Antimicrobial Activity of Euplotin C, the Sesquiterpene Taxonomic Marker from the Marine Ciliate Euplotes crassus

Abstract

Strains of the marine ciliate protist Euplotes crassus produce exclusive terpenoids called euplotins that play an ecological role. Among these derivatives, euplotin C is the main of four secondary metabolites isolated from cultures of this protozoon and represents the sesquiterpene taxonomic marker from E. crassus. Because different terpenoid metabolites of plant origin showed a certain antimicrobial activity, we assessed the compound euplotin C, purified by high-pressure liquid chromatography and solubilized in two solubility enhancers, against the protozoa Leishmania major and Leishmani infantum, the fungus Candida albicans, and nine strains of gram-positive and gram-negative microorganisms. An activity of euplotin C against Leishmania promastigotes was demonstrated (50% lethal doses were 4.6 or 8.1 g/ml depending on the agent used to solubilize the compound), while the effect was less evident on Candida and nearly absent on bacteria. A nonsignificant cytotoxicity (50% lethal dose, >200 g/ml) against the J774 cell line was observed. A leishmanicidal activity was also shown by the living, euplotin-producing cells of E. crassus cultured together with promastigotes; this activity increased with time from 10 min to 6 h of incubation. This study provides an initial rationale for the evaluation of euplotin C and other similar natural products as alternative or possibly synergistic compounds for current antiprotozoon chemotherapeutics.In recent years, several reports have been published on the scientifically confirmed antimicrobial activity of some natural products derived from plants (1,8,13,14,30,34,38,39) or of bioactive metabolites of microbial origin (26,32,33,35,40). Natural products showing effective action of pharmaceutical and medical relevance have also been recovered from marine organisms. Some bioactive antiviral, antitumor, antimicrobial, or generally cytotoxic compounds have been extracted from species of starfish and sponges. For example, extracts from the starfish Asterina pectifera are active against Aspergillus spp. and Cryptococcus neoformans (7), while various sponges exhibit antibacterial properties which are due to the presence of a metabolic product or to host microorganisms (12,25).Recently, four sesquiterpenes-euplotin A, euplotin B, euplotin C (EC), and their biogenetic precursor preuplotinhave been isolated from Euplotes crassus, a eukaryotic unicellular marine ciliate (16,19). All strains of this ciliate morphospecies (taxonomically described by microscopic, outwardly identifying features) produce the same compounds. Notably, euplotins, and in particular EC, are biosynthesized in relative large amounts, suggesting an important ecological role of this substance in the adaptive strategy of the organism. A similar terpenoid (udoteatrial hydrate) was isolated from Udotea flabellum (36), a tropical seaweed belonging to the phylum Chlorophyta that contributes to the structure of coral reefs. Indeed, it has been suggested that strong similarities in anthozoan metabolite biosynthesis reveal the ev...

Antimicrobial activity of the protozoan toxin climacostol and its derivatives

“…These substances include keronopsin isolated from Pseudokeronopsis rubra Ehrenberg, 1836 (Höfle et al 1994), euplotins (Dini et al 1993;Guella et al 1994), raikovenal (Guella et al 1995;Rosini et al 1998) and epoxyfocardin (Guella et al 1996) from Euplotes species, blepharismin from Blepharisma japonicum Suzuki, 1954(Gioffrè et al 1993Checcucci et al 1997), stentorin from Stentor coeruleus Ehrenberg, 1830 (Tao et al 1993;Cameron et al 1995), spirostomin from Spirostomum teres Claparede et Lachmann, 1858(Sera et al 2006, maristentorin from Maristentor dinoferus Lobban et al, 2002(Mukherjiee et al 2006, climacostol from Climacostomum virens Ehrenberg, 1833 (Miyake et al 2003), and monoprenyl-hydroquinone from Spirostomum ambiguum Ehrenberg, 1835 (Buonanno et al 2012). Interestingly, due to its antimicrobial and pro-apoptotic activity in some tumor cell lines, the sesquiterpenoid euplotin C was recently proposed for the design of new drugs (Savoia et al 2004;Cervia et al 2006Cervia et al , 2007. Antiviral and antibacterial activities were also reported for stentorin (Lobban et al 2007) and blepharismin (Pant et al 1997), which are both derivatives of hypericin, i.e., a polycyclic aromatic compound extracted from plants of the genus Hypericum.…”

Antileishmanial activity of HIV protease inhibitors

“…The effects of the two PI, indinavir (Merck; a kind gift of S. Vella, Rome, Italy) and saquinavir (BS 00120083; a kind gift of Roche, Basel, Switzerland), on promastigotes of L. major and L. infantum were assessed by a method similar to that previously described [17]. Promastigotes were counted using a haemocytometer (Thoma chamber) and resuspended in fresh medium to a final concentration of 5 × 10 5 viable (showing motile behaviour and/or lack of staining after vital staining with trypan blue) promastigotes/mL.…”

“…Before use, promastigotes were grown in medium 199 (Invitrogen, CA, USA), modified as previously indicated [17], at 25 • C for 4 days to reach late-log phase growth.…”

Synthesis and antimicrobial activity of dermaseptin S1 analogues

“…Microorganisms were grown in static culture at 37°C in Mueller Hinton Broth (Oxoid) and diluted to 5 Â 10 5 CFU/mL. The MIC was determined using the microdilution serial twofold assay (from 100 lg/mL of each preparation) and performed in sterilized 96-well plates (Nunc) in a final volume of 200 lL 16,37 ; control wells without peptides were included. The MIC was considered the lowest peptide concentration that showed no increase in turbidity after overnight incubation at 37°C.…”

“…Leishmania major (strain MHOM/IL/67/JERICHO-II) promastigotes were grown at 25°C in 'complete medium' 37,40 , comprising medium 199 (Invitrogen, California, USA) supplemented with 20% heat-inactivated FCS (Invitrogen), 100 U/mL penicillin, 100 lg/mL streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 lg/mL hemin (in 50% triethanolamine) and 1 lg/mL 6-biotin (in 95% ethanol) L. major were resuspended in fresh medium to a final concentration of 5 Â 10 5 viable promastigotes/mL and treated with serial dilutions of the different peptides. Protozoal viability was assessed after 3 and 24 h incubation at 25°C under agitation by counting in a haemocytometer (Thoma chamber) after vital staining with trypan blue (dye exclusion method).…”