Interkingdom signaling by structurally relatedcyanobacterial and algal secondary metabolites

Lena Gerwick • Paul Boudreau • Hyukjae Choi • Samantha Mascuch •

Francisco A. Villa • Marcy J. Balunas • Karla L. Malloy • Margaret E. Teasdale •

David C. Rowley • William H. Gerwick

Received: 31 October 2011 / Accepted: 12 May 2012 / Published online: 30 May 2012

� Springer Science+Business Media B.V. 2012

Abstract Several groups of structurally-related

compounds, comprised of either five or six-membered

ring structures with attached lipophilic carbon chains

and in some cases possessing halogen atoms, have

been isolated from various marine algae and filamen-

tous cyanobacteria. The related compounds consid-

ered in the present work include the coibacins,

laurenciones, honaucins, malyngamides and the tumo-

noic acids. Members of all of these compound families

were assayed and found to inhibit the production of

nitric oxide in lipopolysaccharides-stimulated macro-

phages, indicating their anti-inflammatory potential.

In addition, several of these same marine natural

products were found to inhibit quorum sensing

mediated phenotypes in Vibrio harveyi BB120 and/

or Escherichia coli JB525. The mechanism and

evolutionary significance for inhibition of these cel-

lular processes in prokaryotic and eukaryotic systems

are speculated on and discussed.

Keywords Quorum sensing � Anti-inflammatory �Lactone � Acyl chain � Marine natural products

Introduction

Hundreds of marine natural products (secondary

metabolites) have been isolated from cyanobacteria

and macroalgae, making these phyla rich sources for

new compound discovery. In particular, many of these

natural products have been submitted for bioactivity

screening and potential development as anti-cancer

agents (Tidgewell et al. 2010). This emphasis on

cancer could partially result from priorities set by the

funding agencies and partially from the fact that many

natural products are found to be cytotoxic (Nagle and

Paul 1999; Nagarajan et al. 2012). However, many of

the natural products isolated display other types of

bioactivity, for example, quorum sensing inhibition,

anti-microbial activity, anti-inflammatory activity or

inhibition or activation of neuronal receptors or ion

L. Gerwick (&) � P. Boudreau � H. Choi �S. Mascuch � F. A. Villa � K. L. Malloy � W. H. Gerwick

Center for Marine Biotechnology and Biomedicine,

Scripps Institution of Oceanography, University of

California San Diego, 9500 Gilman Dr MC 0212,

La Jolla, CA 92093, USA

e-mail: lgerwick@ucsd.edu

F. A. Villa

Department of Chemistry, Western Arizona College,

Yuma, AZ, USA

M. J. Balunas

Division of Medicinal Chemistry, Department of

Pharmaceutical Sciences, University of Connecticut,

Storrs, Mansfield, CT, USA

M. E. Teasdale � D. C. Rowley

Department of Biomedical and Pharmaceutical Sciences,

University of Rhode Island, Kingston, RI, USA

W. H. Gerwick

Skaggs School of Pharmacy and Pharmaceutical Sciences,

University of California San Diego, La Jolla, CA, USA

123

Phytochem Rev (2013) 12:459–465

DOI 10.1007/s11101-012-9237-5

channels (Choi et al. 2012; Villa et al. 2010; Mo et al.

2009; Li et al. 2001).

Chronic inflammation has been implicated as one of

the causes underlying such common diseases such as

cancer, arthritis, heart diseases, skin diseases, asthma

and inflammatory bowel disease (Adcock et al. 2008;

Grivennikov et al. 2010). However, our supply of anti-

inflammatory treatments is quite limited. At this point,

the corticosteroids and the non-steroidal anti-inflam-

matory drugs (NSAIDs) are the most commonly used;

however, these options can have severe side effects.

More small molecule anti-inflammatory agents, which

have different modes of action, are needed to treat

chronic inflammation, both as a disease prevention and

a cost containment measure.

Quorum sensing (QS) in bacteria is a concentration

dependent process that involves intercellular signal-

ing. Both Gram-negative and Gram-positive bacteria

emit chemical signals that promote or disrupt their

own cellular responses such as sporulation, swarming,

bioluminescence, DNA transfer, biofilm formation,

production or repression of virulence factors and other

secondary metabolites (Pappas and Winans 2003;

Zhang et al. 2002; Ni et al. 2009; Ng and Bassler

2009). Molecules known as autoinducers regulate

gene expression during these QS events. The activa-

tion of the QS physiological response is dependent

upon the concentration of the autoinducer reaching a

certain threshold, below which activity is not induced

(Teng et al. 2011). Biofilm formation and other QS

related responses can increase the pathogenicity of

certain bacteria, and thus, there is a strong interest in

finding inhibitors of the QS response. For example, in

cystic fibrosis (CF), colonization and QS-induced

biofilm formation of the lung by Pseudomonas

aeruginosa leads to chronic pneumonia, a condition

that has few effective treatments at present (Drenkard

and Ausubel 2002). Biofilm formation is also a

persistent problem on the surfaces of indwelling

catheters and QS inhibitors have been used to prevent

these from developing (Thomsen et al. 2011). As

exemplified above, QS inhibitors can be useful

therapeutic agents that inhibit biofilm formation

without inhibiting growth, thus circumnavigating the

pitfalls of developing antimicrobial resistance (Ni

et al. 2009; Galloway et al. 2011).

Several cyanobacterial and algal compounds have

been identified in recent years that possess activity in

both inhibition of QS as well as the production of nitric

oxide (NO) in macrophages. Some of the compounds

that inhibited the NO production were also tested for

potential anti-oxidant activity and none so far has

exhibited this property, hence indicating an intracel-

lular mechanism of action for these compounds. In

comparing this set of anti-inflammatory natural prod-

ucts, it is striking that they possess similar structural

features comprised of either five- or six-membered

rings which have an attached hydrophobic carbon

chain. Intriguingly, the Gram-negative QS modulating

molecules, characterized as acyl homoserine lactones,

are structurally similar to the cyanobacterial and algal

compounds described in this review, and have also

been shown to possess anti-inflammatory properties

(Telford et al. 1998; Kravchenko et al. 2008). As a

result, this structure type has been identified as having

inter-kingdom signaling activity. Below, we describe

several cyanobacterial and algal metabolites which

have similar structural features and biological proper-

ties as modulators of both eukaryotic inflammation

and bacterial quorum sensing.

Laurenciones

Laurencione was first isolated and characterized from

the Oregon red alga Laurencia spectabilis (Bernart et al.

1991). Subsequently, Lowery et al. (2005) determined

that laurencione could induce bioluminescence in the

Vibrio harveyi MM30 mutant system [this mutant is

unable to produce autoinducer-2 (AI-2)] indicating that

laurencione can act as a ligand for LuxP. To further

investigate the biological activity of laurencione, we

synthesized the natural product as well as the mono and

diacetate analogs (Fig. 1). The natural product and

diacetate derivatives were prepared according to liter-

ature procedures, and matched 1H NMR, 13C NMR, and

HR-MS data previously recorded (Bernart et al. 1991;

Aelterman et al. 1997). The monoacetate was obtained

as a minor byproduct of the laurencione one-step

synthesis reported by Aelterman et al. in which glacial

acetic acid replaced dioxane/water as the reaction

solvent [yellow-green oil; IR (neat) vmax 2,970, 1,739,

1,718, 1,421, 1,366, 1,242, 1,090, 1,042, 909, 818,

580 cm-1; 1H NMR (500 MHz, CDCl3) 4.37

(t, J = 5.0), 3.06 (t, J = 5.0), 2.37 (s), 2.03 (s); 13C

NMR (125 MHz, CDCl3) 196.8, 196.1, 171.1, 59.1,

35.5, 23.5, 20.9; HR-ESI-TOF–MS [M ? MeOH ?

Na]? m/z 213.0734 (calcd for C8H14NaO4 213.0733)].

460 Phytochem Rev (2013) 12:459–465

123

Using an assay that measures acyl-homoserine lactone

(AHL) induced green fluorescent protein (GFP) pro-

duction by Escherichia coli JB525 (Anderson et al.

2001; Teasdale et al. 2009), a decrease in the fluores-

cence signal by 50% was observed when *600 lM

laurencione was added. In addition, the two acetate

analogs, laurencione monoacetate and diacetate were

found to be slightly more potent since they reduced

fluorescence in the JB525 strain (IC50 * 150 lM and

*55 lM, respectively) (Table 1). Laurencione and the

mono and diacetate analogs were also assayed for their

potential anti-inflammatory activity by measuring their

ability to inhibit NO production in lipopolysaccharides

(LPS)-stimulated macrophages following the methods

described in Villa et al. (2010). All three of these

compounds inhibited NO production with IC50 values

ranging from 15 to 25 lM (Table 1).

Coibacins

The coibacin series of natural products was isolated

from a marine filamentous cyanobacterium (Oscill-

atoria sp.) collected near the island of Coiba on the

Pacific coast of Panama. In total, four compounds

were isolated, coibacin A–D (Fig. 1). The coibacins

consist of a six-membered lactone ring that is

substituted at C5 with an unsaturated acyl chain that

varies in length (Fig. 1). Coibacins A, B and D were

not assayed using the Vibrio harveyi BB120 quorum

inhibition assay (Teasdale et al. 2009) due to limited

supply of these compounds; however, to our surprise

coibacin C did not show any inhibition of biolumi-

nescence in this assay system. The possibility exists

that the coibacins could be activators of biolumi-

nescence; however, the above mentioned assay

utilizing the Vibrio harveyi MM30 assay is not

currently available to us. Nevertheless, coibacins

A–D were found to inhibit NO production of LPS-

stimulated macrophages with IC50’s of 20, 5, 11 and

21 lM, respectively (Balunas et al. in progress). The

length of the acyl chain with its imbedded cyclo-

propyl ring appears to be important for modulating

the level of activity in this series (Fig. 1). The

coibacins are biosynthetically interesting compounds

due to their variations in acyl chain length, number

and location of double bonds, and terminal cyclo-

propyl rings versus vinyl chloride functionalities

(Gu et al. 2009a, b).

Honaucins

Honaucins A–C were isolated from a bloom of the

cyanobacterium Leptolyngbya crossbyana that over-

grows corals off the coast of the Big Island in Hawaii.

Honaucin A (Fig. 1) was found to inhibit the production

of NO in the macrophage assay with an IC50 of 4.0 lM,

making it one of the most potent natural product

inhibitors that we have identified to date (Choi et al. in

press). In addition, honaucin A potently inhibits the

production of bioluminescence (IC50 5.6 lM) in the

Vibrio harveyi BB120 system (Table 1). We speculate

that honaucin B and C may be artifacts of the isolation

process; however, both of these molecules inhibit

quorum sensing at IC50 values of 17.6 and 14.6 lM as

well as the production of NO with IC50 levels of 4.5 and

7.8 lM, respectively (Choi et al. in press).

Malyngamides

The malyngamide series of natural products have been

isolated from various marine filamentous cyanobac-

teria, mainly from the genus Moorea (formerly

Lyngbya) (Engene et al. 2012). Most of these are

composed of a fatty acyl chain attached to an oxidized

cyclohexyl ring, and these appear to biosynthetically

derive from a mixed Non-Ribosomal Peptide Synthe-

tase (NRPS) and Polyketide Synthase (PKS) pathway.

The first malyngamide structure was isolated from

Lyngbya majuscula in 1979, and since then, at least 28

malyngamide analogs have been isolated from loca-

tions as diverse as Hawaii, Curacao in the Caribbean,

Papua New Guinea, Florida, Puerto Rico and Mada-

gascar (Cardellina et al. 1979; Kwan et al. 2010;

Malloy et al. 2011; Nagarajan et al. 2012). Malynga-

mide C acetate, F, F acetate, H, I, J, K, L and T were

each evaluated in the LPS-stimulated macrophage

assay, but only malyngamide F (5.4 lM) and F acetate

(7.1 lM) inhibited NO production with IC50 values in

the low lM range (Fig. 1, Table 1)(Villa et al. 2010).

The selective inhibition of NO production by just these

two malyngamides revealed the importance of the

hydroxy or acetoxy substituents at C-6 of the cyclo-

hexenone ring [e.g. malyngamide K, which lacks a

substituent at that position, showed no inhibition of

NO (Villa et al. 2010)]. Changes in transcription of

Interleukin 1 (IL1), Interleukin 6 (IL6), tumor necrosis

factor-alpha (TNF-alpha) and inducible nitric oxide

Phytochem Rev (2013) 12:459–465 461

123

synthase (iNOS) were determined in the RAW 264.7

macrophage cell line with and without exposure to

malyngamide F acetate. All of these cytokine and

inflammatory protein transcripts were down-regulated

except for TNF-alpha which was modestly upregu-

lated. Further experiments determined that malynga-

mide F acetate inhibits the MyD88 dependent pathway

(Villa et al. 2010). Of this series, only 8-epi malynga-

mide C has been assessed for quorum sensing

inhibition; it was very modestly active in the E. coli

JM109 pSB1075 assay system (IC50 * 1,000 lM)

(Kwan et al. 2010). However, further testing of

malyngamides F and F acetate in various QS inhibition

assays is needed to determine if these also act as

interkingdom signaling molecules.

Tumonoic acid

The first tumonoic acids were isolated from a mixed

assemblage of Lyngbya majuscula and Schizothrix

calcicola collected at Tumon Bay, Guam, and through

Fig. 1 Structures of the

natural products discussed

462 Phytochem Rev (2013) 12:459–465

123

biological screening, found not to be cytotoxic. Five

tumonoic acids were isolated in total, tumonoic acid A,

B, C, methyl tumonoate A and methyl tumonoate B

(Harrigan et al. 1999). The tumonoic acids, classified as

acyl proline derivatives, structurally resemble the AHL

found in many Gram-negative bacteria. Subsequently,

Clark et al. (2008) isolated tumonoic acids D through I

from a collection of Blennothrix cantharidosmum from

Papua New Guinea. Because of their structural similar-

ity to AHL, these natural products were evaluated for

their ability to inhibit QS regulated bioluminescence

using the Vibrio harveyi BB120 system. Modest

inhibition was determined for all six of the compounds

with tumonoic acid F being the most potent inhibitor

(IC50 = 62 lM; Table 1). More recently, from a survey

of the filamentous marine cyanobacteria found growing

at different locations around the island of Curacao, the

tumonoic acid derivative ethyl tumonoate A was

isolated. This derivative was found to inhibit NO

production in the LPS-stimulated macrophages with

an IC50 of 9.8 lM (Engene et al. 2011).

Conclusion

Several different classes of marine compounds exhib-

iting structural similarities were isolated from cyano-

bacteria and a red alga. These compounds were

assessed for their anti-inflammatory activity and QS

antagonism. The laurenciones, the malyngamides, the

honaucins, the coibacins and the tumonoic acids all

consist of a five or six membered ring that is highly

oxygenated and possesses an acyl chain of varying

length and possessing different substituents such as

halogen atoms. These various natural product classes

show structural resemblance to the AHLs, known QS

signaling molecules that have been isolated from

bacteria such as Vibrio and Pseudomonas sp. Some

AHLs have also been shown to decrease cytokine

production in mice (Kravchenko et al. 2008), hence

indicating their anti-inflammatory properties. How-

ever, it remains uncertain if the compounds isolated

from cyanobacteria function as quorum sensors or

inhibitors in their natural environment. Moreover, it is

unknown if they naturally function as inhibitors of the

innate immune system found in marine invertebrates.

We speculate that for filamentous marine cyanobac-

teria and algae it might be of evolutionary advantage

to produce a single molecule that can interact with

both prokaryotic and eukaryotic life forms, thus being

able to prevent biofilm formation by competing

microorganisms and at the same time down-regulate

the innate immune system of marine invertebrates

with which they may associate. For example on corals,

these compounds might give settling and growth

advantages to the cyanobacteria or algae. This

hypothesis needs to be tested more rigorously by

using purified compounds, bacteria common in the

Table 1 Bioactivity levels

of marine algal and

cyanobacterial natural

products in the anti-

inflammatory and quorum

sensing inhibition assays

* Yet to be assayed# Inhibition of nitric oxide

production in RAW 264.7

macrophages

Compound MW Inhibition of NO

(IC50, lM)#Inhibition of quorum sensing (lM)

Vibrio harveyi

BB120

Escherichia coli

JHB525

Honaucin A 204.6 4 5.6 38.5

Honaucin B 250.1 4.5 17.6 908

Honaucin C 236.0 7.8 14.6 576

Coibacin A 284.4 20 * *

Coibacin B 258.4 5 * *

Coibacin C 266.8 11 * Inactive

Coibacin D 268.8 21 * *

Laurencione 102.1 25 * *612

Laurencione monoacetate 158.2 15 * *150

Laurencione diacetate 200.2 18 *100 *55

Tumonoic acid A 339.2 9.8 * *

Tumonoic acid F 525.3 * 62 Inactive

Malyngamide F 439.0 5.4 *

Malyngamide F acetate 481.0 7.1 * *

Phytochem Rev (2013) 12:459–465 463

123

coral environment, coral larvae and perhaps coral

coelomocytes. However, several of the described

natural products show potent inhibition of QS medi-

ated phenotypes and inhibition of an inflammatory

response. Some of these compounds are being further

evaluated for their mechanism of action and in vivo

efficacy because there exists a great need for devel-

oping biofilm inhibitors as well as new classes of anti-

inflammatory therapeutics.

Acknowledgments This research was partially funded by the

International Cooperative Biodiversity grant (U01 TW006634),

the Ledger Benbough Foundation to L.G, NIH/FIC International

Research Scientist Development Award (IRSDA) to M.J.B.,

NIGMS Training grant in marine biotechnology to S.M., NIH/

NIGMS Institutional Research and Academic Career Develop-

ment Award (IRACDA) fellowship to F.V. and an E.W Scripps

Fellowship to P.B.

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