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Bioactive Compounds from Marine Microbes. Bioactive Compounds from Algae (Part 2) Cyanobacteria (“Blue-Green Algae”) Bioactive Compounds from (Other) Marine Bacteria Bioactive Compounds from Marine Fungi. Algae. Monera (“Prokaryotes”). Protista. Plantae. Fungi. Animalia. Algae.

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Bioactive Compounds from Marine Microbes


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slide1

Bioactive Compounds from Marine Microbes

Bioactive Compounds from Algae (Part 2)

Cyanobacteria (“Blue-Green Algae”)

Bioactive Compounds from (Other) Marine Bacteria

Bioactive Compounds from Marine Fungi

slide2

Algae

Monera

(“Prokaryotes”)

Protista

Plantae

Fungi

Animalia

slide3

Algae

Prokaryotic

Kingdom Monera (Bacteria)

Cyanobacteria (“Blue-Green Algae”)

Eukaryotic

Kingdom Protista

Dinophyta (“Dinoflagellates”)

Raphidophyta (“Raphidophytes”)

Bacillariophyta (“Diatoms”)

Chrysophyta (“Golden Algae”)

Chlorophyta (“Green Algae”)

Phaeophyta (“Brown Algae”)

Rhodophyta (“Red Algae”)

“Microalgae”

“Macroalgae”

slide5

Eukaryotic HAB Toxins

Syndrome Source SpeciesTarget

Domoic Acid “Amnesic Shellfish Pseudonitzschia spp. Glutamate Poisoning” (ASP) Receptors

Saxitoxin “Paralytic Shellfish Alexandrium tamarense Inhibits

Poisoning” (ASP) Sodium Channels

Brevetoxin

“Neurotoxic Shellfish Karenia brevis Activates

Poisoning” (ASP) (and others) Sodium

“Florida Red Tide” Channels

Ciguatoxin

(and others)

“Ciguatera FishGambierdiscus toxicus Activates

Poisoning” (CFP) Sodium

Channels

Pectenotoxin

(and others)

“Diarrhetic Shellfish Dinophysis spp. ?

Poisoning” (CFP)

slide6

Polyketides from Dinoflagellates

“Polyether Ladders”

e.g. PbTx-1

Macrolides

Linear Polyethers

e.g. PTX-1

e.g. Okadaic Acid

slide7

Polyketide Synthases (PKSs) are Modular Enzymes

Enoyl reductase (ER)

NADPH

NADP+

b-ketoacyl synthase (KS)

Dehydrase (DH)

b-ketoacyl reductase (KR)

CO2

H2O

slide8

Polyketide Synthase (PKS)

Acyl

Carrier

Protein

(ACP)

Acyl Transferase (AT)

Enoyl Reductase

(ER)

Dehydratase (DH)

Ketosynthase (KS)

Ketoreductase (KR)

AT

Organism PKSI PKSII

Prorocentrum

lima + +

P. hoffmanianum + -

Karenia brevis + -

Symbiodinium sp. + -

Amphidinium

operculatum + -

KS

AT

ACP

PKSI

Primers

Snyder et al. (2003) Mar. Biotechnol., 5 (1):1-12.

slide9

Prokaryotic Algae

Cyanobacteria

(“Blue-Green Algae”)

Monera

(“Prokaryotes”)

Protista

Plantae

Fungi

Animalia

slide10

Cyanobacteria (“Blue-Green Algae”)

Photosynthetic Bacteria

Oldest Organisms on the Earth

(Fossil Record - 3.5 Billions Years!)

Marine, Freshwater and Terrestrial

Symbiosis - e.g. Lichen

Stromatolites

slide11

Pigments and Algae

PBs4

Chl a1bcdCar. 2Xanth.3PC5PE6

Cyanobacteria x x x

Dinoflagellata x x x x

Bacillariophyta x x x x

Chrysophyta x x x x

Chlorophyta x x x

Phaeophyta x x x x

Rhodophyta x x x x

1Chl = Chlorophyll; 2Car. = Carotenoids; 3Xanth. = Xanthophylls; 4PB = Phycobilins (Phycobiloproteins); 5PC = Phyocyanin; 6PE = Phycoerythrin

slide13

Cyanobacteria as HABs

First Scientific Report of Toxic Cyanobacteria:

George Francis (1878) Nature

Lake Alexandria, Murray River, Australia

“thick scum like green oil paint, some two to six inches thick,

and as thick and pasty as porridge”

“Unwholesome” for cattle and other livestock that drink at the water

Nodularia spumigens

slide14

Cases of Acute Poisoning by Toxic Cyanobacteria in Drinking Water

L. Alexandria, Australia Livestock Poisoning Nodularia spumigens

1931 Charleston, WV Acute Gastroenteritis Unknown

(Ohio River) (9,000 cases/60,000 pop.)

1966 Harare, Zimbabwe Gastroenteritis (children) Microcystis aeruginosa

1975 Sewickley, PA Acute Gastroenteritis Schizothrix calcicola

(62% of 8,000 pop.)

1983 Armidale, Australia Liver damage (elevated M. aeruginosa

g-glutamyltransferase)

1983 Palm Island, Australia Hepatoenteritis C. raciborskii

(139 children) (cylindrospermopsin)

1993 Itaparica Dam, Brazil Gastroenteritis Anabaena, Microcystis

(88 deaths, children)

1996 Caruaru, Brazil Liver failure Aphan., Oscillatoria

(63 deaths) (microcystins)

slide15

Toxins from Cyanobacterial HABs

Neurotoxins

Hepatotoxins

Dermatotoxins

slide16

Neurotoxins from Cyanobacteria: Anatoxin-a

1950s-1960s Paul Gorham and Colleagues

Cultured Anabaena flos-aquae

Isolated “Very Fast Death Factor”

Anatoxin-a

slide17

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

Acetylcholine

Receptor

(AChR)

Synapse

Acetylcholine (in vesicles)

slide18

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

+ + + + + + + +

- - - - - - ++ --

K+

Ca2+

Na+

slide19

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

+ + + + + + + +

- - - - - - ++ --

K+

Ca2+

Na+

slide20

Ca2+

SR

Ca2+

PKC

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

Na+

+ + + + + + + +

- - - - - - ++ --

K+

Ca2+

Na+

slide22

Ca2+

SR

Ca2+

PKC

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

Na+

+ + + + + + + +

- - - - - - ++ --

K+

Ca2+

Na+

slide23

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

Na+

+ + + + + + + +

- - - - - - ++ --

Acetylcholinesterase

K+

Ca2+

Na+

slide24

Ca2+

SR

Ca2+

PKC

Anatoxin-a Irreversibly Binds Acetylcholine Receptors and Inhibits Acetylcholinesterases

Anatoxin-a

Na+

+ + + + + + + +

- - - - - - ++ --

K+

Ca2+

Na+

slide27

Anatoxin-a(s) Inhibits Cholinesterases

LD50 = 20 µg/kg (in mice)

vs.

LD50 of Anatoxin-a = 200 µg/kg (in mice)

slide28

Neurotoxins from Cyanobacteria: Saxitoxin

R4:

R1R2R3

H H H STX GTX5

H H OSO3- GTX2 C1

H OSO3- H GTX3 C2

OH H H NeoSTX GTX6

OH H OSO3- GTX1 C3

OH OSO3- H GTX4 C4

Saxitoxin (STX) and Other “PSP Toxins”

slide29

= STX (or TTX)

STX Binds and Blocks Voltage-Gated Sodium Channels

slide31

Microcystin Structural Diversity

Over 70 Variants of MIcrocystins!

slide32

Microcystin Diversity

Microcystin-LR

slide34

Microcystins (and Nodularin) Accumulate in Liver

Approximately 50% of Microcystin in Liver

ATP-Dependent Carrier-Mediated Transport

slide35

Microcystins and Primary Liver Cancer (PLC)

Nandong District, Jiangsu Province, China

Pond/ditch vs. Well as Drinking Water Supply

~ 24-Fold Higher Rate of PLC with Pond/Ditch Water

100.13 cases per 100,000 (pond/ditch)

4.28 cases per 100,000 (well)

Microcystin Levels:

Pond/Ditch: 60% samples positive, avg. 160 pg/mL

Well: None detected

Calculated 0.19 pg/day Seasonal Intake (4 mo./yr.)

Yu, 1989, Primary Live Cancer, pp. 30-37; Harada et al., 1996, China Nat. Toxins, 4: 277-83; Ueno et al., 1996, Carcinogenesis 17: 1317-21

slide36

Microcystins (and Nodularins) are PP1/2a Ser/Thr Protein Phosphatase Inhibitors

MacKintosh et al. (1990) FEBS Lett. 264: 187-92.

slide37

Non-Ribosomal Peptides are Characteristic of Cyanobacterial Toxins

Methyl-dehydro-Ala

(Mdha)

Iso-D-Glu

Adda

D-Ala

Leu

Arg

Iso-MeAsp

slide38

Non-Ribosomal Peptide Synthetases (NRPSs) are Modular Enzymes

A = Adenylation Domain

C = Condensation Domain

T (PCP) = Thiolation (Peptidyl Carrier) Domain

slide42

Drugs from Cyanobacteria: Curacin-A

Curacin A

Curacin A Binds Colchicine Site of Tubulin and Inhibits Tubulin-Polymerization

slide45

Cryptophycin 52 in Clinical Trials (Phase II)

Clinical Response of Women with Ovarian Cancer Treated with LY355703 (n=24)

ResponseNo.%

Complete Remission 0 0

Partial Remission 3 12.5

Stable Disease 7 29.2

Progressive Disease 14 58.3

D’Agostino et al. (2006) Intl. J. Gyn. Cancer 16: 71-76.

slide47

Bioactive Compounds from Heterotrophic Marine Bacteria

Monera

(“Prokaryotes”)

Protista

Plantae

Fungi

Animalia

slide48

Bacteria: Kingdom Monera

Shape

Cocci = Sphere-Shaped

Bacilli = Rod-Shaped

Spirilla = Spiral-Shaped

“Growth Form”

Staph = Bacteria in Clusters

Strep = Bacteria in Chains

slide49

Classification of the Bacteria

(i.e. Kingdom Monera)

Gram-Negative

Gram-Positive

slide50

Classification of the Bacteria

(i.e. Kingdom Monera)

Eubacteria

Gram-Positive Bacteria w/ Cell-Walls

Gram-Negative Bacteria w/ Cell-Walls

Bacteria w/o Cell-Walls (Mycoplasma)

Archaebacteria

slide51

Cyanobacteria are Eubacteria

Gram-Stained Cyanobacteria - NEGATIVE

slide52

Lipopolysaccharides (LPSs) from Cell-Wall of Gram-Negative Bacteria are “Endotoxin”

“O-Region”: Repeating tri-, tetra- or pentasaccharides (up to 25)

Outside Cell

Polysaccharide “Core”

Cell Wall

Lipid A

Inside Cell

slide53

Actinomycetes: A Rich Source of Bioactive Compounds

Approximately Half of Bioactive Metabolites!!

Approximately 60% of Antibiotics

Streptomyces spp.

slide54

Actinomycetes: A Rich Source of Bioactive Compounds

Antibiotics from Streptomyces

AntibioticSource

Amphotericin B Streptomyces nodosus

Erythromycin S. erythreus

Neomycin S. fradiae

Streptomycin S. griseus

Tetracycline S. rimosus

Vancomycin S. orientalis

Rifamycin S. mediterranei

slide55

Marine Actinomycetes

Salinospora spp.

Salinosporamide A

(NPI-0052)

Feling et al. (2003) Agnew Chem Int Ed Engl, 42:355-7

slide56

Are Salinospora “True” Marine Bacteria?

Jensen et al. (1991) Appl. Environ. Microbiol., 57: 1102-8.

slide57

Salinosporamide A Inhibits the Proteasome

Salinsporamide A

Omuralide

Macherla et al. (2005) J. Med. Chem. 48: 3684-7.

slide58

Salinosporamide A Inhibits the Proteasome

Macherla et al. (2005) J. Med. Chem. 48: 3684-7.

slide59

The Proteasome Degrades Short-Lived and Abnormal Proteins

19S “Caps”

(Regulatory)

26S Proteasome

20S Proteasome

(Proteolytic)

slide60

Proteins Marked for Degradation by Ubiquitinylation

-Helix

-Strands

Ubiquitin (Ubq)

76 Amino Acids (Mol. Mass 8500)

Two C-Terminal Gly -> Bind to Lys of Proteins

Gly Bind Lys of Other Ubq = Polyubiquitinylation

slide62

Salinosporamide A Inhibits NF-kB Activation

Macherla et al. (2005) J. Med. Chem. 48: 3684-7.

slide64

Bioactive Compounds from Salinospora and Marine Actinomycetes

Taken from Jensen et al. (2005)

slide66

Fungi: The First Source of Antibiotics

1928 - Alexander Fleming Discovers Inhibition of Bacteria by Penicillum notatum

Penicillin G

Fleming (1929) Br. J. Exp. Pathol. 10: 226

1939 - Florey and Chain Cure Mice of Bacterial Infection with Penicillin Injection

…by the end of World War II, U.S. companies were making 650 billion units/month

slide67

Antibacterial Compounds from Marine Fungi: Pestalone

Inhibit methicillin- and vancomycin-resistant Enterococcus faecium (MIC = 37 and 78 ng/mL, respectively)

Pestalone

(from Pestalotia sp.)

slide69

Antimalarial Compounds from Marine Fungi: Aigialomycin D

Aigialus parvus

(isolated from mangrove)

Aigialomycin D

slide70

Antimalarial Compounds from Marine Fungi: Aigialomycin D

P. falciparum K1

Compound(IC50, µg/mL)

Aigialomycin A >20

Aigialomycin B >20

Aigialomycin C >20

Aigialomycin D 6

Aigialomycin E >20

slide71

Antimalarial Compounds from Marine Fungi: Ascosalipyrrolidinone A

Ascochyta salicorniae

(symbiont with green alga, Ulva)

Ascosalipyrrolidinone A

slide72

Culture of Bacteria

(and other microbes)

slide73

“Non-Culturability” of Bacteria

Approximately 99% of Heterotrophic Bacteria are Non-Culturable

Required Nutrients, Conditions and Other Factors Unknown

Metagenomics – NRPS, PKS

slide74

Metagenomic Analysis of Bacteria from Discodermia dissoluta

PKS

NRPS

Filamentous

Unicellular

Shirmer et al. (2005) Appl. Environ. Microbiol., 71:4840-9.