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Characterization and Function of the First Antibiotic
Isolated from a Vent Organism: The Extremophile
Metazoan
Alvinella pompejana
Aure
´lie Tasiemski
1
*, Sascha Jung
2
,Ce
´line Boidin-Wichlacz
1
, Didier Jollivet
3
, Virginie Cuvillier-Hot
1
,
Florence Pradillon
4
, Costantino Vetriani
5
, Oliver Hecht
2
, Frank D. So
¨nnichsen
6
, Christoph Gelhaus
7
,
Chien-Wen Hung
8
, Andreas Tholey
8
, Matthias Leippe
7
, Joachim Gro
¨tzinger
2
, Franc¸oise Gaill
9
1Universite
´de Lille1-CNRS UMR8198, Laboratoire GEPV, Ecoimmunology of Marine Annelids (EMA), Villeneuve d’Ascq, France, 2Institute of Biochemistry, Christian-
Albrechts-Universita
¨t, Kiel, Germany, 3Universite
´Pierre et Marie Curie-CNRS UMR7144, Laboratoire AD2M, Adaptation et Biologie des Inverte
´bre
´s en Conditions Extre
ˆmes
(ABICE), Station Biologique, Roscoff, France, 4IFREMER, Centre de Brest, REM/EEP/LEP, Plouzane
´, France, 5Department of Biochemistry and Microbiology and Institute of
Marine and Coastal Sciences, Rutgers University, New Brunswick, New Jersey, United States of America, 6Otto Diels Institute for Organic Chemistry, Christian-Albrechts-
Universita
¨t, Kiel, Germany, 7Institute of Zoology, Zoophysiology, Christian-Albrechts-Universita
¨t, Kiel, Germany, 8Division of Systematic Proteome Research, Institute for
Experimental Medicine, Christian-Albrechts-Universita
¨t, Kiel, Germany, 9Universite
´Pierre et Marie Curie-Muse
´um National d’Histoires Naturelles CNRS BOREA IRD, Paris,
France
Abstract
The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It
relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to
thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic
peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the
peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression
effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first
line of defense against microbial invasion, alvinellacin shapes and controls the worm’s epibiotic microflora. Our results
provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile
eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier
studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this
interaction from the host side.
Citation: Tasiemski A, Jung S, Boidin-Wichlacz C, Jollivet D, Cuvillier-Hot V, et al. (2014) Characterization and Function of the First Antibiotic Isolated from a Vent
Organism: The Extremophile Metazoan Alvinella pompejana. PLoS ONE 9(4): e95737. doi:10.1371/journal.pone.0095737
Editor: Ramy K. Aziz, Cairo University, Egypt
Received December 12, 2013; Accepted March 30, 2014; Published April 28, 2014
Copyright: ß2014 Tasiemski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the CNRS, the MSER, the Universite
´de Lille1 (BQR 2012), the Re
´gion Nord Pas-de-Calais (Emergent 2012) and the
Fondation pour la Recherche sur la Biodiversite
´(VERMER 2013). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: aurelie.tasiemski@univ-lille1.fr
Introduction
Alvinella pompejana is a polychaetous annelid that inhabits active
deep-sea hydrothermal vents along the East Pacific Rise, where it
colonizes the walls of actively venting high-temperature chimneys
[1]. The environment of the worm is characterized by extreme
physicochemical gradients, high pressure and bursts of elevated
temperatures which can be as high as 105uC [2]. To date, the
Pompeii worm is considered as one of the most eurythermal and
thermotolerant metazoan known on Earth [3–6].
One of the striking features of this annelid is its association with
a unique epibiotic bacterial community that forms cohesive hair-
like projections from mucous glands lining the dorsal interseg-
mental spaces [3]. Numerous studies, including metagenomic
analyses, evidenced that the microflora is composed of a
multispecies complex of 12 to 15 phylotypes of which .98% are
Epsilonproteobacteria, a dominating taxonomic group in hydro-
thermal vents [7]. These bacteria have been suggested to provide
Alvinella with a stable source of nutrients and may detoxify the
environment of the worm from reactive heavy metals and free
hydrogen sulfide [1].
Central theme in beneficial bacterial-host interaction is that
hosts must protect themselves against inappropriate colonization
and replication of the symbiotic flora [8]. Various mechanisms are
employed to control the symbionts without compromising host
vitality. Amongst them, beneficial partnership between symbiotic
bacteria and the immune reactions of the host has been widely
invoked in mammals and insects [8]. The molecular interactions
between the two partners of the association seem to modulate host
immunity, and in turn the immune system shapes the composition
of the microbiota.
Antimicrobial peptides (AMPs) are small sized molecules
naturally produced by bacteria, protists, fungi, plants and animals.
Their large distribution in nature within both unicellular and
multicellular organisms suggests that they are crucial immune
effectors which presumably have evolved under positive selection
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for a long period of time [9]. Recently, Login et al demonstrated
that coleoptericin A, an AMP produced by the beetle belonging to
the Sitophilus genus, keeps endosymbionts under control within the
bacteriocytes [10]. By comparison with the number of AMPs
isolated from terrestrial invertebrates (<1500), relatively few
AMPs (<40) have been characterized from marine organisms
[11]. Yet, marine animals are permanently in close contact with
very high densities of microbes (10
5
to 10
7
per ml) suggesting that
their immune effectors are effective in microbial growth inhibition
and killing [12]. Although AMPs have been found in numerous
marine invertebrate taxa such as Cnidarians, Annelids, Mollusks,
Arthropods, Tunicates and Echinoderms [11], there is no evidence
of active AMPs in organisms living in the deep-sea. To date, the
aspect of AMP coevolution under selective pressures associated
with the abyssal environment has never been investigated, whereas
many life forms, in such an extreme habitat, rely on a symbiotic
association.
Here, we describe the nature of the first abyssal AMP found in a
symbiotic animal, its common origin with AMPs of coastal
annelids as well as the manner in which an extremophile eukaryote
uses it to interact with the particular microbial community of the
hydrothermal vent ecosystem.
Material and Methods
Biological materials
Animal collection. Alvinella pompejana were collected from the
bio9 and P Vent sites (EPR 9u509N, 2.500 m depth) on board of
the R/V L’Atalante using the telemanipulated arm of DSV
Nautile (MESCAL Cruises 2010, 2012). Animals were brought
back to the surface inside an insulated basket and directly dissected
upon recovery. Although not subjected to specific property
regulations (international water areas), authors have obtained
permission to use samples for any analysis from both chief-
scientists. This study did not involve endangered or protected
species.
Primary cell culture. Freshly harvested coelomic cells were
cultured in Leibovitz L-15 medium under sterile conditions on
board. For microbial treatment, cells were separately incubated in
500 mL of medium containing 10 mL of killed bacteria, for 12 h.
Incubations without bacteria were performed under the same
conditions as controls.
Microorganisms. The bacterial strains used in this study are
listed in S.1 in Material and Methods S1. Epibionts were scraped
with a thin razor from 1 cm
2
of the tegument of Alvinella freshly
harvested and were suspended in 4 mL of sterile seawater.
Primary enrichment cultures were obtained shipboard by adding
an aliquot of epibionts or fragments of Alvinella tubes to 10 mL of
modified SME media prepared as previously described and
followed by incubation at 30 and 50uC [13], [14].
Peptide purification and identification
A purification guided assay (see S.2 in Material and Methods S1
for details) was performed from the coelomic liquid of Alvinella.
After three steps of chromatography (Reverse Phase-HPLC), the
purity of the antimicrobial fractions was assessed by mass
spectrometry (MS) analyses (DE STR PRO; Applied Biosystems)
and homogeneous material was subjected to protein sequencing
via Edman degradation (pulse liquid automatic peptide sequena-
tor, Beckman Coulter).
Three dimensional structures
NMR spectroscopy. (see S.3 in Material and Methods S1 for
details). The renaturated alvinellacin was submitted to NMR
measurements on a Bruker Avance III 800 MHz spectrometer.
The chemical shift data were deposited in the University of
Wisconsin Biological Magnetic Resonance Bank database under
the accession number 18085. All spectra were processed with the
program NMRPipe [15] and analyzed with the program
NMRView [16]. Models of the three dimensional structures of
capitellacin were generated using the solution NMR structure of
alvinellacin as template. Structure calculations were performed
using the program CYANA [17]. The 10 best structures were
selected as the final structural ensemble and were deposited (PDB
accession code 2LLR).
Assignment of disulfide bridges in alvinellacin by Mass
Spectrometry. (see S.4 in Material and Methods S1 for details)
Alvinellacin was incubated with the endoproteinase Lys-C. For
sequential Lys-C and tryptic digestion, trypsin was added to the
Lys-C digestion. During the incubation, aliquots were taken from
the digest at different time points in order to monitor the enzyme
digestion efficiency. MS experiments were performed on an offline
nanoESI-LTQ Orbitrap Velos mass spectrometer with ETD
option (Thermo Fisher Scientific, San Jose, CA). Spectrum
interpretation and disulfide bridge assignment were performed
manually.
Alvinellacin activities
Antimicrobial assays. The minimal inhibitory concentra-
tion (MIC) and minimal bactericidal concentration (MBC) of the
synthetic peptide (diluted in acidified water 0.05% acetic acid)
against bacterial growth were determined by a microdilution
susceptibility assay in microtiter plates as previously described
[18]. Permeabilization of membranes of viable bacteria and pore-
forming activity towards liposomes were measured as previously
described in details [19], [20]. Alamethicin, cecropin P1, and
magainin II were purchased as synthetic peptides from Sigma.
Shipboard antimicrobial assays against
epibionts. Epibionts were scrapped with a thin razor from the
tegument of freshly harvested Alvinella. They were incubated in
presence or absence (control) of the alvinellacin peptide for 4 h
and were subsequently fixed in 3% glutaraldedyde on board of the
ship. In the laboratory, samples were directly placed on copper
grids and counterstained with uranyl acetate and lead citrate.
Morphological changes on epibionts were detected and damaged
versus intact bacteria were counted on a Hitachi H 600 electron
microscope.
Gene characterization and gene expression
The nucleotidic sequence coding the preproalvinellacin precursor
was obtained by blasting the amino acid sequence of Alvinellacin
to the Alvinella EST database (TERA 00513) [21].
Gene structure
nellacin (Genbank accession number KJ489380) was obtained
from the cloning of PCR-products coming from the nested
amplification of a series of A. pompejana gDNA using specific
primers targeted on the 59and 39ends of the cDNA. Used primers
are as followed: AP_alvinellacinF starting from the first methio-
nine codon: 59-ATG ACG TAT TCT GTA GTT GTG ACG
CTG GTC-39, AP_alvinellacinR1 (in the 39UTR region): 59-TAG
GCA GGA CGG AGC CGC CAG ATC A-39, and AP_alvi-
nellacinR2 (starting on codon stop): 59-CTC AGT GAA ATG
AAG CAG GTG AGT TAT G-39. PCR amplifications were
obtained following 40 cycles of 96uC for 45 s, 60uC for 45 s and
72uC for 4 min after a first denaturation of gDNA at 96uC for
4 min and a final elongation of 10 min. Putative splicing sites
(ACEs and ISEs) and both mobile and regulatory elements were
detected using ACESCAN2 web server (http://genes.mit.edu/
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.The complete gene sequence of the preproalvi-
acescan2/index.html) and modules of the geneinfinity (http://
www.geneinfinity.org/sp/sp_coding.html), webgene (http://www.
itb.cnr.it/webgene/) and the TE tools (ergmanlab.smith.man.a-
c.uk/?page_id = 295) platforms. The complete gene sequence of
preprocapitellacin was obtained by blasting the preproalvinellacin
in the Capitella teleta genome database (http://genome.jgi-psf.org/
Capca1/Capca1.home.html).
Quantitative Reverse Transcription PCR. RNA from cells
were extracted (Qiazol, Qiagen) and used for cDNA synthesis with
an oligodT according to the protocol of the manufacturer
(SuperScript II; Invitrogen). The primers used for quantification
were designed with the Primer3 Input software (http://frodo.wi.
mit.edu/cgi-bin/primer3/primer3 www.cgi).
-Alvinellacin primers: forward: 59-TGACATCGTGAAG-
GAACTCG-39; reverse: 59-CCGTTCCTACCAACTTTCCA-39
-Ribosomal Protein 26S primers (Alvinella database [21]:
TERA01523): forward: 59-CCGGCTAGTTCAAGATGACC-
39; reverse: 59-AGCTGCTGCCTCCACTATGT-39.
The RP26S was used as the reference gene. Real Time
reactions were conducted on a CFX96 qPCR system (BioRad)
using a hot start, then 40 cycles at 94uC, 15 s; 56uC, 30 s; 72uC,
30 s., and a final extension step at 72uC for 3 min. Analysis of
relative gene expression data was performed using the DDCt
method. For each couple of primers, a plot of the log cDNA
dilution versus DCt was generated to validate the qPCR
experiments (data not shown). Reference and target were
amplified in separated wells.
Alvinellacin production sites
Polyclonal antiserum. The alvinellacin antiserum was
raised in two New Zealand White rabbits (Saprophyte pathogen-
free). The chemically synthesized peptide was coupled to OVA
and used for the immunization procedure according to the
protocol described previously [22]. The reactivity of the antibody
was tested by Dot Immunobinding Assay (DIA) using 1 mL of the
RP HPLC fractions [23].
Immunocytochemistry and immunohistochemistry. Cells
or tissues were fixed on board in 4% paraformaldehyde. Later, the
SHANDON Cytospin 3 was used to spin cell suspension onto poly-
lysine slides (8 min, 2,000 rpm). Immunocytochemistry and immu-
nohistochemistry were performed with the rabbit anti-alvinellacin
(1:100) and the FITC-conjugated anti-rabbit secondary antibody
(1:100; Jackson Immunoresearch Laboratories) according to a
protocol already described by our group [21]. Samples were
examined using a confocal microscope (Zeiss LSM 510).
Coelomocyte structure. The coelomocytes were collected,
and immediately fixed in 3% glutaraldehyde according to the
protocol previously described [24]. Coelomocytes were observed
on a Hitachi H 600 electron microscope.
Results and Discussion
Nature of Alvinella AMP and evolutionary link with AMPs
from coastal species
To date, only one AMP isolated from marine species belongs to
an AMP family already characterized in terrestrial species [11].
Figure 1. Alvinellacin is evolutionary linked to a family of AMPs from coastal annelids. (A) Molecular organization of the alvinellacin
precursor and sequence alignment of alvinellacin with arenicin, an AMP produced by Arenicola marina, and capitellacin, a sequence so named by our
group, issued from the analysis of the Capitella teleta genome. N and C denote N- and C-termini. (B) Comparison of the three-dimensional structures
of the three AMPs. Disulfide bonds are depicted as yellow balls and sticks.
doi:10.1371/journal.pone.0095737.g001
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The majority of marine AMPs presents novel structures and is
confined to certain taxa or even species, as observed for AMPs of
polychaeta [25]. In order to identify peptides with antibiotic
activity from the Pompeii worm, a biochemical approach was
combined with the analysis of the Alvinella EST database [22]. The
anatomy of annelids is characterized by the presence of a coelom,
a compartment that includes mobile cells, named coelomocytes
that sterilize the coelomic fluid by releasing humoral factors such
as antimicrobial peptides (AMPs) [26]. We purified and identified
a cationic peptide composed of 22 amino-acid residues which we
named alvinellacin, from the coelomocytes of Alvinella (Figures S1
and S2). As for most AMPs from all invertebrate phyla, mature
alvinellacin is processed from a larger precursor molecule
containing a signal peptide and an anionic proregion [27]
(Figure 1A).
Following a BLAST search, the mature peptide did not display
any similarity in its primary structure with other known proteins.
However, the proregion had ,33% identity to the proregion of
AMPs from coastal annelids: arenicin from Arenicola marina and
capitellacin, a putative peptide inferred from the genome sequence
of Capitella teleta (Figure S3). Pfam analysis of the proregions
revealed the presence of a conserved BRICHOS domain. So far,
this 100 amino acids domain has never been reported in other
AMP precursors than preproarenicin [28].
Despite the lack of an obvious similarity between the primary
structures of alvinellacin, arenicin and capitellacin, we analyzed
and compared their three-dimensional structures. Using NMR
spectroscopy and mass spectrometry, we determined the tertiary
structure of alvinellacin (Figures S4 and S5, Tables S1 and S2) and
compared it to the solved and predicted structures of arenicin and
capitellacin, respectively (Figure 1B). Alvinellacin and capitellacin
are stabilized by two disulfide bonds, whereas arenicin possesses
only one cystine. Like capitellacin, alvinellacin folds into a double-
stranded antiparallel beta-sheet resembling the structure of
arenicin [29]. Consequently, the three AMP precursors i.e.
preproalvinellacin, preprocapitellacin and preproarenicin harbor
the conserved pattern of almost all the BRICHOS containing
proteins: a hydrophobic domain (here, the signal peptide), a linker
region, the BRICHOS domain itself and a C terminal region with
b-sheet propensities (here, the AMP) (Figure 1A) [28].
To date, proregions of AMP precursor are essentially known to
be implicated in cell chemotaxy and/or protection against the
cytotoxic activities of certain AMPs [30]. The BRICHOS domain
has been found as a constituent of proteins associated with a wide
variety of human diseases such as dementia, respiratory distress
and cancer [31].
Recent data evidence that BRICHOS participates in the
complex post-translational processing of proteins, and functions
as an intramolecular chaperone domain that can bind bhairpin
motifs and prevents them from bsheet aggregation and amyloid
fibril formation [28]. Because of their strand-loop-strand structure,
it seems reasonable that alvinellacin like the two other AMPs
interacts with BRICHOS. Coastal and, even more, hydrothermal
annelids are naturally submitted to strong hypoxic and thermal
stresses. We hypothesize that the presence of the BRICHOS
domain might be an evolution-driven adaptation of the worms to
warrant the correct folding of their AMP under extreme
conditions such as hypoxia and/or eurythermality. All these
suggestions should be experimentally tested: BRICHOS might
also have a novel function in A. pompejana that remains
undiscovered.
As a conserved gene structure constitutes a convincing evidence
for evolutionary relatedness between protein families, we also
characterized the complete gene sequence of alvinellacin and
compared it to the capitellacin gene [32] (Figure S6). Both genes
display a 5 introns/6 exons structure with nearly all conserved
intron-splicing positions. Given the taxonomic position of Capitella
and Alvinella [33], their gene structure along with the proregion
sequence identity and the three-dimensional peptide structure,
strongly indicate that alvinellacin and capitellacin presumably
together with arenicin, share an ancient origin and are evolution-
ary correlated since hundred millions of years. Further detailed
comparisons of the proregions showed a high level of amino acid
changes in the first part of the propieces; that may be also
attributable to an adaptive ‘hot spot’ of mutations (functional
change) in the face of the very long period of time since divergence
between the two polychaeta species. The low amino acid
Figure 2. Bactericidal activity of alvinellacin. (A) Membrane
permeabilization of viable bacteria induced by alvinellacin compared
with that induced by other antimicrobial peptides. The percentage of
bacteria with compromised membranes was monitored fluorometrically
using the membrane-impermeable SYTOX green dye. Bacteria were
incubated with varying concentrations of alvinellacin, cecropin P1 and
magainin II at pH 7.4. Induction of membrane permeabilization was
monitored against B. megaterium after 10 min (closed symbols) and
against E. coli after 2 h (open symbols). (B) Time course of pore
formation induced by alvinellacin. The dissipation of a valinomycin-
induced diffusion potential in vesicles of asolectin after addition of
alvinellacin (0.5 nmol), control peptide alamethicin (0.1 nmol), and the
peptide solvent (0.05% acetic acid) were recorded. Pore-forming activity
is reflected by the increase of fluorescence as a function of time. The
arrow marks the time point at which the peptide is added.
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conservation in the AMP sequences compared to the proregions
suggests that they might have evolved independently. To the best
of our knowledge, AMP proregions are not known to interfere with
components of the external environment as AMPs do by
interacting with microbes. Thus, the mature AMP presumably
evolved to respond to the specific microbial communities
(hydrothermal or coastal habitats) as well as to the specific lifestyle
(symbiotic or not) of the worms while the proregion did not. This is
consistent with the observation that the C-terminal propeptides of
the interstitial collagen of Alvinella and Arenicola are similar, while
the helical domain of the mature protein, which is located in the
extracellular matrix and is presumably more exposed to environ-
mental conditions, is not. [34].
Alvinellacin in the first line of defense towards
environmental microbes
In general, microbial invasion into the host causes bacterial
infection which prompts an immune response such as the release
of AMPs to eliminate invaders. Since alvinellacin was isolated
from the coelomocytes, these cells are likely to produce and secrete
the AMP into the coelomic fluid where it exerts its antibacterial
activities. The presence of a signal peptide in the alvinellacin
precursor (Figure 1A) together with the results obtained by
immunocytochemistry (see below) corroborates this assumption.
The antimicrobial activity of alvinellacin was then evaluated
(Figure 2 and Table 1). As the worm’s coelomic fluid composition
is not very different from seawater, assays were performed at salt
concentrations mimicking this environment [35]. Under these
conditions, alvinellacin’s activity was constant primarily against
Gram-negative bacteria. This may represent an adaptation of the
worm to its associated microorganisms, which have been shown to
be predominantly Gram-negative e-proteobacteria [3,36]. We
then wondered whether an exposure to various microorganisms
might have differential impacts on the synthesis of alvinellacin.
Usually, to investigate the immune response of an organism,
animals are submitted to experimental infections and variations of
immune markers are quantified. Since Alvinella precludes in vivo
investigation because of lethal decompression effects upon
sampling [6,37], we developed an ex vivo model by establishing
primary culture of cells obtained from freshly harvested animals.
Despite being subjected to 250 bars decompression, cells were
alive and morphologically preserved (Figure 3A). Primary cell
cultures were then initiated and maintained on board. To mimic a
systemic infection, coelomocytes were incubated in presence of
either Alvinella epibionts or of different vent bacterial strains
(Figure 3B). Quantitative RT-PCR experiments showed a selective
induction of the gene encoding alvinellacin upon exposure of the
worm to the vent bacteria. Both epibionts, which are highly
represented by e-proteobacteria [7], and bacterial enrichments
obtained shipboard from Alvinella tubes using culture conditions
that support growth of vent e-proteobacteria, appear to be better
inducers than pure cultures of c-proteobacteria. These results
show that Alvinella coelomocytes can sense different microorgan-
isms and that alvinellacin synthesis might be the outcome of the
adaptation of Alvinella’s immune defense system against the specific
microorganisms present in its environment. To date, the role of
archaea in activating the host’s immune system and the ability of
its immune receptors to detect their presence has never been
investigated. Interestingly, Alvinella tubes are inhabited by an
extremely dense population of archaea related to the Thermo-
coccales, including members of the two major genera, Thermococcus
and Pyrococcus, the former being more prevalent in Alvinella tubes
than the latter [38,39]. We investigated the ability of these
hyperthermophilic microbes to induce the expression of the
alvinellacin gene in our cell cultures. Remarkably, only archaea
belonging to the Thermococcus genus induced the expression of the
alvinellacin gene. These data indicate that Alvinella can selectively
recognize specific archaea and in turn induces an immune
response, suggesting for the first time the existence of pattern
recognition receptors in an eukaryote organism able to recognize
and discriminate archaeal microbe-associated molecular patterns
[40].
Alvinellacin controls and shapes the epibiotic flora
While a key role of AMPs in fighting infections is well described,
very recent studies also evidenced that these effector molecules can
be employed to regulate/control the symbiotic microflora [10,30].
Table 1. Antimicrobial activity of alvinellacin.
MIC, mM MBC, mM
Gram-negative bacteria
Escherichia coli D31 0.012–0.024 0.048
Escherichia coli D31 (300 mM NaCl) 0.012–0.024 0.048
Escherichia coli D31 (500 mM NaCl) 0.012–0.024 0.048
Pseudomonas sp.* 0.001–0.003 0.012
Vibrio diabolicus* 0.048–0.096 .0.19
Vibrio MPV19 0.012–0.024 0.024
Gram-positive bacteria
Bacillus megaterium 0.012–0.024 0.024
Bacillus megaterium (300 mM NaCl) 0.024–0.048 0.048
Bacillus megaterium (500 mM NaCl) 0.048–0.096 0.096
Staphylococcus aureus 0.048–0.096 .0.19
Assays were performed against bacteria routinely used for antimicrobial assays or having a medical incidence, and against the scarce hydrothermal strains (asterisk*)
cultivable under the conditions of a microbial assay. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) are expressed as final
concentration in mM. .denotes no activity detected at the given concentration. The MBC and MIC values are the same, indicating that the bacterial growth inhibition is
due to the killing of bacteria.
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The strong and vital relationship between Alvinella and its epibionts
prompted us to investigate such an alternative function of
alvinellacin. Immunohistochemistry experiments showed that
alvinellacin is expressed constitutively by epithelial cells of the
tegument associated with the epibiotic microflora, i.e. the dorsal
but not the ventral epidermis (Figures 4A vs 4B). This observation
supports the idea that alvinellacin may prevent bacterial entrance
and/or keep epibionts under control. Accordingly, we determined
the antimicrobial potency of alvinellacin against epibionts
(Figure 5). As epibionts have not been cultivated so far, we carried
out a shipboard antimicrobial assay aimed at detecting epibiont-
cell damage in response to exposure to alvinellacin. Interestingly,
alvinellacin significantly targeted epibionts that correspond to
filamentous bacteria (epibiont types 5 to 9). In particular,
alvinellacin killed 100% of the two most abundant morphotypes
within this group (types 6 and 7). In contrast, the presence/
absence of alvinellacin on epibionts types 1 and 4 did not have
distinguishable effects and other epibionts (types 2 and 3) are not
affected by alvinellacin. Altogether, the data suggest that
alvinellacin controls epibiosis by selectively killing the most
dominant part of the filamentous bacteria found on the dorsal
part of the worm.
That is reminiscent of the role of the defensin HD5 in shaping
the composition of the symbiotic microflora of the digestive tract in
Figure 3. Selective induction of the gene encoding alvinellacin in cœlomocytes exposed to vent bacteria. (A) Electron-microscopic
image showing the intact structure of Alvinella cœlomocytes despite 250 bars decompression. C: cœlomocytes. H: blood cells. (B) RT-qPCR on primary
culture of coelomocytes incubated in the presence (t = 12 h) or not (control) of Alvinella epibionts, enrichment cultures obtained from Alvinella
samples, and different pure cultures of bacteria and archaea isolated from hydrothermal vents. Graphics represent the results of two independent
experiments; p-values from Student’s tests were calculated versus the control treatment, based on the experimental measures performed in triplicates
(*p,0.05).
doi:10.1371/journal.pone.0095737.g003
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mammals [41]. We hypothesize that high production of
alvinellacin by epidermal cells selects and shapes the epibiotic
microflora and prevents microbiota from over proliferating and
subsequently penetrating the underlying tissue. To test this
hypothesis, an accidental invasion was simulated by incubating
coelomocytes with epibionts (Figure 4C). The distribution of
alvinellacin-immune reactivity was compared by immunofluores-
cence in unchallenged versus challenged cells. Under basal
conditions (t = 0), the AMP was strongly detectable inside the
cells, suggesting that this active compound is stored after synthesis.
One hour after the bacterial infestation, the immune staining
inside the cells faded, evidencing that alvinellacin is secreted
rapidly when the cells are challenged by microorganisms. The
induction of transcription observed by RT-qPCR in the cells
incubated for 12 h with epibionts probably contributes to the
renewal of the alvinellacin peptide stock (Figure 3B). Overall, these
Figure 4. Alvinellacin is produced by tissues or cells in contact with epibionts. (A) Picture of Alvinella showing the distribution of epibionts.
(B) Immunohistochemistry data evidence that alvinellacin peptide accumulates in tissue hosting epibionts i.e. the dorsal but not the ventral
tegument. (C) Accidental entrance of epibionts stimulates the secretion of alvinellacin by circulating cells. Images of immunodetection of alvinellacin
in coelomocytes incubated with epibionts. After one hour of exposure, the signal was reduced evidencing an extracellular secretion of the peptide.
Control is performed with preimmune serum 1: FITC fluorescence, 2: transmission, 1+2: overlay.
doi:10.1371/journal.pone.0095737.g004
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results reinforce the role of alvinellacin in keeping symbionts under
control.
Conclusion
Altogether, the data indicate the production of an original AMP
from a deep-sea animal that endorses a durable relationship with
Epsilonproteobacteria and possibly archaea in the face of the
hostile vent habitat. Alvinellacin appears to act as a first line of
defence against microbial invasion. The specificity of the gene
induction along with the selective anti epibiotic activity and the
expression in tissues exposed to the environment suggest that
alvinellacin is actively participating in the surveillance of the
epibiotic community. The conservation of the proregion and the
gene structure of alvinellacin with AMPs of coastal annelids,
suggest a common origin of the molecules. To draw a decisive
conclusion regarding the gene evolution of alvinellacin, we plan to
search for related genes in more than 30 annelid species living in
various habitats. Such phylogenetic analysis will aim at determin-
ing whether the amino acid sequences of the antimicrobial part of
the precursor diverged between species in order to face (i)
contrasted temperatures, (ii) different microbial environments
and/or (iii) to allow the establishment of epibioses.
Supporting Information
Figure S1 Alvinellacin purification and molecular iden-
tification. Material eluting at 60% acetonitrile (ACN) upon solid
phase extraction was loaded onto a C18 column (25064 mm,
Vydac). Elution was performed with a linear gradient of
acetonitrile in acidified water (dotted line), and absorbance was
monitored at 225 nm. Each individually collected fraction was
tested for its antimicrobial activity (white bar) and its immunore-
activity to the alvinellacin Ab by DIA (grey bar). Fractions
containing antimicrobially active alvinellacin were further purified
by two additional RP-HPLC purification steps. Asterisk shows the
active final fraction containing alvinellacin.
(TIF)
Figure S2 MS spectrum of native alvinellacin. Analysis of
purified alvinellacin by MALDI TOF-MS shows a m/z value of
2,600.35 MH+which perfectly matches the theoretical mass of the
peptide including two disulfide bonds.
(TIF)
Figure S3 Sequence alignments of the precursors of
alvinellacin, capitellacin, and two arenicin isoforms.
(TIF)
Figure S4 Intact protein MS spectrum of alvinellacin
measured by nanoESI-Orbitrap MS. (A) Full range MS
survey spectrum. (B) Zoom-in of the [M+5H]5+charge state
species in a. A small species (indicated as asterisk) found next to the
major component was identified as the methionine oxidation
product of alvinellacin. The experimentally determined mono-
isotopic MW of alvinellacin was 2,599.2221 Da. (C) Display of
theoretical MW (2,599.2067 Da) of alvinellacin and its isotope
distribution at charge state 5. The results indicated that all four
cysteines are involved in the formation of disulfide bonds.
(TIF)
Figure S5 Time-course analysis of the proteolytic
cleavage of alvinellacin. The products of alvinellacin digestion
were analyzed by nanoESI-Orbitrap MS. (A) Peptide MS survey
spectra of alvinellacin digested with Lys-C at 35uC (overnight). (B)
Subsequent digestion of the Lys-C-digest with trypsin after
Figure 5. Alvinellacin activities against epibionts. (A) Freshly collected epibionts were incubated alone or in the presence of alvinellacin for
4 h, and after fixation observed under the electron microscope. The number of damaged bacteria was estimated among nine different morphotypes
(numbered from 1 to 9) clearly distinguishable in all our preparations. (B) Bacterial lesions are visible at high magnification as the formation of
membrane blebs and the release of cytoplasmic material (arrow).
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30 min; (C) after 2 h; (D) after 18 h at 37uC. The identities of the
peptides are summarized in Table S2.
(TIF)
Figure S6 Alvinellacin and capitellacin gene structures.
(A) As opposed to CDS (648 bp), the alvinellacin gene is rather
long (1949 bp from the initial methionine to the stop codon) with a
5 introns/6 exons structure and a first large intron of 442 bp.
Introns are all inserted in phase 0 with the exception of the last one
in phase 1. (B) Alignment of the translated regions of the
alvinellacin and capitellacin genes. The intron splicing positions
(triangles) are nearly conserved. The BRICHOS domains are
shaded and the AMP sequences are in bold type.
(TIF)
Material and Methods S1
(DOCX)
Table S1 Structural statistics for the 10 best structures
of alvinellacin showing the lowest target functions. None
of the distance constraints was violated by more than 0.5 A
˚in any
structure.
(DOCX)
Table S2 Disulfide-connected peptide fragments of
alvinellacin observed after proteolytic cleavage. Peptides
with oxidized cysteines were successively digested using the
proteases Lys-C and trypsin. The resulting peptides were analyzed
by offline nanoESI-Orbitrap MS/MS as shown in Figure S2. The
results unambiguously indicated two disulfide linkages between
C1–C4 and C2–C3.
(DOCX)
Acknowledgments
We thank the captain of the crew of the R/V Atalante, the DSV Nautile
group (IFREMER), along with N. Lebris and F. Lallier chief scientists of
the MESCAL 2010 and 2012 cruises. We also thank C. Slomianny and N.
Barois for access to the confocal laser microscope and electron microscope,
respectively, M.A. Cambon for deep sea microbial strains and H.
Liebegang for technical help during membrane-activity testing.
Author Contributions
Conceived and designed the experiments: A. Tasiemski ML JG SJ FG.
Performed the experiments: A. Tasiemski CBW DJ VCH SJ CV CWH A.
Tholey CG ML JG FDS OH. Analyzed the data: A. Tasiemski SJ CV DJ
JG ML. Contributed reagents/materials/analysis tools: FP. Wrote the
paper: A. Tasiemski SJ CV DJ JG ML.
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