The interaction of the ribotoxin α-sarcin with complex model lipid vesicles.

Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal ß-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane.

Low peripheral blood chemokine (C-C motif) ligand 5 and tumor necrosis factor α gene expression is associated with unfavorable progression of respiratory syncytial virus bronchiolitis in infants.

OBJECTIVES: We aimed to analyze whether the expression of inflammatory and antiviral genes in respiratory syncytial virus (RSV)-infected infants' peripheral blood is associated with bronchiolitis progression. METHODS: We conducted a prospective study on 117 infants between 2015 and 2023. The expression levels of nine genes were quantified by quantitative polymerase chain reaction. Infants were classified according to their clinical evolution during hospital admission: (i) non-progression (n = 74), when the RSV bronchiolitis severity remained stable or improved; (ii) unfavorable progression (n = 43), when the RSV bronchiolitis severity increased. The association analysis was performed by logistic regression, adjusted by age, gender, prematurity, and RSV bronchiolitis severity in the emergency room. RESULTS: Infants were 57.3% male, and the median age of the study population was 61 days. Thirty-five infants (30.7%) were admitted to the intensive care unit after hospital admission. Univariate logistic models showed that tumor necrosis factor (TNFα) and chemokine (C-C motif) ligand (CCL5) gene expression at baseline were inversely associated with unfavorable progression, which was confirmed by multivariate analyses: TNFα (adjusted odds ratio = 0.8 [95% confidence interval = 0.64-0.99], P-value = 0.038) and CCL5 (adjusted odds ratio = 0.76 [95% confidence interval = 0.62-0.93], P-value = 0.007). CONCLUSIONS: An inadequate immune response to RSV, characterized by reduced gene expression levels of CCL5 and TNFα in peripheral blood, was associated with an unfavorable progression of RSV bronchiolitis.

Production and characterization of monoclonal antibodies for the detection of the hepatitis C core antigen.

Background: Despite highly effective treatments to cure hepatitis C, almost 80% of chronically HCV-infected people are not treated, as they are unaware of their infection. Diagnostic rates and linkage to care must be substantially improved to reverse this situation. The HCV core antigen (HCVcAg) is a highly conserved protein that can be detected in the blood of HCV-infected patients and indicates active infection. Aim: To produce murine monoclonal antibodies against HCVcAg suitable for rapid and inexpensive tests to detect HCV infection. Methods: BALB/c mice were sequentially inoculated with purified recombinant HCVcAg from Gt1a, Gt3a, Gt4a, and Gt1b genotypes. Hybridomas producing the desired monoclonal antibodies were selected, and the reactivity of antibodies against HCVcAg from various genotypes was tested by Western blotting and dot blotting. The binding kinetics of the antibodies to purified HCVcAg was analyzed by surface plasmon resonance (SPR), and their ability to detect HCVcAg was tested by double antibody sandwich ELISA (DAS-ELISA). Results: Four specific monoclonal antibodies (1C, 2C, 4C, and 8C) were obtained. 1C, 2C, and 4C recognized HCVcAg of all genotypes tested (Gt1a, Gt1b, Gt2a, Gt3a, and Gt4a), while 8C did not recognize the Gt2a and Gt3a genotypes. Based on SPR data, the antibody-HCVcAg complexes formed are stable, with 2C having the strongest binding properties. DAS-ELISA with different antibody combinations easily detected HCVcAg in culture supernatants from HCV-infected cells. Conclusion: Specific and cross-reactive anti-HCVcAg monoclonal antibodies with strong binding properties were obtained that may be useful for detecting HCVcAg in HCV-infected samples.

Sticholysin recognition of ceramide-phosphoethanolamine.

Actinoporins are pore-forming toxins produced by sea anemones. They exert their activity by binding to the membranes of target cells. There, they oligomerize, forming cation-selective pores, and inducing cell death by osmotic shock. In the early days of the field, it was shown that accessible sphingomyelin (SM) in the bilayer is required for the activity of actinoporins. While these toxins can also act on membranes composed solely of phosphatidylcholine (PC) with a high amount of cholesterol (Chol), consensus is that SM acts as a lipid receptor for actinoporins. It has been shown that the 2NH and 3OH moieties of SM are essential for actinoporin recognition. Hence, we wondered if ceramide-phosphoethanolamine (CPE) could also be recognized. Like SM, CPE has the 2NH and 3OH groups, and a positively charged headgroup. While actinoporins have been observed to affect membranes containing CPE, Chol was always also present, with the recognition of CPE remaining unclear. To test this possibility, we used sticholysins, produced by the Caribbean Sea anemone Stichodactyla helianthus. Our results show that sticholysins can induce calcein release on vesicles composed only of PC and CPE, in absence of Chol, in a way that is comparable to that induced on PC:SM membranes.

Determination of the boundary lipids of sticholysins using tryptophan quenching.

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane.

Sea Anemones, Actinoporins, and Cholesterol.

Spanish or Spanish-speaking scientists represent a remarkably populated group within the scientific community studying pore-forming proteins. Some of these scientists, ourselves included, focus on the study of actinoporins, a fascinating group of metamorphic pore-forming proteins produced within the venom of several sea anemones. These toxic proteins can spontaneously transit from a water-soluble fold to an integral membrane ensemble because they specifically recognize sphingomyelin in the membrane. Once they bind to the bilayer, they subsequently oligomerize into a pore that triggers cell-death by osmotic shock. In addition to sphingomyelin, some actinoporins are especially sensible to some other membrane components such as cholesterol. Our group from Universidad Complutense of Madrid has focused greatly on the role played by sterols in this water-membrane transition, a question which still remains only partially solved and constitutes the main core of the article below.

Sticholysin I-II oligomerization in the absence of membranes.

Sticholysins are pore-forming toxins produced by the sea anemone Stichodactyla helianthus. When they encounter a sphingomyelin-containing membrane, these proteins bind to it and oligomerize, a process that ends in pore formation. Mounting evidence indicates that StnII can favour the activity of StnI. Previous results have shown that these two isotoxins can oligomerize together. Furthermore, StnII appeared to potentiate the activity of StnI through the membrane-binding step of the process. Hence, isotoxin interaction should occur prior to membrane encounter. Here, we have used analytical ultracentrifugation to investigate the oligomerization of Stns in solution, both separately and together. Our results indicate that while StnI seems to be more prone to oligomerize in water solution than StnII, a small percentage of StnII in StnI-StnII mixtures promotes oligomerization.