INTEGR. COMP. BIOL., 43:313–322 (2003)
Natural Peptide Antibiotics from Tunicates: Structures, Functions and Potential Uses1
ROBERT I. LEHRER,2*,† J. ANDREW TINCU,‡ STEVEN W. TAYLOR,‡,§ LORENZO P. MENZEL,*
AND
ALAN J. WARING*
*Department of Medicine and
†Molecular Biology Institute, Department of Medicine, UCLA School of Medicine, 10833 LeConte Avenue,
Los Angeles, California 90095
‡Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego,
La Jolla, California 92093
§Present Address of Steve W. Taylor is MitoKor corporation, 11494 Sorrento Valley Road, San Diego, California 92121
SYNOPSIS. Because tunicates rely on innate immunity, their hemocytes are important contributors to host
defense. Styela clava, a solitary ascidian, have eight hemocyte subtypes. Extracts of their total hemocyte
population contained multiple small (2–4 kDa) antimicrobial peptides. When purified, these fell into two
distinct families that were named styelins and clavanins.
Styelins A-E are phenylalanine-rich, 32 residue peptides with activity against marine bacteria and human
pathogens. They show considerable sequence homology to pleurocidins, antimicrobial peptides of the flounder, Pseudopleuronectes americanus. Styelin D, one of the five styelins identified by peptide isolation and
cDNA cloning, was remarkable in containing 12 post-translationally modified residues, including a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines (DOPA), four dihydroxylysines
and one dihydroxyarginine. These modifications enhanced Styelin D’s bactericidal ability at acidic pH and
high salinity. A novel histochemical stain for DOPA suggested that Styelin D was restricted to granulocytes.
Clavanins A-E are histidine-rich, 23 residue peptides that are C-terminally amidated and most effective
at acidic pH. Clavaspirin is a newly described family member that also has potent cytotoxic properties. By
immunocytochemistry, clavanins were identified in the granules of five eosinophilic granulocyte subtypes
and in macrophage cytoplasm.
Transmission and scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA)
and E. coli that had been treated with Styelin D and clavaspirin suggested that both peptides induced osmotic
disregulation. Treated bacteria manifested cytoplasmic swelling and extrusion of cytoplasmic contents
through their peptidoglycan cell wall. The diverse array of antimicrobial peptides in S. clava hemocytes
constitutes an effective host defense mechanism.
Styela clava is a cosmopolitan solitary tunicate
(‘‘sea squirt’’) whose tadpole-shaped larvae display a
constellation of features (notochord, pharyngeal gill
slits, dorsal tubular nervous system, and post-anal tail)
that is characteristic of the Phylum Chordata. These
attributes are obliterated when it undergoes metamorphosis into a sessile polypoid adult. Because T-cells
and immunoglobulin-producing B cells first arose in
cartilaginous fish (Bartl et al., 1997; Lee et al., 2000),
tunicates—like other invertebrates—rely on their innate immune system for host defense. The ability of
S. clava and other tunicates to thrive in microbe-laden
waters suggests that they possess a robust innate immune system.
The importance of white blood corpuscles (leukocytes) in innate host defense was established by the
investigations of Metchnikoff in the 19th century. In
recent decades, much has been learned about the antimicrobial mechanisms of mammalian leukocytes. As
background for our studies on tunicates, this work will
be summarized briefly and very superficially. Mammalian phagocytes are of two general types, granulocytes and mononuclear phagocytes. The cytoplasmic
‘‘granules’’ of granulocytes are membrane bounded
storage organelles that contain multiple peptides and
proteins. Humans have four types of granulocytes.
These were named over a century ago to reflect how
their cytoplasm stained with aniline dyes. Three granulocyte types (neutrophils, eosinophils and basophils)
circulate in the blood, and the fourth (mast cells) is
restricted to tissues.
The cytoplasmic granules of human neutrophils
(PMNs) are of several types. Azurophil (primary)
granules contain large amounts of defensins, ;3.5 kDa
cysteine-rich antimicrobial peptides (Lehrer et al.,
1993). In addition, they contain several neutral proteases (elastase, cathepsin G and proteinase 3); a hemecontaining enzyme called myeloperoxidase, lysozyme,
and at least three antimicrobial proteins (azurocidin,
B/PI and lysozyme). Although some of the contents of
azurophil granules are released extracellularly when
neutrophils (PMN) encounter microbes, most of their
contents are discharged into phagocytic vacuoles
formed when microbes are ingested. At least two additional types of cytoplasmic granules (secondary and
tertiary) also exist in human PMNs. The secondary
granules release their contents extracellularly in response to a variety of signals associated with the presence of microbes. In addition to lysozyme, the secondary granules of human PMNs also contain lactoferrin and the precursor of an alpha-helical peptide
called LL-37 (Cowland et al., 1995).
Because of their abundance in blood and their im-
1 From the Symposium Comparative Immunology presented at the
Annual Meeting of the Society for Integrative and Comparative Biology, 2–6 January 20002, at Anaheim, California.
2 E-mail:[email protected]
313
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R. I. LEHRER
FIG. 1. Sequences of five S. clava Clavanins. aKey to symbols.
Standard single letter code is used, except that Ý denotes a modified
tyrosine and * signifies amidation.
portance in host defense, the microbicidal mechanisms
of human PMNs have been examined closely. Mammalian PMNs use three general mechanisms to kill ingested microbes. The relative contributions of these
mechanisms differ considerably from species to species. While one mechanism utilizes peptides and proteins, two of the mechanisms are oxidative in nature.
One of these oxidative modes involves the assembly
of a multi-component NADPH oxidase complex that
reduces molecular oxygen univalently to superoxide
(O22) anions (Nauseef, 1999; Babior, 1999). The other,
which is mediated by an inducible, multi-component
nitric oxide synthase (iNOS), is less prominent in human PMNs than the NADPH oxidase system (Murray
and Nathan, 1999; Nathan and Shiloh, 2000). We are
unaware of data concerning the presence or absence
of NADPH oxidase and iNOS in tunicate hemocytes.
Phenoloxidase, an enzyme that can generate H2O2, is
present in the ‘‘morula cells’’ of Styela plicata (Cammarata et al., 1997) and of Botryllus schlosseri (Ballarin et al., 1998).
The superoxide anions generated by NADPH oxidase are unstable and undergo secondary reactions, including dismutation to hydrogen peroxide. H2O2 is
more stable than superoxide and has modest antimicrobial properties, but it can be degraded rapidly by
catalase, an enzyme found in erythrocytes and many
bacteria. However, PMNs transform H2O2 into more
potent oxidants in several ways, one of which involves
myeloperoxidase. When provided with chloride and
H2O2, myeloperoxidase catalyzes the formation of
molecules indistinguishable from the hypochlorite
found in Clorox bleach (Klebanoff, 1999). This nascent bleach can also react with primary or secondary
amines to form longer lasting, antimicrobial chloramines (Weiss et al., 1983). In addition to its reactions
with myeloperoxidase, H2O2 can interact with divalent
iron in a Fenton reaction that generates hydroxyl radicals (Wardman and Candeis, 1996). The highly reactive hydroxyl radicals are potent microbicides.
In certain species, especially rodents, macrophages
have an inducible nitric oxide synthase complex
(iNOS) that can generate large amounts of nitric oxide—a strong oxidant with antimicrobial properties. In
many rodent models, the ability to produce nitric oxide
is critical in allowing macrophages to deal with diverse
pathogens, organisms ranging from trypanosomes
(Murray and Nathan, 1999) to mycobacteria (Bekker
et al., 2001). Because macrophages lack storage pools
ET AL.
FIG. 2. Comparison of clavaspirin and clavanin A. The signal sequences, anionic propieces and C-terminal extensions of Clavanin
A (ClavA) and Clavaspirin (Cvspn) are on top, and the sequences
of the mature clavanin A and clavaspirin peptides are below. Identical residues are connected by a vertical line, similar residues are
denoted by a 1. In both mature peptides, the C-terminal glycine (G)
is represented by an amide group.
of dedicated antimicrobial proteins and peptides, they
may depend on NADPH oxidase and nitric oxide synthase more than PMNs or other granulocytes.
Although eosinophils have been studied mostly with
respect to their activity against various helminths, their
cytoplasmic granules contain at least three molecules
that can also kill bacteria—eosinophil peroxidase, major basic protein and eosinophil cationic protein (Lehrer et al., 1989).
While the following caveat may not surprise any
readers who have perused the voluminous older literature on tunicate hemocytes, there is no direct correspondence between the various white blood cell subtypes of tunicates and mammals. Indeed, using light
and electron microscopy, we could identify six different subtypes of S. clava granulocytes rather than the
four types known in humans. Moreover, when we examined the hemocytes of several other tunicates (e.g.,
Styela plicata, Styela montereyensis and Pyura spp.),
we found decided differences among them. In our
studies of S. clava cells, we generally made no effort
to separate the various hemocyte subtypes. Instead,
FIG. 3. Possible Homologues of Styelins. A data base search performed on the signal sequence and mature peptide of Styelin C
retrieved several antimicrobial peptides. These included numerous
cecropins, including cecropin 1 (AF416602) of the housefly, Musca
domestica and pleurocidin (AF301512), an alpha-helical peptide of
the flounder, Pseudopleuronectes americanus. A search done only
on the styelin signal sequence retrieved the signal peptides of two
additional antimicrobial peptides: attacin A (AF220544) of the fruitfly, Drosophila melanogaster, and myticin A (AF162334) of the
mussel, Mytilus galloprovincialis.
TUNICATE ANTIMICROBIAL PEPTIDES
315
FIG. 4. Morphology of S. clava hemocytes. The large panel on the left was stained with Mallory’s trichrome stain. The small inset shows
two adherent granulocytes stained with a mixture of methyl blue and orange G. The rightmost panel shows an alkaline nitroblue tetrazolium
stain that detects redox-active residues, such as DOPA (dihydroxyphenylalanine) and TOPA (trihydroxyphenylalanine). Abbreviations: G,
granulocyte; M, macrophage, L, lymphocyte-like cell, U, uropod. The arrow shows a granulocyte within a macrophage.
mixed hemocyte populations were harvested and extracted into acetic acid. These extracts were fractionated by gel chromatography, preparative electrophoresis and RP-HPLC. Peptide purification was monitored by gel overlay and radial diffusion assays to
identify fractions with antimicrobial activity.
Figure 1 shows the sequences of the five clavanin
peptides, Clavanins A-E, that we isolated from a
mixed population of S. clava hemocytes (Lee et al.,
1997a). Each peptide contained 23 amino acid residues, of which 18 (78.2%) were identical. Clavanins
C and D contained a post-translationally modified tyrosine residue that we tentatively identified as methyltyrosine. However, given the findings on Styelin D described below, these could also be 3,4-dihydroxyphenylalanine (DOPA) residues instead. Each Clavanin
peptide contained 4 or 5 phenylalanine residues. At
neutral pH, their amidated C-terminus plus the presence of 1 or 2 arginines/lysines imparted a net charge
12 to 13, making them moderately cationic. At an
acidic pH, protonation of their 3 or 4 histidines more
than doubled this net charge and increased their antimicrobial potency (Lee et al., 1997c). CD spectrometry revealed that clavanins assumed an alpha-helical
conformation in the membrane-like environments,
such as were provided by phospholipid micelles or trifluoroethanol.
We also cloned Clavanins A-E from a cDNA library
that was prepared from the pharyngeal tissues of S.
clava (Zhao et al., 1997a). The peptide precursors had
a distinctive architecture, with a 19 residue signal sequence followed in turn by a 10-residue polar propiece, the peptide, and a 27 residue anionic C-terminal
extension. The sequence (LEERKSEEEK) of the propiece was invariant and that of the 27 residue C-terminal extension was highly (96.3–100%) conserved.
While performing cloning experiments, we found
mRNA for the precursor of clavaspirin. This mRNA
was highly homologous to proclavanins A-E except in
the mature peptide domain (Fig. 2), where only 7/23
(30.4%) residues were identical. In contrast, the signal
sequences, propieces and C-terminal extensions of
these precursors were identical in 50/56 (89.3%) residues (Lee et al., 2001). This is reminiscent of the
situation found in cathelicidins (Zanetti et al., 1995).
In this structurally diverse group of antimicrobial peptides, all of the precursors contain a conserved ‘‘cathelin’’ pro-domain.
An anionic propiece exists in other antimicrobial
peptides, including pleurocidin (discussed below), ranalexin and other frog peptides, and mammalian alpha
defensins. It has been speculated that electrostatic interactions between the anionic pro-domains and the
cationic antimicrobial domains may protect host cells
from self-inflicted damage while moving the peptides
from their synthesis sites to their storage organelles.
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R. I. LEHRER
ET AL.
FIG. 5. Characteristics of five granulocyte subtypes in S. clava. Type 1, 2 and 3 granulocytes were previously described (Sawada et al.,
1993). Types 4 and 5 granulocytes are also eosinophilic, but their granules differ distinctly from the type 2 and 3 granules. Type 1 granulocytes
are also called basophils. A full report of this study will appear elsewhere (Menzel et al., 2002).
In addition to the five clavanins, we purified two
larger antimicrobial peptides from S. clava hemocytes,
Styelins A and B (Lee et al., 1997b). Although their
post-translational modifications made sequencing difficult, we obtained enough data to design probes for
screening the S. clava branchial cDNA library (Zhao
et al., 1997b). In this manner, we cloned mRNAs for
three prepropeptides that were designated Styelins C,
D and E. They had identical 22 residue signal sequences and contained a 26 residue C-terminal anionic domain in which 21/26 (80.8%) residues were identical.
Their 31 or 32 residue peptide domains resided prox-
TUNICATE ANTIMICROBIAL PEPTIDES
317
FIG. 6. Scanning electron micrographs. E. coli ML-35p was treated at room temperature for 30 minutes with 50 mg/ml of Styelin D (StyD,
panels A, B) or 50 mg/ml of clavaspirin (cspn, panels C, D). The arrows point to cytoplasmic extrusions. The Styelin-treated organisms have
a bumpy surface, while the clavaspirin-treated cells are smooth.
imal to the C-terminal propiece. The peptide domains
of Styelins A, B and C were identical in at least 17/
20 (85%) N-terminal residues. Styelins D and E were
identical to each other in 80/81 residues (98.8%), differing only in position 25, which was a lysine in Styelin D and a glutamine in Styelin E.
Styelin D was remarkable in containing 12 posttranslationally modified residues (Taylor et al., 2000).
These included a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines
(DOPA), four dihydroxylysines and one dihydroxyarginine. Its sequence was: GW➅ LR➁ K➁ AAK➁
SVGK➁FY➀Y➀K➁HK➀Y➀Y➀IK➀AAWQIGKHA
L-NH(2), where W➅ is 6-bromotryptophan, R➁ is dihydroxyarginine, Y➀ is 3,4-dihydroxyphenylalanine
(DOPA), K➀ is 5-hydroxylysine, and K➁ is dihydroxylysine. These modifications enhanced Styelin D’s
bactericidal ability at acidic pH and high salinity. The
presence of multiple hydroxylated residues in Styelins
could impart other properties to them. Among these,
are an ability a) to bind metals, b) participate in processes that generate free radicals, and c) form multiple
pro-adhesive hydrogen bonds. All of these possibilities
are attractive subjects for future research.
In an earlier report on Styelins, we commented on
an apparent sequence homology between the precursors of styelins and cecropins (Zhao et al., 1997b).
Cecropins are a family of alpha helical peptides found
in diptera (e.g., flies, mosquitoes, lepidoptera, etc.). In
a more recent data base search, we found a possible
homologue of Styelins in a vertebrate—the winter
flounder, Pseudopleuronectes americanus. In Figure 3
the sequence of Styelin D is aligned with that of Pleurocidin. The program (Blast 2.2) has inserted three
gaps with a total of eight residues to maximize alignment. Overall, 19/62 residues (30%) are identical and
32/62 (51.6%) are either identical or conservatively
replaced. To continue our ‘‘fishing expedition,’’ we
also searched for any antimicrobial peptides with a signal sequence that resembled Styelin D’s. This exercise
was not necessarily frivolous, because the signal sequences of some antimicrobial peptides (e.g., defensins
(Zhao et al., 2001) and frog skin peptides (Clark et
al., 1994; Amiche et al., 1994) can be more highly
conserved than the associated peptides. As shown in
Figure 3, two antimicrobial peptides met our search
profile: attacin A from the fruit fly Drosophila melanogaster, and myticin C from the mussel, Mytilis galloprovincialis. The signal sequences of attacin A and
Styelins C-E were identical in 15/23 (65.2%) residues
and similar in 17/23 (73.9%). The signal sequence of
myticin C was identical to Styelins C-E in 11/14 residues (78.6%) and similar in 13/14 (92.9%). Although
such resemblance could have arisen by chance, they
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R. I. LEHRER
ET AL.
FIG. 7. Effects of Styelin D on E. coli ML-35p. Panel A shows untreated E. coli controls. Panel B (a negative image) shows E. coli exposed
to 50 mg/ml of Styelin D for 15 minutes. The fine arrows indicate areas of cytoplasmic rarefaction (loss of electron density) and the larger
arrowheads point to the outer membrane, which has expanded and displays multiple small folds. Panel C (a positive print) shows other
organisms treated with Styelin D for 15 min. The large arrows call attention to the multiple small outer membrane folds. Cytoplasmic rarefaction
is present in most bacteria and one organism shows transmural cytoplasmic extrusion. Panel D, taken after 30 minutes of exposure, shows
two largely empty bacteria with little residual cytoplasmic content. The arrow points to an area of extruded cytoplasm.
may represent traces of exon shuffling in the evolutionary histories of these peptides.
Which hemocytes of S. clava contain clavanins and
styelins? The main panel of Figure 4 shows a mixed
population of hemocytes that had been gently centrifuged onto a glass slide, fixed, and stained with Mallory’s trichrome—a mixture of orange G, methyl blue
and acid fuchsin. Three general types of cells can be
seen. About half of them are eosinophilic granulocytes
(G), cells with multiple red-stained cytoplasmic granules. The remainder are mostly macrophages (M) (relatively large cells whose cytoplasm appears devoid of
granules) and small cells (L) that superficially resemble lymphocytes. The inset displays two granulocytes
that were stained with a mixture of orange G and
methyl blue. These cells adhered to the slide spontaneously under unit gravity, rather than being centrifuged onto it, and have a more ‘‘life-like’’ appearance.
One of these granulocytes has a trailing ‘‘uropod’’ (arrow), a cytoplasmic protrusion opposite to its direction
of motion. The cytoplasmic granules of both cells are
oval-shaped, uniform in size and distinctly orange.
These features exemplify the Type 5 granulocyte
shown in Figure 5 and described below.
Close to the bottom of the panel, one macrophage
contains a granulocyte (arrow) that it may have just
ingested. In other fields, we noted occasional macrophages with orange-staining cytoplasm, perhaps the
residue from some earlier feast of this nature. Electron
microscopy allowed us to examine the cytoplasmic
granules of S. clava hemocytes more closely. We observed six distinct granulocyte subsets, three of which
(Types 1, 2 and 3) had earlier been defined (Sawada
et al., 1993). Information about five of these six granulocyte subsets is shown in Figure 5. Immunocytochemical studies revealed clavanins in the cytoplasmic
granules of Type 2–5 eosinophilic granulocytes, as
well as in the cytoplasm of macrophages and the large
spherical vacuoles of Type 6 granulocytes (Menzel et
al., 2002). These Type 6 cells may correspond to the
‘‘morula cells’’ described by other investigators. Styelins and clavanins were very abundant, and constituted
at least 10–20% of the total extractable protein in our
crude starting extracts. They may be largely respon-
TUNICATE ANTIMICROBIAL PEPTIDES
319
FIG. 8. Effects of Clavaspirin on E. coli ML-35p. These thin sections were viewed by transmission electron microscopy. The organisms had
been treated with 50 mg/ml of clavaspirin and were nonviable. Their outer membranes were smooth, and organisms with extruded cytoplasm
(ec) were rare. The inset shows a 33 enlargement of the area in the white box. The white arrow points to surface fuzz, which was seen on
some cells and the black arrows point to small electron-dense precipitates which contained lead and uranium when analyzed by electron
dispersive spectroscopy.
sible for the eosinophilic staining of the granules that
contain them.
It is known that DOPA residues react with nitroblue tetrazolium (NBT) under alkaline conditions to
produce an insoluble blue formazan dye (Taylor et al.,
1995, 1997). Since Styelin D has 4 DOPA residues,
we used this reaction as a histochemical test to determine which hemocyte types contained DOPA-rich
peptides (Fig. 4, right panel). Neither the macrophages
nor the small lymphocyte-like cells were stained by
alkaline NBT. In contrast, most granulocytes were rendered distinctly blue. We consider this presumptive evidence that styelins are components of granulocytes
rather than macrophages.
How do clavanins and styelins kill bacteria? Electron micrographs of treated Escherichia coli and
Staphylococci provide useful clues. Figure 6 shows
four scanning electron micrographs of E. coli cells that
were exposed to 50 mg/ml of Styelin D (Panels A, B)
or clavaspirin (Panels C, D) for 30 or 60 minutes.
Many bacteria have extruded their cytoplasm beyond
the confines of the cell wall (arrows) and the outer
membrane of bacteria treated with Styelin D has a cobblestone appearance quite different from the relatively
normal appearance of the surface of clavaspirin-treated
bacteria.
Figure 7 shows transmission electron micrographs
of E. coli treated with Styelin D. Panel A shows untreated controls. Panel B shows Styelin-treated cells
and is printed as a negative. The heavy arrowheads
call attention to the outer membrane, which is thrown
into multiple small folds, and the fine arrows point to
areas of cytoplasmic rarefaction. Panel C is a conventional positive print of Styelin-treated bacteria. The
cells appear swollen and contain areas of cytoplasmic
rarefaction (loss of electron density). One cell appears
to have extruded some of its cytoplasm beyond the cell
wall, consistent with findings in the scanning micrographs. In Panel D, a later stage, a fine arrow points
to an area of extruded cytoplasm. Note that two cells
near the bottom have lost most of their cytoplasmic
contents. Although its antimicrobial mechanism requires additional study, the appearance of styelin-treated organisms is reminiscent of protegrin-treated bacteria.
Protegrin-treated bacteria sustain a rapid influx of
water that triggers a massive efflux of potassium. The
water influx is driven by osmotic forces and causes the
bacteria to swell. This swelling induces secondary
membrane permeability changes and can extrude portions of the cytoplasmic membrane through small
openings (tesserae) in the peptidoglycan. Extruded cytoplasmic protrusions are osmotically fragile and easily ruptured. This overall mechanism was named ‘‘hydro-osmotic trans-tesseral extrusion and rupture’’ and
given the acronym ‘‘HOTTER.’’ (Orlov et al., 2002).
In contrast to the membrane ruffling found with
Styelin D treated E. coli, those treated with clavaspirin
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R. I. LEHRER
ET AL.
FIG. 9. Scanning electron micrographs. A methicillin-resistant strain of Staphylococcus aureus (MRSA ATCC 33591) was treated with 50
mg/ml of Styelin D (Panels A, B) or 50 mg/ml of clavaspirin (Panels C, D). Incubation times are noted on each panel. The arrows point to
cytoplasmic extrusions.
show only modest swelling in Figure 8. The inset of
an area enlarged further shows a fuzzy outer membrane, likely due to the sectioning angle, and small
deposits of electron dense material noted by the small
arrows. On similar spots, lead and uranium were determined responsible for the electron density suggesting that clavaspirin may have an affinity for these
heavy metals.
Staphylococci are affected similarly by these peptides. Styelin D has caused many of these cells shown
in the scanning electron micrographs of Figure 9 panels A and B to extrude parts of their cytoplasm. Different stages are illustrated in the two panels, 15 minutes and 60 minutes of exposure in A and B respectively. Panels C and D show that the morphological
effects of clavaspirin on staphylococci are nearly identical to the Styelin D treated ones. That these peptides
act rapidly is illustrated in Panel A, which shows representative staphylococci after 5 minutes of exposure
to 50 mg/ml of clavaspirin.
Figure 10 illustrates two of the three ways by which
S. clava can engage bacteria; by delivery to phagocytic
vacuoles or following discharge at the hemocyte’s
membrane. The third way (secretion into the animal’s
hemolymph) does not lend itself to illustration by electron microscopy. Panel A of Figure 10 shows a phagocytic Type 3 granulocyte (recognized by its character-
istic granules) whose cytoplasmic vacuole (vac) contains an ingested marine bacterium, Psychrobacter immobilis (p), and the debris of a second organism
(arrow). Panel B shows two Psychrobacter cells attached to the surface of another hemocyte by a myriad
of filamentous ties. Two of the hemocyte’s granules
(g) can be seen adjacent to plasma membrane and the
lower organism. Near these granules are several vesicular structures that could represent granules that have
discharged their contents. The outer membranes of
both bacteria show multiple tiny folds (arrowhead), resembling those noted in styelin-treated E. coli (Fig. 7).
Are there potential uses for antimicrobial peptides
from tunicates? There could be many, both for the environment (as ‘‘green’’ anti-pollutants and microbicides) and in medical practice. The introduction of antibiotics was perhaps the major medical advance of the
twentieth century. However, in recent years many bacteria have become resistant to conventional antibiotics
and there is an urgent need for new antimicrobial
agents (Hancock and Lehrer, 1998). Although most antibiotics are secondary metabolites of fungi, streptomycetes or bacteria, a few are peptides (e.g., nisin and
other lantibiotics), lipopeptides (e.g., polymyxin B) or
glycopeptides (e.g., vancomycin or teichoplanins). In
general, peptide antibiotics have different mechanisms
and targets than the antibiotics now in medical use.
TUNICATE ANTIMICROBIAL PEPTIDES
321
FIG. 10. S. clava hemocytes and Psychrobacter immobilis. Panel A, shows Psychrobacter immobilis (P), a Gram-negative marine bacterium,
within a phagocytic vacuole (vac) that also contains some bacterial fragments (arrow). This hemocyte’s granules have an electron-dense core
(c) and an electron-lucent sheath (s). Panel B shows two Psychrobacter immobilis bound to the membrane of another hemocyte. Only a few
cytoplasmic granules (g) remain visible inside the hemocyte, whose cytoplasm contains multiple small vesicles. The fine arrows point to zones
of adherence, and the large arrowhead to the extensively folded bacterial outer membrane.
Consequently, many are highly effective against resistant organisms, including Pseudomonas aeruginosa
and methicillin-resistant S. aureus. Technologies exist
to produce peptides by recombinant or direct synthesis,
and they are likely to improve in the future. Tunicates
and their hemocytes, peptides and depsipeptides provide an accessible, renewable resource that could reward wider exploration.
ACKNOWLEDGMENTS
We thank Birgitta Sjostrand for her expertise and
assistance with electron microscopy. Our work on tunicate peptides was supported, in part, by the National
Sea Grant College Program of the U.S. Department of
Commerce’s National Oceanic and Atmospheric Administration under a NOAA Grant: project number R/
MP-93 through the California Sea Grant College Program. The views expressed herein do not necessarily
reflect the views of any of these organizations.
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