Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense

Abstract

a b s t r a c tCertain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca 2þ influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca 2þ ions even during blockade of Ca 2þ channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex selfprotective mechanism that still needs to be addressed.

Nutrient starvation effects on the allelochemical potency of Alexandrium tamarense (Dinophyceae)

“…The same might be the reason for a reduced lytic activity upon N starvation; however, nothing is yet known of the elemental composition of Alexandrium allelochemicals. What we know is that at least one group of lytic allelochemicals produced by A. tamarense comprises a suite of large non-proteinaceous and probably nonpolysaccharide compounds between 7 and 15 kDa (Ma et al 2011a, b).…”

Differential inimical effects of Alexandrium spp. and Karenia spp. on cleavage, hatching, and two larval stages of Japanese pearl oyster Pinctada fucata martensii

“…The same study reported that dissolved STX, A. tamarense cell content, and cellfree medium did not affect the hatching of scallops, but resuspended algal cell and cell fragments did, also showing that the negative effects on egg hatching are associated with uncharacterized cytotoxic compounds located on the cell surface of A. tamarense, rather than exuded compounds. In more recent reports, the lytic activity of Alexandrium against protists seemed to act on external cell membranes (Ma et al, 2009), as was the case of lytic compounds from A. tamarense that increased membrane permeability to Ca 2+ ions without specifically binding to calcium ion channels (Ma et al, 2011), and lytic compounds from three strains of A. tamarense complex whose toxicity were related to reactive oxygen species (ROS) and/or to secondary compounds produced by ROS-induced lipid peroxidation (Flores et al, 2012); however, cytotoxicheatlabile exotoxin (Lush et al, 2001), hemolytic proteinaceous exotoxin (Emura et al, 2004), and a polysaccharide-based cytotoxic compound (Yamasaki et al, 2008) were recently isolated from Alexandrium minutum, A. taylori, and A. tamarense, respectively, showing that both exudates and cell contact are possible mechanisms for the observed deleterious effects of Alexandrium spp. that could be strain-specific, and both mechanisms could have been implicated in the observed effects of A. affine and A. catenella on embryos and trochophores in the present study.…”

Growth- and nutrient-dependent gene expression in the toxigenic marine dinoflagellate Alexandrium minutum

“…In order to further illuminate the factors influencing synthesis and accumulation of the lytic compounds of Alexandrium spp., highly targeted datasets combining a high number of measurements of allelochemical activity with finely graded variations of the physiological parameters seem most promising. The elucidation of the chemical composition and structure of those compounds is currently under way (Ma et al, 2009(Ma et al, , 2011. …”