Characterization and lytic activity of allelochemicals produced by the toxigenic marine dinoflagellate Alexandrium tamarense

Abstract

In this thesis, allelochemistry of Alexandrium tamarense, a well-known producer of saxitoxin and its analogues was investigated in an attempt to interpret the chemical characteristics and mode of action of the allelochemicals produced. As the response of target cells was lysis, the allelochemicals were thereafter designated as lytic compounds. One A. tamarense clonal strain, Alex2, with potent lytic activity, was chosen as the producer of the lytic compounds. Another A. tamarense clonal strain from the same geographical population, but without measurable lytic activity was introduced as a negative control. First, a cryptophyte Rhodomonas salina bioassay was used to guide the isolation and eventual structural characterization of the unknown lytic substances. Highest lytic activity per cell of A. tamarense cultures was achieved in the stationary growth phase, however, the allelopathic potency per cell remained constant throughout the entire exponential phase. The supernatant of stationery cell cultures prepared via centrifugation was chosen as the major source of lytic compounds for research through out the whole thesis. Further isolation and purification of the supernatant often resulted in major loss or even disappearance of lytic activity, prompting the physico/chemical characterization of the lytic compounds. Although temporal stability of the lytic compounds was high, in that the lytic activity was stable over wide ranges of temperatures and pH and was refractory to bacterial degradation (at least of the bacteria consortium present in the cultures), substantial losses were evident during purification. The lytic compounds possessed high adsorption to various materials including polycarbonate and other plastics, glass, etc. Additionally, the lytic activity was reduced when stored at low concentration, in deionized water, or in vessels with large surface area/volume ratios. Similar to the bioactivity of allelochemicals produced among other harmful algal bloom species, A. tamarense produced a suite of metabolites acting as lytic compounds. At least two groups of lytic compounds with various polarities were separated and quantified via reversed phase solid phase extraction (SPE). Emphasis was put on the less polar fraction because of its higher lytic activity. Further purification of this SPE fraction was performed via both C8 high performance liquid chromatography (HPLC) and hydrophilic interaction ion-chromatography (HILIC). Through matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) masses of the spectra between the lytic and non-lytic strain in SPE, C8 HPLC, and HILIC fractions were compared. Two small unique masses, 1061.6 Da and 1291.6 Da were found in lytic but not in non-lytic SPE fractions, however, they were later excluded to be related to lytic compounds by both HPLC and HILIC. Trypsic digestion and trypsic digestion-coupled size exclusion chromatography (SEC) suggested that the lytic compounds are large non-proteinaceous compounds less than 22.3 kDa. Although the large masses range from 7 to 15 kDa found only in HILIC lytic fraction haven t been proved to be lytic, the mass range deduced from SEC suggest that they are probably related to the lytic compounds. Total sugar content analysis suggested the percentage of sugar in dry mass equivalent in the lytic fraction was rather low, indicating the major composition of the lytic compounds may not be related to sugar. Alexandrium tamarense causes lysis of a broad spectrum of target protistan cells, and since the process is rapid, direct membrane disruption of target cell was suspected to be the mode of action. The lytic compounds increased permeability of the cell membrane for calcium ion from buffer external to the outer cell membrane, and such increase was not inhibited even during blockade of calcium ion channels with cadmium. This indicates that the lytic compounds lyse membrane directly, and the molecular targets are not ion channels. Membrane sterols were then suspected as the molecular target of lytic compounds. Adding sterols to a lysis bioassay with the R. salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent affinity for free sterols. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. However, 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. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. It is therefore concluded that the molecular targets of the lytic compounds are not, or are not exclusively, sterol components of the membranes.