Action mechanisms of the secondary metabolite euplotin C: signaling and functional role in Euplotes.

Abstract

Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca(2+) and Na(+), suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

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Action Mechanisms of the Secondary Metabolite Euplotin C: Signaling and
Functional Role in Euplotes
FRANCESCA TRIELLI,aDAVIDE CERVIA,b,cGRAZIANO DI GIUSEPPE,cCHIARA RISTORI,cTHOMAS KRUPPEL,d
BRUNO BURLANDO,eGRAZIANO GUELLA,fALDO VIARENGO,ePAOLA BAGNOLI,c
MARIA UMBERTA DELMONTE CORRADOaand FERNANDO DINIc
aDipartimento per lo Studio del Territorio e delle sue Risorse, Universita ` di Genova, 16132 Genova, Italy, and
bDipartimento di Scienze Ambientali, Universita ` della Tuscia, 01100 Viterbo, Italy, and
cDipartimento di Biologia, Universita ` di Pisa, 56126 Pisa, Italy, and
dAG Zoophysiologie, FB Biologie/Chemie, Universita ¨t Osnabru ¨ck, 49069 Osnabru ¨ck, Germany, and
eDipartimento di Scienze dell’Ambiente e della Vita, Universita ` del Piemonte Orientale, 15100 Alessandria, Italy, and
fDipartimento di Fisica, Laboratorio di Chimica Bioorganica, Universita ` di Trento, and CNR, Institute of Biophysics, 38050 Trento, Italy
ABSTRACT.
ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its
cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin
C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with
E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca21and Na1, suggesting a
generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the
electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling
of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of
phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results
suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite
to face the challenges of the interstitial environment.
Key Words. Action potentials, cation homeostasis, ciliated protists, lysosomes, marine microorganisms, oxidative stress, phagocytosis,
sesquiterpenoids.
Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine,
E
against environmental challenges. Some of these compounds are
known to function as intracellular signal transducers and regula-
tors of complex processes (Blunt et al. 2007; Pietra 1997; Zhang et
al. 2005). The sesquiterpenoids euplotin A, B, and C and their
putative biogenetic precursor preuplotin are a class of lipophilic
metabolites uniquely produced by the strains characterizing the
cosmopolitan, marine, interstitial, ciliate morphospecies Euplotes
crassus (Dini et al. 1993; Guella et al. 1994). The structure of
euplotin C has been elucidated at the absolute configuration level
(Guella, Dini, and Pietra 1996; Guella et al. 1994, 2004). Euplotin
C represents the most abundant secondary metabolite along the
biosynthetic pathway leading to terpenoids in E. crassus, and it is
the most powerful agent among the other related products. In par-
ticular, euplotin C mediates its cytotoxic effects in non-producer
Euplotes strains, including E. vannus, by altering the cell cycle,
ciliary motility, and cell shape, and thus allowing E. crassus to
broaden its niche size (Dini et al. 1993; Guella et al. 1994, Guella,
Dini, and Pietra 1996; Savoia et al. 2004). However, intracellular
signaling mechanisms mediating the ecological role of euplotin C
are largely unknown. In recent years, the spectrum of cytotoxic
actions of euplotin C has been broadened to microorganisms other
than ciliates, including parasitic protists, yeasts, and some bacte-
rial strains (Savoia et al. 2004). In addition, signaling mechanisms
(i.e. Ca21) coupled to the cytotoxic actions of euplotin C have
been elucidated in neuroendocrine tumoural cells of mammals
(Cervia et al. 2006, 2007). A similar scenario may also occur in
the marine ecosystem because intracellular systems in bacteria,
unicellular eukaryotes, and metazoa share common features
(Christensen et al. 1998).
UKARYOTIC, marine microorganisms produce unique sec-
ondary metabolites as adaptative tools to survive and defend
In ciliated protists, the importance of Ca21as an intracellular
mediator has been well established. Indeed, Ca21plays a role in
the regulation of many processes, such as exocytosis/endocytosis
(phagocytic activity), chemosensory responses, ciliary motility/
regeneration, cell contraction, and nuclear migration/mitotic
division (Plattner and Klauke 2001). Ciliated protists are excit-
able cells displaying Ca21-dependent membrane action potentials
(Machemer 1995; Plattner and Klauke 2001). A correlation
between Ca21and Na1homeostasis has been demonstrated in
Euplotes (Burlando et al. 1999; Kruppel 1993; Kruppel and
Lueken 1990). The notion that, in higher eukaryotes, Ca21medi-
ates cell damage has been well established, as well as its cross-talk
with different pathways regulating oxidative stress (Camello-
Almaraz et al. 2006; Orrenius, Zhivotovsky, and Nicotera
2003). In this respect, imbalance in the steady-state of reactive
oxygen species (ROS), mostly formed as side products of oxida-
tive phosphorylation in mitochondria, are deleterious and even
lethal (Ryter et al. 2007). Interaction between Ca21and ROS also
occurs in protists (Ridgley, Xiong, and Ruben 1999). At the
functional level, the stability of lysosomes, the cell organelles
which are involved in compartmentalization and degradation of
nutrients, particulate material, and xenobiotics ingested during
phagocytosis, may be affected by different signals, including
Ca21and ROS (Andrews 2002; Dondero et al. 2006; Plattner
and Kissmehl 2003; Plattner and Klauke 2001; Roussi et al. 2007;
Zhao, Wang, and Zhang 2005). In protists, including Euplotes,
different compounds with ecological activity, including many
toxic substances, appear to influence lysosome homeostasis which
is, therefore, used as a valuable indicator of cellular damage and of
compromisedbioticintegrity(Donderoetal.2006;Triellietal.2007).
In the present study, we investigated intracellular signaling
mechanisms and their functional correlates mediating the ecolog-
ical role of euplotin C in E. vannus as a representative of a sample
of marine, interstitial ciliate diversity. Among Euplotes morpho-
species, which do not produce euplotin C, E. vannus is one of the
most sensitive to euplotin C (Guella et al. 1994, Guella, Dini, and
Corresponding Author: Fernando Dini, Dipartimento di Biologia,
Universita ` di Pisa, via A. Volta 4, 56126 Pisa, Italy—Telephone num-
ber: 139 050 2211399; FAX number: 139 050 2211399; e-mail:
fdini@biologia.unipi.it
365
J. Eukaryot. Microbiol., 55(5), 2008 pp. 365–373
r 2008 The Author(s)
Journal compilation r 2008 by the International Society of Protistologists
DOI: 10.1111/j.1550-7408.2008.00335.x