Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation

Abstract

Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA₂ inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.

Keywords: Cell membrane; Cytotoxicity; Envenoming; Phospholipase; Snakebite; Toxin.

Graphical abstract

Fig. 1

The concept of using fluorescently dyed phospholipid vesicles as a proxy for membrane disruption. Graphical overview of the concept of the VD assay. RFU = relative fluorescence units. Image created using www.biorender.com.

Fig. 2

Assay validation using a panel of snake venoms to study vesicle degradation. The degradation of egg yolk vesicles was monitored over 12 h (data captured every 10 min) at 37 °C, with six concentrations of venom (100–0.4 μg/mL). Ultrapure water was added as a negative control, and 2% Triton-X was added as a positive control. Readings were performed on a CLARIOstar Plus Microplate reader (using bottom optic reading); data was measured in triplicate; error bars show standard deviation.

Fig. 3

Overview of the neutralising effects of varespladib on vesicle degradation evoked by different snake venoms, relative to negative control. The degradation of egg yolk vesicles after 12 h of incubation at 37 °C, with and without varespladib, is shown in the green and red bars, respectively. Results from each venom are represented in a graph, in which the vesicle degradation of six venom concentrations (100–0.4 μg/mL) is given for which the venoms are either pre-incubated with 20 μM of varespladib or, in the absence of varespladib. Negative control values were 5078 ± 145 RFU (i.e., no venom, no varespladib) and 4795 ± 43 RFU (i.e., no venom, with varespladib control). Readings were performed on a CLARIOstar microplate reader (using bottom optic reading). Data was measured in triplicate; error bars show standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Identification of bioactive fractions of C. rhodostoma venom (1 mg/mL injected) by correlating bioactivity data with high throughput venomics data. i: bioactivity chromatograms of the VD assay after 8 h of incubation of egg yolk emulsions with venom fractions at 37 °C. Peaks with negative minima indicate the presence of bioactive fractions; ii and iii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the tryptically digested and bottom-up proteomics analysed well fractions. NB. The PSCs in ii represent PLA₂s correlating in retention time frame with the bioactivity peaks, and the PSCs in iii represent other protein hits. The two vertical dotted lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks. The last three data points (38.1–38.3 min) show the positive control (2% Triton-X). Measurements represented by the bioactivity chromatograms were performed in triplicate; error bars show standard deviation. The corresponding CSV and Excel files can be found in the Supplementary Information folders “CSV files” and “Excel files”.

Fig. 5

Identification of bioactive fractions of N. mossambica venom (1 mg/mL injected) by correlating bioactivity data with high throughput venomics data. i: bioactivity chromatograms of the VD assay after 8 h of incubation of egg yolk emulsions with venom fractions at 37 °C. Peaks with negative minima indicate the presence of bioactive fractions; ii - iii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the tryptically digested and bottom-up proteomics analysed well fractions. NB. The PSCs in ii represent PLA₂s hits, iii these represent 3FTx correlating in retention time frame with the bioactivity peak, and in iv these represent other protein hits. The two dotted lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks. The last three data points (38.1–38.3 min) show the positive control (2% Triton-X). Measurements represented by the bioactivity chromatograms were performed in triplicate; error bars show standard deviation. The corresponding CSV and Excel files can be found in the Supplementary Information folders “CSV files” and “Excel files”.