Table Of ContentLessons in Immunity:
From Single-Cell
Organisms to Mammals
Edited by
Loriano Ballarin
University of Padova, Padova, Italy
Matteo Cammarata
University of Palermo, Palermo, Italy
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List of Contributors
Luigi Abelli University of Ferrara, Ferrara, Italy
Vizzini Aiti University of Palermo, Palermo, Italy
Claudio Alimenti University of Camerino, Camerino, Macerata, Italy
Vincenzo Arizza University of Palermo, Palermo, Italy
Loriano Ballarin University of Padova, Padova, Italy
Silvia Battistella University of Trieste, Trieste, Italy
Kornélia Bodó University of Pécs, Pécs, Hungary
Francesco Buonocore University of Tuscia, Viterbo, Italy
Paola Caicedo CIDEIM, Cali, Valle del Cauca, Colombia
Matteo Cammarata University of Palermo, Palermo, Italy
Laura Canesi University of Genova, Genova, Italy
Heather Coatsworth Simon Fraser University, Burnaby, BC, Canada
Maria R. Coscia Institute of Protein Biochemistry, National Research Council
of Italy, Naples, Italy
Małgorzata Cytryńska Maria Curie-Sklodowska University, Lublin, Poland
Parrinello Daniela University of Palermo, Palermo, Italy
Magda de Eguileor University of Insubria, Varese, Italy
Stefania Domeneghetti University of Padova, Padova, Italy
Francesco Drago University of Lille – Science and Technology,
Villeneuve D’Ascq, France
Péter Engelmann University of Pécs, Pécs, Hungary
Antonella Franchini Modena and Reggio Emilia University, Modena, Italy
Nicola Franchi University of Padova, Padova, Italy
Michela Furlan University of Trieste, Trieste, Italy
François Gagné Emerging Methods Section, Aquatic Contaminants Research
Division, Environment Canada, Montréal, QC, Canada
Marco Gerdol University of Trieste, Trieste, Italy
Stefano Giacomelli Institute of Protein Biochemistry, National Research
Council of Italy, Naples, Italy
Piero G. Giulianini University of Trieste, Trieste, Italy
xiii
xiv List of Contributors
Annalisa Grimaldi University of Insubria, Varese, Italy
Yuya Hayashi Aarhus University, Aarhus, Denmark; Karlsruhe
Institute of Technology (KIT), Karlsruhe, Germany
Teresa Jakubowicz Maria Curie-Sklodowska University, Lublin, Poland
Christophe Lefebvre University of Lille – Science and Technology, Villeneuve
D’Ascq, France
Simonetta Lorenzon OGS (National Institute of Oceanography and Experimental
Geophysics), Sgonico (TS), Italy
Carl Lowenberger Simon Fraser University, Burnaby, BC, Canada
Pierangelo Luporini University of Camerino, Camerino, Macerata, Italy
Davide Malagoli University of Modena and Reggio Emilia, Modena, Italy
Annalaura Mancia University of Ferrara, Ferrara, Italy
Chiara Manfrin University of Trieste, Trieste, Italy
Valerio Matozzo University of Padova, Padova, Italy
Cammarata Matteo University of Palermo, Palermo, Italy
László Molnár University of Pécs, Pécs, Hungary
Parrinello Nicolò University of Palermo, Palermo, Italy
Clara Ocampo CIDEIM, Cali, Valle del Cauca, Colombia
Umberto Oreste Institute of Protein Biochemistry, National Research Council
of Italy, Naples, Italy
Enzo Ottaviani University of Modena and Reggio Emilia, Modena, Italy
Alberto Pallavicini University of Trieste, Trieste, Italy
Maria G. Parisi University of Palermo, Palermo, Italy
Carla Pruzzo University of Genova, Genova, Italy
Giuseppe Scapigliati University of Tuscia, Viterbo, Italy
Domenico Schillaci University of Palermo, Palermo, Italy
Cole Schonhofer Simon Fraser University, Burnaby, BC, Canada
Marco Scocchi University of Trieste, Trieste, Italy
Nidhi Sharma University of Padova, Padova, Italy
Gianluca Tettamanti University of Insubria, Varese, Italy
Adriana Vallesi University of Camerino, Camerino, Macerata, Italy
Gerardo R. Vasta University of Maryland School of Medicine, Baltimore, MD,
United States
Paola Venier University of Padova, Padova, Italy
Jacopo Vizioli University of Lille – Science and Technology,
Villeneuve D’Ascq, France
Iwona Wojda Maria Curie-Sklodowska University, Lublin, Poland
Preface
Animals constitute the greatest part of eukaryotic biodiversity, with more than 2
million known species grouped in approximately 35 phyla.
The exploitation of only a limited part of this great variety of species was
fundamental for the advancement of immunobiology, starting with the original
experiments of Elie Metchnikoff with sea star larvae, which posed the basis for
the phagocytosis theory, up to the recent studies on Toll receptors in Drosoph-
ila by Jules A. Hoffmann, which led to comprehension of the role of Toll-like
receptors in innate immunity.
Animals have evolved a wide range of approaches to cope with foreign, poten-
tially pathogenic organisms, and today, the increasing need to extend our knowledge
of immune responses requires new, suitable, and simple model organisms for the
study of the variety of defense strategies present in metazoans, besides the few spe-
cies investigated so far. This is of general biological interest and might reveal new
adaptive solutions and unknown recognition and effector mechanisms, useful for
further progresses in immunology. It also has an applied interest, as a solid knowl-
edge of invertebrate immunity is fundamental for setting up biological methods to
control invertebrates, which are vectors of diseases or pests for crops. In addition,
a better knowledge of the immune system of farmed species can be of great help
in the optimization of the rearing strategies in cattle breeding and aquaculture.
Furthermore, focusing on mammals only, as most immunologists do, does not allow
for the study of the evolution of immune defense and host–parasites coevolution.
This book stems from the activity of the Italian Association of Develop-
mental and Comparative Immunobiology, born in 1997, which coordinates the
activity of many research teams on the common theme of immune responses in
no mouse, no man models through periodic scientific meetings. We invited part
of its members and other international collaborators with consolidated com-
petence in their respective fields to write on specific aspects of their research.
The outcome is a series of short chapters (lessons), in the form of overviews, in
which a wealth of new and reviewed information, concerning many aspects of
immunity in invertebrates and vertebrates, can be found. The aim is to provide
scientists and teachers with an easy and updated tool reporting, in an evolution-
ary perspective, the state of the art in relevant fields of immunobiology.
We thank all the authors and reviewers for their important contributions and
their patience with the process that, finally, brought this book to fruition.
Matteo and Loriano
xv
Chapter 1
Ciliate Pheromones: Primordial
Self-/Nonself-Recognition
Signals
Adriana Vallesi, Claudio Alimenti, Pierangelo Luporini
University of Camerino, Camerino, Macerata, Italy
INTRODUCTION
Independently of their prokaryotic or eukaryotic nature, cells are evolu-
tionarily driven to socialize to acquire the temporary or stable multicellular
organization necessary to increase their size and engage in functional spe-
cialization and partition of labor.1 This ancient impulse to socialize implies
a very early evolution of the cell’s ability to discriminate between self and
nonself, which is a sine qua non condition for pursuing effective cell–cell
communication, cooperation, and structure–function integration. In its most
basic aspects, this ability is primarily directed to the recognition of self-
signaling molecules, as exemplified by the quorum-sensing phenomena in
which bacteria perceive their environmental population densities and activi-
ties and, consistently, vary their behavior and metabolism.2,3 It then under-
goes vast functional diversification among the unicellular eukaryotes in which
it governs impressive phenomena of cell aggregation based on kin discrimi-
nation in social amoebas.4,5 Its maximal complexity is eventually shown by
animals with the evolution of innate and adaptive immunity systems that are
primarily directed to recognizing and processing nonself molecules for the
defense of body integrity.6,7
This chapter focuses on the biology and structure of diffusible protein
signals, nowadays known as pheromones and earlier as mating-type factors/
substances or gamones, that control self/nonself recognition in protist cili-
ates. Together with Dinoflagellates and Apicomplexa, ciliates (Ciliophora)
form the group of Alveolata, which clusters with those of Stramenopiles
(diatoms) and Rhizaria (foraminifers and radiolarians) into the so-called SAR
supergroup.8
Lessons in Immunity: From Single-Cell Organisms to Mammals
http://dx.doi.org/10.1016/B978-0-12-803252-7.00001-1 1
Copyright © 2016 Elsevier Inc. All rights reserved.
2 Lessons in Immunity: From Single-Cell Organisms to Mammals
PHEROMONE IDENTIFICATION
Ciliate pheromones are the chemical markers of genetically distinct vegetative
cell classes—only two of the same sex in some species or multiple with indefi-
nite numbers in others9,10—that have been described as mating types because
their mixing determines a ciliate-specific mating phenomenon of conjugation.
Ciliate conjugation is a sexual phenomenon as it involves a temporary union in
mating pairs between cells, which mutually exchange gamete nuclei, undergo
fertilization, and develop new micro- and macronuclei from the synkarya. How-
ever, it has nothing to do with the general phenomena of fertilization between
gametes of opposite sexes. One major reason for opposing this parallelism is
that many species of ciliates may form mating pairs between genetically differ-
ent cells (heterotypic pairs), as well as between genetically alike (clonal) cells
(homotypic mating pairs). Although destined to generate self-fertilization, these
homotypic pairs are fully fertile (the Blepharisma case excepted) just like the
heterotypic ones.
It is precisely by virtue of this unique capability of ciliates to form homo-
typic (intraclonal or selfing) mating pairs that ciliate pheromones were iden-
tified more than half a century ago.11 By investigating mating interactions in
Euplotes patella—the first Euplotes species used to study the Mendelian genet-
ics of ciliate multiple mating systems controlled by a series of alleles codomi-
nantly expressed at a single genetic locus (annotated as mat locus)—Kimball11
observed that an experimental condition sufficient to induce the formation of
homotypic mating pairs was the simple suspension of cell cultures with cell-free
filtrates from other cultures of different mating types. From this observation it
became evident that in E. patella, and ciliates in general, the mating-type factors
(pheromones) can be freely released into the extracellular environment and that
the presence of these molecules in solution can be promptly detected by assay-
ing the mating-induction activity of cell-free filtrates.
By applying this mating-induction assay to cell-free filtrates, pheromones
have been identified in species of Blepharisma, Dileptus, Oxytricha, Ephelota,
and Tokophrya in addition to other Euplotes species (Refs 12, 13, as reviews).
However, studies of structure–function characterization have been carried out
to varying degrees of complexity only on pheromones isolated from Blepha-
risma japonicum, Euplotes raikovi, Euplotes octocarinatus, Euplotes nobilii,
and Euplotes crassus. These studies are reviewed in this chapter with particular
attention to the cross-reactions that some Euplotes pheromones show with the
signaling system components of more modern organisms.
PHEROMONE STRUCTURES
The first two pheromones to be purified and structurally characterized belong
to the pink-colored freshwater ciliate, B. japonicum. Although they are of two
mating types (I and II) defined as “complementary,” of which nothing is known
about their genetic determination, their molecular structures are chemically
Ciliate Pheromones Chapter | 1 3
unrelated. The pheromone isolated from the type-I cells (originally designated
as gamone 1 or blepharmone) is an unstable glycoprotein of 272 amino acids
and 6 N-linked sugars.14 It is particularly rich in polar and aromatic amino
acids (Asn 10.3%, Ser 10.3%, Tyr 8.1%, Trp 5.1%), synthesized in the form of
a cytoplasmic precursor, and active in inducing mating between type-II cells at
picogram concentrations. In contrast, the second pheromone distinctive of type-
II cells (also designated as gamone 2 or blepharismone) is a very stable tryp-
tophan derivative, namely a calcium-3-(2′-formyl-amino-5′-hydroxybenzoyl)
lactate.15 This has also been obtained by chemical synthesis and is active at
nanomolar concentrations in attracting type-I cells besides those for inducing
their mate.16
Quite different is the picture that emerges for the pheromone structures deter-
mined in four species of Euplotes, namely E. raikovi, E. nobilii, E. octocarinatus,
and E. crassus. In all these species, pheromones have been shown to form species-
specific families of structurally homologous proteins. Among these pheromone
families, a more in-depth structural knowledge has been obtained for E. raikovi
and E. nobilii, which secrete comparatively larger amounts of pheromones. Up
to 200 μg of pure protein per liter can be recovered from E. raikovi cell-free fil-
trates.17 This has greatly facilitated chemical and genetic approaches to the deter-
mination of significant numbers of pheromone primary amino acid sequences.
It subsequently opened up the way to NMR and crystallographic analyses of the
pheromone three-dimensional structures based on the use of proteins at a natural
isotope composition purified from cell-free filtrate preparations.
In E. raikovi, nine distinct amino acid pheromone sequences in their form of
cytoplasmic precursors (pre/propheromones) and six three-dimensional struc-
tures of the secreted molecules are known18–24 (Fig. 1.1). In contrast to the tight
structural conservation of the strongly hydrophobic presegment (or signal pep-
tide) of 19 amino acids and (to a slightly lesser extent) of the prosegment of
16–18 amino acids, the secreted pheromones of 37–40 amino acids (51 only in
pheromone Er-23) show minimal sequence conservation. Only the positions of
six cysteines are fully conserved, and the sequence dissimilarity between any
two pheromones may be as high as 74%. In any case, despite this wide vari-
ability in the amino acid sequence, all the pheromones (pheromone Er-23 in
part excepted) mimic one another in their three-dimensional conformation. This
is based on a fold of three nearly parallel, right-handed α helices (increased to
five with the addition of two single-turn helices in Er-23) that remain tightly
associated with each other by the close proximity and strategic disposition of
their disulfide bonds. As is usually required for waterborne signaling molecules
that must maintain long-term activity in the environment, the stability of the
globular conformation of E. raikovi pheromones is such that even heating these
proteins up to 95°C does not cause unfolding and disruption of their secondary
structures.25 However, in spite of showing closely comparable molecular archi-
tectures and equivalent stabilities, each pheromone has its own unmistakable
cell type structural earmarks that are likely deputed to confer specificity to its
4 Lessons in Immunity: From Single-Cell Organisms to Mammals
(cid:11)(cid:36)(cid:12)
(cid:11)(cid:37)(cid:12)
FIGURE 1.1 Euplotes raikovi pheromone family. (A) Alignment of nine distinct sequences of
pheromone precursors, as deduced from their DNA-coding sequences. Sequence alignment is based
on the Clustal W algorithm and optimized manually by gap insertions. Residues conserved among
all sequences, or all but one sequence, are shadowed. The positions of the cysteine residues are
highlighted in bold. The numbers of residues of the pre, pro, and secreted regions are reported on the
right of the sequences. (B) Ribbon presentations (as visualized by the program MOLMOL) of the
six pheromone structures (side view) that have been determined by NMR analysis of native protein
preparations. The Protein Data Bank codes are as follows: 1erc (Er-1), 1erd (Er-2), 1erp (Er-10),
1ery (Er-11), 1hd6 (Er-22), and 1ha8 (Er-23). The amino and carboxyl termini of each molecule are
labeled N and C, respectively, and the sulfur atoms involved in the disulfide bonds are indicated as
spheres. The three more regular α-helices common to all six molecules are labeled h1–h3 starting
from the N-terminus.
Ciliate Pheromones Chapter | 1 5
(cid:11)(cid:36)(cid:12)
(cid:11)(cid:37)(cid:12)
FIGURE 1.2 Euplotes nobilii pheromone family. (A) Alignment of seven distinct sequences
of pheromone precursors, as deduced from their DNA-coding sequences. (B) Ribbon presentations
(as visualized by the program MOLMOL) of the four pheromone structures that have been deter-
mined by NMR analysis of native protein preparations. The Protein Data Bank codes are as follows:
2nsv (En-1), 2nsw (En-2), 2jms (En-6), and 2kk2 (En-A1). Indications and symbols as in Fig. 1.1.
signaling activity. These earmarks are imposed by the evolution of variations
that are most apparent for the shape, extension, and spatial arrangement of the
carboxy-terminal domain, as well as for the geometry of the interhelix loops of
the molecules.
The overall structural organization of E. nobilii pheromones strictly matches
the E. raikovi pheromone organization with the close phylogenetic kinship that
links these two species.26,27 Nevertheless, E. nobilii pheromones, for which
seven amino acid sequences of the precursor forms28 and four NMR struc-
tures have been determined29–31 (Fig. 1.2), have longer sequences (from 52
to 63 amino acids), include eight instead of six cysteines, and, more impor-
tantly, show structural specificities that appear closely correlated with the dif-
ferent ecology that distinguishes E. nobilii from E. raikovi. While E. raikovi
thrives in temperate waters, E. nobilii has colonized the Antarctic and Arctic
seas.32,33 Hence, E. nobilii pheromones are cold-adapted, psychrophilic pro-
teins. With respect to E. raikovi pheromones, their overall contents of polar
