The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation

The Important Role of Membrane Fluidity on the Lytic Mechanism of the α-Pore-Forming Toxin Sticholysin I

Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action

Efecto agregante plaquetario de 2 citolisinas: Sti y Stii purificadas de la anémona marina Stichodactyla Helianthus Platelet aggregating effect of 2 cytolysins: StI and StII purified from the sea anemone Stichodactyla helianthus

Las citolisinas Sticholysina I (St I) y Sticholysina II (St II) inducen la agregación plaquetaria en el plasma rico en plaquetas en el rango de concentraciones ensayadas (0,5 a 10 µg/mL). Para ambas citolisinas se obtienen porcentajes de agregación plaquetaria superiores al 90 % con menos del 50 % de lisis celular. La agregación plaquetaria se mantiene elevada aún cuando la lisis celular disminuye a menos del 20 %. El EDTA 2 mM/L y el verapamilo 100 mM/L inhiben significativamente la agregación inducida por StI, lo que evidencia que el calcio extracelular tiene una función importante en este proceso y probablemente esta citolisina tiene una función similar a la de un ionóforo de calcio. Con StII no se obtuvo inhibición significativa de la agregación en presencia de EDTA y verapamilo. La agregación inducida por ambas citolisinas no está influida por el aumento del AMPc intracelular y es independiente de la formación de tromboxano A2 en la plaquetaCytolysing Sticholysina I (St I) and Sticholysina II (St II) induce platelet aggregation in the plasma rich in platelets in the range of assayed concentrations (0.5 to 10 m g/mL). Percentages of platelet aggregation over 90 % with less than 50 % of cellular lysis were obtained for both cytolysins. The platelet aggregation is high even when the cellular lysis decreases to less than 20 %. EDTA 2 mM/L and verapamyl 100 mM/L significantly inhibit the aggregation induced by StI, which shows that extracellular calcium has an important function in this process and that probably this cytolysin plays a role similar to that of a calcium ionophore. With StII there was no significant inhibition of the aggregation in the presence of EDTA and verapamyl. The aggregation induced by both cytolysins is not influenced by the increase of intracellular AMPc and it is independent of the formation of thromboxane A2 in the platele

The presence of sterols favors Sticholysin I - membrane association and pore formation regardless of their ability to form laterally segregated domains

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT. As for actinoporins, it has been proposed that the presence of cholesterol (Chol) and the coexistence of lipid phases increase binding to the target membrane and pore-forming ability. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, and the presence of lipid domains) on the activity of actinoporins or which regions of the membrane are the most favorable for protein insertion, oligomerization, and eventually pore formation. To gain insight into the role of membrane properties on the functional activity of St I, we studied its binding to monolayers and vesicles of phosphatidylcholine (PC), sphingomyelin (SM), and sterols inducing (ergosterol -Erg and cholesterol -Chol) or not (cholestenone - Cln) membrane phase segregation in liquid ordered (Lo) and liquid disordered (Ld) domains. This study revealed that St I binds and permeabilizes with higher efficiency sterol-containing membranes independently of their ability to form domains. We discuss the results in terms of the relevance of different membrane properties for the actinoporins mechanism of action, namely, molecular heterogeneity, specially potentiated in membranes with sterols inducers of phase separation (Chol or Erg) or Cln, a sterol noninducer of phase separation but with a high propensity to induce nonlamellar phase. The role of the Ld phase is pointed out as the most suitable platform for pore formation. In this regard, such regions in Chol-containing membranes seem to be the most favored due to its increased fluidity; this property promotes toxin insertion, diffusion, and oligomerization leading to pore formation.Fil: Pedrera Puentes, Lohans. Universidad de La Habana; CubaFil: Gomide, Andreza. B.. Universidade de Sao Paulo; BrasilFil: Sanchez, Rafael E.. Universidad de La Habana; CubaFil: Ros Quincoces, Uris. Universidad de La Habana; CubaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Pazos, Fabiola. Universidad de La Habana; CubaFil: Lanio, María E.. Universidad de La Habana; CubaFil: Itri, Rosangela. Universidade de Sao Paulo; BrasilFil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Álvarez Valcárcel, Carlos Manuel. Universidad de La Habana; Cub

Improved purification and enzymatic properties of a mixture of Sticholysin i and II: Isotoxins with hemolytic and phospholipase A2 activities from the sea anemone Stichodactyla helianthus

Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI–StII was relatively high (1.85 U/mg) and dependent of Ca2+. The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40 mM Ca2+ at pH 8.0. This Ca2+ concentration did not exert any effect on binding of StI–StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.The authors wish to thank INFORMATICA ddmm, Bergamo, Italy and CYTED (ENZNUT network 108RT0346), for kindly provided financial support to A. del Monte, and thanks to Havana University, Cuba, and CONACyT, México, for scientific grants.Peer Reviewe

Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis

A more effective vaccine against tuberculosis (TB) is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb), the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms), could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL) or nonadjuvanted (LMs) showed significant reductions in bacterial load (P<0.01) compared to the negative control group (animals immunized with phosphate buffered saline (PBS)). Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG). Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P<0.01) and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB