Evaluation of Antifungal Activity of Naja pallida and Naja mossambica Venoms against Three Candida Species

Abstract

In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis, affected cell cycle of C. albicans, inhibited C. tropicalis-based biofilm formation and promoted oxidative stress in C. albicans, C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.

Keywords: Candida albicans; ammonia signaling; cobra venoms; colony development and aging; phospholipase secretion.

Figure 1

The quantitative analysis of two cobra venom proteins: N. pallida venom (NPV) (a); and N. mossambica venom (NMV) (b). The identified protein groups with protein content less than 1% of total protein were classified as “other”. Detailed description of “other” category can be found in the Supplementary Materials. Values have been rounded up to the nearest integer. 3FTx, three-finger toxin family; PLA₂, phospholipase A₂; VNGF, venom nerve growth factor; CRISP, cysteine-rich secretory protein; LAAO, L-amino acid oxidase; SVMP, snake venom metalloproteinase; Ig-like, Ig-like superfamily SSF48726.

Figure 2

NPV- and NMV-mediated changes in cell viability (a) and cell cycle (b) of three Candida species. (a) Metabolic activity and cell viability were assessed using resazurin assay. To emphasize snake venom action, a red horizontal line is added. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01 compared to the control (ANOVA and Dunnett’s a posteriori test). (b) DNA-based cell cycle analysis was conducted using an Amnis^® FlowSight^® imaging flow cytometer and IDEAS software. Representative histograms (normalized frequency vs intensity) are presented. NPV, N. pallida venom; NMV, N. mossambica venom.

Figure 3

NPV- and NMV-mediated changes in biofilm formation based on C. albicans, C. glabrata and C. tropicalis cells. (a) Biofilm formation was visualized using methylene blue staining and densitometry analysis was performed using GelQuantNET software. Biofilm formation at standard growth conditions was considered as 1.0. To emphasize snake venom action, a red horizontal line is added. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01 compared to the control (ANOVA and Dunnett’s a posteriori test). (b) Representative photographs are presented. NPV, N. pallida venom; NMV, N. mossambica venom.

Figure 4

NPV- and NMV-mediated increase in the levels of mitochondrial ROS in: C. albicans cells (left); C. glabrata cells (middle); and C. tropicalis cells (right). Mitochondrial ROS levels were measured using ROS-specific fluorescent probe MitoTracker^® Red CM-H₂Xros. Fluorescence intensity due to oxidation of reduced MitoTracker^® to oxidized MitoTracker^® was measured using a Tecan Infinite^® M200 fluorescence mode microplate reader. Mitochondrial ROS levels at standard growth conditions (untreated control) are considered as 100%. To emphasize snake venom action, a red horizontal line is added. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the control (ANOVA and Dunnett’s a posteriori test). NPV, N. pallida venom; NMV, N. mossambica venom.

Figure 5

NPV- and NMV-mediated changes in the secretion of extracellular hydrolases, namely phospholipases (a) and secreted aspartyl proteinases (Saps) (b) by three Candida species. To reveal the secretion of extracellular hydrolases, dedicated plate-based assays were used. The phospholipase activity was judged as the formation of an opaque zone around the yeast colonies (a) and the proteinase activity as the formation of a transparent halo around the yeast colonies (b). Representative photographs are presented. Secreted phospholipase activity was measured as a diameter of an opaque zone around the yeast colonies and phospholipase activity at standard growth conditions was considered as 1.0. To emphasize snake venom action, a red horizontal line is added. Bars indicate SD, n = 3, *** p < 0.001, ** p < 0.01, * p < 0.05 compared to the control (ANOVA and Dunnett’s a posteriori test). NPV, N. pallida venom; NMV, N. mossambica venom.

Figure 6

NPV- and NMV-mediated changes in ammonia pulses during colony development and aging in three Candida species. Candida cells were cultured onto bromocresol purple plates for 21 days and the phases of acidification (pH ~ 5.2, yellow color) and alkalization (pH ~ 6.8, violet color) were monitored after 1, 5, 8, 13 and 21 days of culture. Representative photographs are presented (both front and back plate view). NPV, N. pallida venom; NMV, N. mossambica venom.