterologous antigens are expressed under the control of strong inducible promoters to ensure a high level of synthesis. Although high expression levels of the antigen can improve the immunogenicity of the vaccine, they might be toxic to the Salmonella carrier. Expression problems could be avoided by the use of promoters with specific characteristics with respect to strength and timing of expression. To study the expression of ten selected promoters, translational promoter-green fluorescent protein (GFP) fusions were analyzed in three attenuated Salmonella strains, Ty21a, SL3261 and PhoPC. Promoter expression was evaluated both in vitro and in intracellular conditions using flow cytometry and confocal microscopy, with specific focus on the levels and timing of expression. We identified one major candidate promoter (Pasr) that could be used to express antigens specifically during in vivo conditions, without impairing bacterial growth during in vitro vaccine production.

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.

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.