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1.
FASEB J ; 34(1): 1665-1678, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31914676

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/metabolismo , Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/patología , Membrana Celular/metabolismo , Membrana Celular/patología , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patología , Calcio/metabolismo , Muerte Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Humanos , Células Jurkat , Monocitos/metabolismo , Monocitos/patología , Células Mieloides/metabolismo , Células Mieloides/patología , Streptococcus pneumoniae/patogenicidad , Células THP-1 , Células U937
2.
J Nanobiotechnology ; 19(1): 46, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33588835

RESUMEN

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.


Asunto(s)
Liposomas/farmacología , Liposomas/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Antibacterianos/uso terapéutico , Toxinas Bacterianas , Diagnóstico Tardío , Proteínas Hemolisinas , Humanos , Streptococcus , Streptococcus pyogenes
3.
FASEB J ; 33(1): 275-285, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29979630

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/farmacología , Membrana Celular/metabolismo , Proteínas de Choque Térmico/farmacología , Proteínas Hemolisinas/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Estreptolisinas/farmacología , Proteínas Bacterianas/farmacología , Transporte Biológico , Sistemas CRISPR-Cas , Calcio/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Supervivencia Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Humanos , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
5.
Cells ; 11(1)2022 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-35011729

RESUMEN

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.


Asunto(s)
Citotoxinas/toxicidad , Leucocitos/metabolismo , Liposomas/química , Streptococcus pyogenes/metabolismo , Streptococcus/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Colesterol/metabolismo , Humanos , Leucocitos/efectos de los fármacos , Pruebas de Neutralización
6.
Toxins (Basel) ; 13(2)2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33572185

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/toxicidad , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Monocitos/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Toxinas Biológicas/toxicidad , Muerte Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/patología , Resistencia a Medicamentos , Genes Reporteros , Humanos , Monocitos/metabolismo , Monocitos/patología , Células THP-1 , Células U937
7.
PLoS One ; 13(2): e0192507, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29408864

RESUMEN

The recent rise of multidrug-resistant Gram-negative bacteria represents a serious threat to public health and makes the search for novel effective alternatives to antibiotics a compelling need. Bacteriophage (Phage) lysins are enzymes that hydrolyze the cell wall of bacteria and represent a promising alternative to tackle this ever-increasing problem. Despite their use is believed to be restricted to Gram-positive bacteria, recent findings have shown that they can also be used against Gram-negative bacteria. By using a phage genome-based screening approach, we identified and characterized a novel lysin, PlyE146, encoded by an Escherichia coli prophage and with a predicted molecular mass of ca. 17 kDa. PlyE146 is composed of a C-terminal cationic peptide and a N-terminal N-acetylmuramidase domain. Histidine-tagged PlyE146 was overexpressed from a plasmid in Lactococcus lactis NZ9000 and purified by NI-NTA chromatography. PlyE146 exhibited in vitro optimal bactericidal activity against E. coli K12 (3.6 log10 CFU/mL decrease) after 2 h of incubation at 37°C at a concentration of 400 µg/mL in the absence of NaCl and at pH 6.0. Under these conditions, PlyE146 displayed antimicrobial activity towards several other E. coli, Pseudomonas aeruginosa (3 to 3.8-log10 CFU/mL decrease) and Acinetobacter baumannii (4.9 to >5-log10 CFU/mL decrease) strains. Therefore, PlyE146 represents a promising therapeutic agent against E. coli, P. aeruginosa and A. baumannii infections. However, further studies are required to improve the efficacy of PlyE146 under physiological conditions.


Asunto(s)
Colifagos/metabolismo , Bacterias Gramnegativas/metabolismo , Antibacterianos/farmacología , Western Blotting , Glicósido Hidrolasas/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Microscopía Electrónica de Transmisión
8.
Diagn Microbiol Infect Dis ; 90(1): 7-10, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29146285

RESUMEN

The Rapid Polymyxin NP test has been recently developed to rapidly detect polymyxin resistance in Enterobacteriaceae. Here we evaluated this test for detecting MCR-1/MCR-2-producing Enterobacteriaceae using a collection of 70 non-redundant strains either recovered from the environment, animals, or humans. Sensitivity and specificity were found to be 100%.


Asunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/genética , Polimixinas/farmacología , Animales , Enterobacter aerogenes/efectos de los fármacos , Enterobacter aerogenes/genética , Enterobacter aerogenes/aislamiento & purificación , Enterobacter cloacae/efectos de los fármacos , Enterobacter cloacae/genética , Enterobacter cloacae/aislamiento & purificación , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Humanos , Klebsiella oxytoca/efectos de los fármacos , Klebsiella oxytoca/genética , Klebsiella oxytoca/aislamiento & purificación , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genética
9.
Front Immunol ; 9: 1688, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30100903

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/inmunología , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/inmunología , Transducción de Señal , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inmunidad , Inmunomodulación , Inmunofenotipificación , Modelos Biológicos , Monocitos/inmunología , Monocitos/metabolismo , Fenotipo
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