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Apicomplexan Biochemistry Basics Toxoplasma

Good cell biology model Genome sequencing not completed Virtual pathways

Cryptosporidium The “strange one” Genome sequence completed Virtual pathways

Plasmodium Best biochemistry data Numerous studies over many years Genome sequencing completed!

Glycolysis - Plasmodium

Lactate

Pentose Pathway

Pyruvate

PEP

Glucose-6-P

Pyruvate Metabolism

Erythrocytic stage Glucose is major substrate

Infected RBC’s utilize 75%more glucose than uninfected

Lactate is major end product 85% of glucose lactate

High glycolytic rate Enzyme utilize ATP (not PPi) Overall 2 ATP produced for

every glucose utilized

Role of mitochondrion???

Pentose PhosphatePathway

Salvagepathway

Essential biosynthetic

pathwayShikimatepathway

Glycolytic intermediate isutilized (G-6-P)

Reducing equivalentsgenerated

Products utilized forNucleotide metabolism

Products utilized forshikimate pathway

Regenerates someglycolytic intermediates

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Shikimate Pathway

Important pathway that isabsent from animals! Folate synthesis Aromatic amino acid

synthesis Essential for apicomplexan

parasites Large body of data on

biochemistry Bacteria, fungi, plants

Glycolysis

PPP

FolateSynthesis

UbiquinoneSynthesis

TryptophanSynthesis

Mitochondrial Function Simplified View???

Historically thought to bequiescent

Few if any cristae Developmental changes

Asexual stages Single mitochondrion tubular

network Gametocytes

Multiple organelles Basics - Asexual stage

Cytosolic glycolysis Complete complement of TCA

cycle enzymes Electron transport subunits Alternative oxidase

Mito Oxidative Phosphorylation

Apicomplexans

Trypanosomatids

Cryptosporidium

Toxoplasma

Plasmodium

TrypanosomaBloodstream form

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Puzzle of Pyruvate Metabolism

Acetyl-CoA

Pyruvate Dehydrogenase Complex Multienzyme complex (PDC)

E1 - pyruvate dehydrogenase E2 - dihyrolipoyl transacteylase E3 - dihydrolipoyl dehydrogenase

Convert Pyruvate to Acetyl-CoA,generate reducing equivalents

Typically the regulated step committingto TCA cycle

PDC is absent from mitochondriaPDC is present in apicoplastE3 subunits present in mitochondria

Endocytosis of Host Cytoplasm

Pinocytosis Cytostome Endocytic pathway Food Vacuole

Hemoglobin digestion

• heme destabilizes and lysesmembranes• hydrolases released into

parasite cytoplasm• parasite dies

Heme β-hematin

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The Plasmodium Food Vacuole

hemoglobin

+heme globinfragments

small fragments(6-8 amino acids)

hemozoinATP

ADP

ATP

ADP

H+

Fe3+

Fe2+O2

H2O2

H2O + O2

-O2 •O2superoxidedismutase?

catalase?

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plasmepsin

falcipain

plasmepsin

falcilysin

Pfmdr-1?

aminoacids

A specialized lysosome - acidic compartment

Malaria pigmentMalaria pigment

The Power of Genomics Hugely difficult to isolate intact

apicoplasts in pure form. Why?

How do you study the function?

Virtual pathways!

Confirmation!!!!!

Delayed Death Phenotype Ciprofloxacin

inhibited apicoplast DNA replication(T. gondii)

Treatment resulted in: Loss of apicoplast DNA Cell growth inhibited after 1 round of

cell division

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Apicoplast functions - Simplified!

Apicoplast is an essential organelle - delayed death phenotype

Non-photosynthetic!

Transporters:Plant-like

1-deoxy-D-xylulose-5-phosphateDOXP pathway:

Bacteria, chloroplasts

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An Unusual DNA Polymerase

Fatty Acid Synthesis Iterative elongation of

acyl chains Growth of chain by 2 C

Type I (Eukaryotic) Multiple enzymatic

activities on a singlelarge multifunctionalprotein

Type II (Prokaryotic) Each activity is on a

separate polypeptide

Dr. Kim Paul

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Inhibition of Type II FAS

Initially thought: no FAS Type II - prokaryotic

Separate subunits ACC - acetyl CoA carboxylase

Aryloxyphenoxyproprionateherbicides (FOPS)

ACP - acyl carrier protein KAS - keto-acyl ACP synthase

Thiolactomycin Cerulenin

ENR - enoyl reductase Triclosan

The Good, the Bad, the Ugly

What purpose do Triclosan and the Copolymerserve?

Colgate Total is the only toothpaste to contain triclosanwith a copolymer.Triclosan is an effective antibacterial ingredient, whichis effective in controlling a wide range of bacteria.The copolymer is used in the formulation to prolongretention of triclosan on oral hard and soft tissues andin plaque.

Without the copolymer, triclosan would be rapidly lostfrom the mouth, reducing its clinical effect.

Bottom line: (numerous studies) Antibacterial ingredient triclosan degrades rapidly

when exposed to chlorinated tap water, producingpotentially toxic byproducts (chloroform).

The really uglyWhen flushed into water systems, and exposed toultra violet radiation, triclosan converts to a dioxin.The amount of dioxins created is small, however,it's an extremely toxic and stable chemical thatpersists in the environment and is eliminatedslowly from the body.

Sewage sludge - commonly used on farmer’s fields

Isoniazid

1st line anti-tuberculosisInhibits mycolic acid synthesis (mycobacterial cell wall)

Drug resistance is a big problem.Never use Isoniazid alone.

Mycolic acids consist of long-chainalpha-alkyl-beta-hydroxy fatty acids that are

produced by successive rounds of elongation catalyzed by a type II fatty acid synthase (FAS-II).

Apicoplast functions - Simplified!

Apicoplast is an essential organelle - delayed death phenotype

Non-photosynthetic!

Transporters:Plant-like

1-deoxy-D-xylulose-5-phosphateDOXP pathway:

Bacteria, chloroplasts

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Presence of Plastid Fatty acid biosynthesis

Cytosol - animals Plastid

Location of Haembiosynthesis

Close association betweenplastid and mitochondrion

ALA = aminolevulinic acid Early precursor of haem

biosynthesis Mitochondrial!!!!!