Localisation of bioactive metabolites in marine sponges

7 Pages·1999·2.1 MB·

by  D J Faulkner

LOCALISATION OF BIOACTIVE METABOLITES IN MARINE SPONGES
D. JOHN FAULKNER, MARY KAYHARPER, CHRISTINE E. SALOMONAND ERIC W. SCHMIDT
Faulkner, D.J., Harper, M.K., Salomon, C.E. & Schmidt, E.W. 1999 0630: Localisation of
bioactivemetabolites inmarinesponges. MemoirsoftheQueenslandMuseum44: 167-173.
Brisbane. ISSN 0079-8835.
Marinenatural productchemistshaveoftenproposedthatbioactivespongemetabolitesare
produced by symbiotic micro-organisms. This paper discusses the rationale for these
proposals, reviewsthe strengths and weaknesses ofmethods that areavailabletotestsuch
hypotheses and reports some experimental studies. The conclusion reached from the
research to date is that it is too early to make generalisations concerning eitherthe role of
symbiontsinthebiosynthesisofspongemetabolitesoreventhebesttechniquesforstudying
G
the cellular localisation of bioactive metabolites. Porifera, bioactive metabolites,
cyanobacteria,filamentous eubacteria, symbiosis, Aplysinafistularis. Dysidea herbaeea,
Theonellaswinhoei, Oceanapiasagirtaria, Jaspissplendens. '
D.JohnFaulkner(email;[email protected]), MatyKayHarper, ChristineE. Salomon&
Eric W, Schmidt, Scripps Institution ofOceanography, University ofCalifornia at San
Diego, LaJolla, CA, 92093-0212, USA; 22December'}998.
Sponges are exceptionally good synthetic unexpected structural motifs. Within this group,
chemists. They can make chemicals ofextreme spongeshaveprovidednotonlythebestsourceof
toxicity and/or deterrent value that have novel compoundsbutalso thegreatestoccurrence
undoubtedly contributed to their survival over ofpotentialpharmaceuticals(Faulkner, 1998, and
the ages. But they may not always have acted references therein). However, when chemists
alone. We now know that some sponges harbor compared the structures of sponge metabolites
populations of symbiotic micro-organisms that with those ofcompounds in the literature, they
produce the chemicals thought to defend the found many structures that were very similar to
spongefromcompetitionorpredation. However, those ofmicrobial metabolites. When chemists
it is clear thatthis situation is less common than sawr scanning electron micrographs of sponges
themarinenaturalproductsliteraturewouldhave that contained large numbers of
us believe. This paperreviews the methods used micro-organisms, theyfeltjustified in proposing
to determine the cellular location of natural that compounds resembling microbial
products in sponges and presents some recent metabolites were in fact of microbial origin.
results fromourlaboratorythateitherconfirm or Furthermore, when closely related or identical
deny the production of'sponge metabolites' by compounds were found in different phyla and
symbiotic microbes. there was no evidence of transmission of the
chemicals throughthe food chain,they proposed
SYMBIOSIS AS SEEN FROM THE that these compounds might be produced by the
VIEWPOINT OF CHEMISTRY. The history of same or similar micro-organisms endemic to
naturalproductschemistryhasbeendrivenbythe hostsofdifferentphyla.Thesehypothesessetthe
use of natural products to treat diseases. First stage for a careful investigation of the role of
came an interest in plant products such as symbiotic microbes in the production of'sponge
digitalis and morphine, but this was superseded metabolites'.
inthesecondhalfofthiscenturybythediscovery'
ofaplethoraofimmensely important antibiotics LOCALIZATIONOFSPONGEMETABOLITES.
and other drugs obtained by the fermentation of There aretwobasic strategies fordeterminingthe
microbes. Chemists became indoctrinated with location of specific metabolites in sponges:
the concept that micro-organisms could provide detectionofcompoundsusingmicroscopy,orcell
the needs ofthe pharmaceutical industry, which separation followed by chemical analysis ofeach
for a long period oftime was not far from the cell fraction. The strategy selected generally
truth. Then came the discovery that marine depends on the molecular properties of the
organisms could provide many new classes of compound to be investigated. Compounds that
natural products that incorporated new and containheavvelementssuchasbromineoriodine
168 MEMOIRS OF THE QUEENSLAND MUSEUM
OMe OMe dissociated cells can then be fixed, which
stabilises the cells during the period between
collection and analysis. It is a relatively simple
matter to separate cyanobacteria using a cell
sorter to distinguish fluoresecent from
non-fluorescent cells but this method does not
H H distinguishbetweenspongeandeubacterialcells.
Cell types canalso be separatedbydensity using
either differential centrifugation or Ficoll or
o o Percoll density gradients. It has been our
experience that repeated centrifugation is
(A) n = 4, aerothionin requiredtoproducefractionsofreasonablepurity
(B) n = 5, homoaerothionin and that the different sponge cell types are
difficult to separate on the basis of density.
FIG. 1. Tetrabrominated metabolites. A, aerothionin. Nonetheless,filamentousbacteria,mixedsponge
B, homoaerothionin. cells andmixed unicellularbacterial cells canall
be enriched to ca. 90% purity using
can be detected by using an energy dispersive centrifugation. To detect the compounds of
spectroscopy (EDS) detector on a scanning interest, each cell fraction is then extracted
electron microscope (SEM) or a scanning individually and analyzed using two or more of
transmission electron microscope (STEM). In the following techniques: mass spectrometry
theory, one could use the same technique to (MS), which can be combined with high
determine the location ofcompounds containing performance liquid chromatography (HPLC) or
chlorine or sulfur but, in practice, the levels of gas chromatography (GC), HPLC using a diode
chloride and sulfate ions in seawaterpreclude its array detector to measure the UV spectrum, and
use with marine specimens. Fluorescent 'H NMR spectrometry.
compounds can be conveniently detected by
fluorescence microscopy and by laser scanning RESULTS AND DISCUSSION
confocal microscopy, but this technique is
susceptable to problems caused by background The tetrabrominated metabolites aerothionin
fluorescence duetophotosyntheticpigmentsand (Fig. 1A) and homoaerothionin (Fig. IB), which
general autolluorescence ofcells. Themethodof occur as a 10:1 mixture in a shallow-water
immunostaining using polyclonal antibodies to specimen ofAplysinafistularis from La Jolla,
bind to a specific compound is common in were ideal candidates for study using energy
cellular biology but prior to a report at this dispersive spectroscopy because the molecules
symposium (llan, 1998) and one other recent contain such a high concentration of bromine.
paper(Perry etal., 1998) had notbeen applied to Analysis of the STEM images using energy
study sponge metabolites. Finally, there is the dispersive X-ray analysis revealed a 20-fold
possibility that specific compounds may be larger concentration of bromine in spherulous
detected in cell preparations usingsecondary ion cellsthaninbacterialorotherspongecells,which
mass spectrometry in conjunction with tandem were both at background levels. We therefore
mass spectrometry. The lattertwomethods, both argued that the brominated metabolites (Fig.
ofwhich can be fine-tuned to detect individual 1A-B) were produced and stored in spherulous
compounds, couldofferconsiderableadvantages cells (Thompson et al., 1983).
over methods that rely on detecting a class of
There are two major chemotypes ofDysidea
compounds.
herbacea\ one contains both sesquiterpenes and
Cell separationmethods take advantage ofour metabolitesbiosynthesisedfrompolychlorinated
ability to analyse the chemical content of fixed amino acids, the other produces only
cells but suffer from the disadvantage that polybrominated biphenyl ethers and lacks
fractions containing only a single cell type may terpenes. Very significant populations of
be difficult to prepare. Sponge tissues can be cyanobacteriaare found in both chemotypes and
dissociated by enzymatic digestion or in both cases, the cyanobacterium is considered
mechanical disruption in calcium-magnesium to be Oscillatoria spongelliae. The fluorescent
free seawater using squeezing, sieving, simple cyanobacterial cells were separated from all
mincing, vigorous stirring, or even ajuicer. The othernon-fluorescentcellsusingacell sorterand
BIOACTIVE METABOLITE LOCALIZATION 69
1
AcO 16S rRNA sequences of
representative samples to
CCI:
determine whether the
cyanobacteria represent two
different strains of O.
sponge!Iiaeordifferentcyano-
bacterial species, which are
(B) spirodysin
twoofseveralpossibleexplan-
ations of the chemical
diversity. The diversity of
(A)13-demethylisodysidenin chemistry assigned to Dysidea
spp. may also provide a good
rationale for a sponge
taxonomist to re-examine the
genus and particularly the
chemists' voucher specimens.
The lithistid sponge
Theonella swinhoei has
provided chemists with an
almost unequalled source of
(C)herbadysidolide
highly bioactive chemicals
FIG. 2. Metabolites from two major chemotypes ofDysidea herbacea. A (Bewley & Faulkner, 1998).
13-demethylisodysidenin. B, spirodysin. C, herbadysidolide. D Ourinterestinthisspongewas
polybrominated biphenyl ether. piqued by the structural
the chemical content of each cell type was similarity between swinholide
analysed by !HNMR spectroscopyand GC-MS. A (Fig. 3A), which had previously been isolated
InaspecimenofD. herbaceafromHeron Island, from T. swinhoei, and the cyanobacterialproduct
13-demethylisodysidenin (Fig. 2A), a scytophycin C (Fig. 3B) and by the fact that the
polychlorinated amino acid derivative, was cyclic peptides of T. swinhoei, such as
extracted from the cyanobacterial cell fraction theopalauamide (Fig. 3C)(Schmidt et al., 1998),
contain aromatic 3-amino acids similar to those
whilethesesquiterpenesspirodysin(Fig.2B)and
found in some cyanobacterial cyclic peptides
herbadysidolide (Fig. 2C) were detected in the
f(rUanctsioonnth&atcFoanutlaiknneedr,spo1n9g9e3a)n.dbGaacrtesroianl caelnlds B(Ieswhlibeaysh&i Fetauallk.n,er1,98169;98K)i.tagawa et al., 1990;
coworkers recently separated the sponge cells This led to a suggestion that the prominent
using aPercoll density gradient and showedthat filamentousmicro-organismsin T.swinhoeiwere
the sesquiterpenes were located in a fraction cyanobacteria that produced both groups of
containing archaeocytes and choanocytes compounds (e.g. Kobayashi & Ishibashi, 1993;
(Flowers et al., 1998). A similar analysis of a Fusetani & Matsunaga, 1993). We had reason to
specimen of D. herbacea from Palau revealed suspect that this assumption was incorrect
thatthepolybrominatedbiphenyl ether(Fig. 2D) because the sponges were often found in caves,
was located not only in the cyanobacterial the filaments were found in the interior of the
traction but also as crystals situated just below sponge, away from the light, and extracts ofthe
the surface ofthe sponge (Unson et al., 1994). endosomal tissueofthe sponge did not appearto
contain sufficient chlorophyll pigments. In a
The sponge cellswere consideredtobe devoid specimen ofT. swinhoei from Palau, there were
of brominated metabolites, although it was unicellular cyanobacteria (Aphanocapsa
possible to detect a very low level ofthe poly- feldmannt) in the ectosome, which was peeled
brominated biphenyl ether (Fig. 2D), which was away and examined separately. The ectosomal
consistentwiththepresenceofasmallnumberof tissues were dissociated and the cyanobacteria
cyanobacterial cells that remained as were separated using differential centrifugation,
contaminants inthe sponge cell fraction. Having but they did not contain the metabolites of
shown that cyanobacteria are responsible forthe interest. Theendosomaltissuesweredissociated,
halogenatedchemicals in thetwochemotypes of fixedinamixtureofformalinandglutaraldehyde
D. herbacea, there is now a need to analyse the in artificial seawater, and separated using
170 MEMOIRS OF THE QUEENSLAND MUSEUM
OMe
OHC.
OMe OMe
(A) swinholideA (B)scytophycin C
OOQ
"^
ft*"
(C) theopalauamide
FIG. 3. Metabolites from the lithistid sponge Theonellaswinhoei. A, swinholide A, which partially resembles
scytophycin C. B, cyanobacteriai productscytophycinC. C, theopalauamide.
differential centrifugation into three fractions analysed by 'H NMR and HPLC.
containing mixed sponge cells, a filamentous Theopalauamide(Fig.3C)wasfoundtobepresent
bacterium, and mixed unicellular bacteria. The in about 4% dry weight in the filaments, which
cell fractions were thoroughly washed, then were examined by TEM and found not to be
extracted to obtain crude extracts that were cyanobacteria, since they lacked the thylakoid
BIOACTIVF METABOLITE LOCALIZATION
N^
acid vapor. A similar pH dependency was noted
when sections were observed using fluorescence
microscopy, but there was so much fluorescence
from cells that were out-of-plane that it was
impossible lo clearly image the cells containing
dercitamide. Examination ofboth thick sections
and clinched cell fractions using a confocal
(A)pyridoacridlne NMCOEt microscope under both neutral and acidic pit
(B) dercitamide conditions led tothe conclusion thai dercitamide
was localised in sponge cells containing between
i KJ.4 a. pyridDacritfineskeleton- R. dercitamide. ten and Iw*etrty spherical inclusions.
Transmission electron microscopy
i
structures that house the photo$ynthette employedtoshow that therewerenointracellular
apparauisofey;niobacteria{Bev\lcy cl al., 1996). bacteria thai could be responsible for the
Wc are currently characterising the eubacterial chemistry (Fig.6). The ikicitamide-coiilaimng
Jilaments using 16S rRNA analysis. Swinhohde celf- were characterised by ll:M analysis
A (Fig. 3A) was extracted from the unicellular although theyhavenotbeenclassified as apnrtic-
bacterial fraction Which contained many ivpe of sponge cell. Tins appears to be the
a
morphologically distinct bacteria. A recent first time that eonfneal microscopy has been
re-examination ofthe '11 NMR spectrum ofthe employed to locate marine naiura) products on
unicellularbacterial fractionrevealedthepresence the basis oftheirautolluorescence. Research is in
of the 4-meth\lene sterols that are typical of progress to determine ihe cellular location of
Tlietwella spp., but wc need to reconfirm that pyridoaeridine alkaloids in aseidians.
resultbecause sterols are not usually produced by Not every study has resultedman unambiguous
cultured bacteria. Both the sponge cells andthe ii-ation of metabolites. The cyclic
cyanobactcrium Aphanocapsa jeUiwanni depsipeptide jaspamide (Fig. 5) is a cytotoxic
appeared to be devoid ofbioaetivc metabolites. metabolite ofJaspi& spfendens (De Laubenfels,
1954), referred to as Jwpfc sp. in our earlier
theThseapmyeridUonadeerrildyiinnegaltkeatlroaicdysc,liwchiacrhomaalltipcossTeisnsg chemistry paper(Zabnskie et al.. 19S6), that has
been proposed bothasachcmotaxonornic market
system (Fig. 4A), are examples of a class of
and lo be ofmicrobial origin, h is interesting to
metabolitesthat have been foundin fourdifferent note that jaspamide (Fig 5) was also isolated
marine phyla, but predominantly in sponges and
aseidians(Molinski, I 9931.They ha\c frequently cfornoSmU'atcctQom(pClreteewlsyetdiaflf..ere1n9t94s).poTnhgee,sApuolnegtetu cl.
been proposed to be metabolites ofundesignated
dissociated in a juicer, a technique previously
microbial populations that might occur as
symbionts m the different phyla. We felt that used successfully on T. swinhaei^ followed by
there might be an alternative explanation hased Ho
on the evolution ofsimilar biosyntheiie schemes
-ocr°
in different phyla, in part because polyaromatic
compounds arc ihe most stable products that can
arise from their presumed mode ol biosynthesis
(StefTan etaf, 1993). Dercitamide (Tig. 4BJ has
HrsL >,0
been reported from both sponges and aseidians
(: ninaua—rdana cl al.. 1992; Carroll & Sebeuer,
|9go) the latter authors referring to
dercitamide as luianoniamine C - and we have
isolated it as the major metabolite ot the sponge
Oceafiapm sagiltarfQ (Salomon & Faulkner,
1906). Dercitamide isan mlerestingpigmentthat
changes color from yellow in neutral or basic-
solution(piI 7) tored in acidicsolution(piI<6) jaspamide
and has a fluorescence spectrum that is also piI
dependent. Using a light microscope, one can
observeachange inthecolorofthespongetissue FIG. 5. ( \clic depsipeptide i GrOttl
when a section is acidified using trilluoroacetic \endens*
172 MEMOIRS OF THE QUEENSLAND MUSEUM
2Mm
inm
FIG. 6. Micrograph of a dercitamide-containing FIG. 7. Micrograph of dissociated cells of Jaspis
inclusional sponge cell from Oceanapia sagittaria splendens. Arrows indicate the unidentified
thatshowsthe absence ofintracellularsymbionts. symbiontsthatdo notcontainjaspamide.
tixation and cell separation using differential unicellularbacterium asthesourceofabioactive
centrifugation. Jaspamide was not detected in compound. The latter will undoubtedly require
extracts of an unidentified extracellular theculture ofsymbioticmicro-organisms,which
'symbiont' (Fig. 7) or associated micro- isthegoal ofseveral research groups. In orderto
organisms, which represented a large proportion accomplishthisgoal,wehaveproposedastrategy
ofthewhole spongebiomass, butwas isolatedin thatinvolves identificationofthe symbionts from
nearly 4%yield from afraction containing small their 16S rRNA sequence (Brantley et al., 1995)
(ca. 500nm) orange bodies and cellular debris. before attempting to culture them using media
Theidentityoftheorangebodiesisuncertain,but that are suitable for culturing their nearest
mweayhahvaeveevirduepntcuertehdattthhee sdipsosnocgieaticoenllpsrowcietshs rseylamtbivieos.ntWshile the ulttihmaatte goal is tporcoudlutucree
concomitant release of the orange bodies.
Examination ofnewly acquired sponge material pharmacologically-active sponge metabolites in
order to speed their pharmaceutical
by light microscopy revealed the presence of
numerous small orange inclusions within the development, information gained from cellular
sponge cells. We now believe that jaspamide localisation studies can also be useful in
(Fig. 5) is locatedwithin spongecells andfurther chemotaxonomy and the understanding ofbio-
research is in progress to testthis hypothesis. synthetic pathwaysthatmay have influencedthe
evolution ofsymbioses within sponges.
CONCLUSIONS
ACKNOWLEDGEMENTS
The major conclusion that we have reached
duringourstudies oftherole ofsymbionts inthe
production of sponge metabolites is that it is The earlier studies reported above were taken
extremely dangerous to make any general
from thethesisresearchof JaniceE. Thompson,
statements about the sources of bioactive
MiaUnsonand CaroleA. Bewleyandhavebeen
metabolites. Inessence,eachcompoundofinterest reported in detail elsewhere. We thank the
requires an individual study to determine its
source. The results that we and others have Republic of Palau and the State of Koror for
generated indicate that it is possible to detect permission to collect specimens and the Coral
specific compounds or classes ofcompounds in Reef Research Foundation, Koror, Palau for
either symbiont or sponge cell fractions andthat providing logistical support and laboratory
the concentrations of secondary metabolites in facilities. We also thank Mary Garson for
isolated cell fractions can be spectacularly high. providingus withthe opportunity to do research
However, it is often difficult to determine which atHeron Island. This research program hasbeen
spongecell typeproducesthemetaboliteanditis generously supported by the National Science
nearly impossible to define a particular Foundation (CHE 95-27064).
:
BIOACTIVE METABOLITE LOCALIZATION 173
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