amoeba lecture

Entamoeba histolytica

Wrap up nematodes with Dracunculus

Intro into protozoa

Entamoeba, cell biology, disease, and treatment

Why do some amoebae cause disease and others not?

From Exercitationes de Vena Mediensis et de Vermiculis Capilaribus Infantium by G.H. Velschius, published in Augsburg, Germany 1674

dracunculiasis

Guinea worm infection caused by Dracunculus mediensis

3 million cases in 1992 prior to start of eradication program

Now down to 10 thousands, most of them in the Sudan

dracunculiasis

dracunculiasis

Formation of a blister which can burn or itch strongly

The blister bursts and gives rise to an ulcer with the female protruding (triggered by cooling through water contact)

Frequent secondary bacterial infections

dracunculiasis

Embryos are squeezed out in response to cold water

dracunculiasis

dracunculiasis

Drancunculiasis is a preventable disease:

No reservoir Prevent contact of infected

people with water sources Filter water to remove

intermediate host Relatively low cost educational

campaign Go to the web site of the

Carter Center to learn more about this highly successful control/eradication campaign

protozoa

Primary unicellular eukaryotes, often also called protists

Many important human and veterinary pathogens

It is important to understand that protozoa is mostly a historic grouping and not a cohesive biological group that contains closely related organisms

A very diverse group with a vast variety of morphological and biochemical adaptations to almost any ecological niche

the tree of life(Ernst Haeckel, 1874)

protozoa

reptiles

molluscs

man

crustaceans

whales

fish

worms

carnivoresungulates The tree of life (who is

related and how did they evolve) was initially based on morphological characteristics

“Complex” organisms were viewed as derived and highly evolved “simple” organisms as primitive

This scheme puts protozoa as a cohesive group to the bottom of the tree

The three kingdoms of life(Mitch Sogin’s 16s RNA tree)

While the protozoan phyla as evolutionary groups are well defined how they related to each other is not well understood

The most recent models of eukaryotic evolution propose an early split giving rise to animals, fungi and amoeba on one side and plants and most of the other protozoans on the other

Who came first on this tree, who is the most primitive and what the relationships among the phyla are is not well defined

amoebae the most primitive eukaryotes?

Classical taxonomy puts amoebae at the very base of the eukaryotic tree as they lack many derived morphological characters

No special organelles for locomotion (cilia, flagella)

No mitochondrion and no typical mitochondrial enzymes

A fermentative “bacteria-like” anaerobic metabolism

It was assumed that amoeba represent the stage of early eukaryotes before the endosymbiosis event that let to the mitochondrion

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Is the absence of mitochondria a primary of secondary trait?

The genomes of most important protozoan parasites are now fully sequenced

This provides the opportunity to hunt for ‘molecular fossils’

Most proteins that do their job in the mitochondrion are actually encoded in the nucleus and are imported from the cytoplasm

Genome searches in Entamoeba identified several genes for proteins targeted to the mitochondrion in other organisms

Antibodies generated against these proteins identified a small highly reduced mitochondrion - the mitosom

This suggest amoebae had full-fledged mitochondria in the past but reduced them when they moved to an anaerobic environment

For the moment we don’t know a eukaryote featuring a primary lack of mitochondria



what is amoeboid about amoebae?

Amoeboid movement

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Acanthamoebahttp://cmgm.stanford.edu/theriot/movies.htm#Hits

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what is amoeboid about amoebae?



Uroid

Pseudopodium

Hyaline ectoplasm

Endoplasm (sol)

While the endoplasm (sol) is ‘liquid’ andfilled with organelles the ectoplasms appearsmore solid (gel) and clear.

(gel)

Amoeboid movement is not limited to amoeba

Neutrophil chasing a bacterium

What could be the engine and gears powering this movement?

Muscle: actin provides structure

but myosin is the motor

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Ameboid movement is driven by actin

Amoeboid movement depends on the actin cytoskelleton

Earlier models were based on cortical actin/myosin squeezing the cytoplasm to the leading edge (toothpaste tube model), this was thought to be accompanied by cytoplasmic gel/sol transformations

More recent data strongly support actin polymerization as the force generating step (at least for the best understood part of protrusion)

Actin dynamics in amoeboid movement are complex and not easily dissected -- can just polymerizing actin really drive motility?

Listeria as a model to demonstrate and study actin polymerization motility

http://cmgm.stanford.edu/theriot/movies.htm#Hits

Listeria in host cell (150x)

Listeria in Xenopus extract (right panel

Phase contrast, left panel actin-GFP fluorescence)

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The actin polymerization model is based on cell free reconstitution of the movement of intracellular bacteria

These studies allowed to identify the factors involved in the initiation of actin filament polymerization

Entamoeba histolytica

Fedor Alexandrewitch Lösch describes amoebae associated with severe dysentery in a patient in 1873

He transferred amoebae to a dog by rectal injection, which became ill and showed ulceration of colon

Patient who died from infection showed similar ulcers upon autopsy

trophozoites and cysts


multiple well defined pseudopodia often extended eruptively

Differentiation into endo- and ectoplasm

Spherical nucleus (4-7 m) with small central nucleolus and characteristic radial spokes


Trophozoites 20-40 m diameter

Ribosomes arranged in helical patterns

Tissue forms often contain phagocytized red blood cell


Trophozoites encyst and cysts mature as they travel through the colon

Only mature cysts are infective


Chromidial bodies and bars are semicrystalline arrays of riobosomes

Round (10- 16 m), 4 nuclei

150 nm cyst wall with fibrillar structure

Entamoeba cysts (light microscopy)

E. coli E. histolytica

Human infection

Major sources for human infection are contamination of drinking water and vegetables (fertilization with material containing or contaminated with human feces)

Patients without any symptoms might nevertheless shed large amounts of cysts

If kept cool and moist (water or soil) cysts can stay infectious for up to a month

Cysts are fairly resistant to chlorination of drinking water (10 mg/l versus 0.1 - 1.0 mg/l for enteric bacteria)

Humans harbor numerous amebae (most are nonpathogenic)

Colitis is the most common form of disease associated with amoebae

Gradual onset of abdominal pain, watery stools containing mucus and blood

Some patients have only intermittent diarrhea alternating with constipation

Fever is uncommon Formation of ulcers

Colitis is the most common form of disease associated with amoebae

Amoeba invade mucosa and erode through laminia propria causing characterisitic flask shaped ulcers contained by muscularis

Ulceration can lead to secondary infection and extraintestinal lesions

Extraintestinal amebiasis

Amebic liver abscess

Most common form of extraintestinal amebiasis

Fast growing abscess filled with debris, amoebae are found only at borders

Lead symptoms are are right upper quadrant pain and fever

Acute as well as chronic illness, with gradual or sudden onset

Amebic liver abscess

30-50% of patients with liver abscess show also pneumonic involvement

Rupture is again a major thread, especially rupture into the pericardium

Draining abscesses is today only performed in extreme cases when rupture is feared

Responds well to chemotherapy

Metronidazole is the drug of choice for extra-intestinal amebiasis

Several drugs are available to clear symptomatic and asymptomatic enteric (luminal) infection (e.g. dichloroacetamides which have unknown mode of action)

Metronidazole (Flagyl) is the drug of choice for invasive amoebiasis (and should be combined with a lumen acting drug as it is not fully effective on luminal stages)

Metronidazole is a prodrug which is activated by an enzyme involved in the microaerobic fermentation metabolism of E. histolytica

Amoebae use fermentation

“La fermentation est la vie sans l’air” (Louis Pasteur)

Entamoeba lacks a functional Krebs cycle and oxidative phosphorylation

Final endproducts of E. histolytica fermentation are CO2, acetate, ethanol and alanine

Metronidazole is activated by PFOR

acetate

CO2

ADPATP



Entamoeba uses a pyruvate ferredoxin oxidoreductase (PFOR) to break down pyruvate

This process depends on the absence (or low level) of oxygen

This enzyme system is limited to anaerobic bacteria and some protozoa and humans lack this enzyme

PFOR and ferredoxin can transfer an electron to metronidazole producing a highly toxic nitroradical

Drugs which are not toxic but have to be activated into a toxic compound are called prodrugs (look at this as a suicide substrate)

Epidemiology of Entamoeba

480,000,000 people harbor Entamoeba36,000,000 develop clinical symptoms40,000 - 100,000 deaths per year

(Walsh, 1986, Rev. Infect. Dis., based on 1981 data, no significant change since then)

Less than 10% of the people infected show disease. Several hypotheses have been put forward to explain this differential pathogenesis.

Commensal hypothesis

E. histolytica usually is a benign gut commensal as many other amoebae (minuta form)

A certain stimulus (gut flora, diet, host immune status …) transforms the organism into a pathogen (magna form, Kuenen, 1913)

This has been the accepted view for most of the 20th century

Two species hypothesis There are two morphologically

indistinguishable species: E. histolytica and E. dispar. Only one of them (hystolytica) causes disease while the other is benign (Brumpt, 1928)

This theory was discounted and ridiculed

Recent molecular data have revived the two species hypothesis

We now know that most people are infected with the apathogenic E. dispar

Emile Brumpt 1877-1951

Genetic evidence for two species

Species specific isoenzyme patternsMultiple antibodies specific for either the

pathogenic or apathogenic speciesNumerous genes sequenced which show clear

differencesRepetitive DNA elements are differentGenomic organization of conserved gene loci

like actin is differentRibosomal RNA (2.2% difference)

Pathogenic amoeba show contact dependent killing

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Pathogenic amoeba show contact dependent killing

Entamoeba pathogenesis factors

What are pathogen proteins (and genes) that are required to cause disease?

Several candidates have been studied for their involvement in contact dependent cell killing by amoeba:

The surface lectins: These are proteins that allow the amoeba to bind to sugars on the surface of cells and establish tight contact

Proteases: several protein degrading enzymes have been linked to tissue penetration and liver abscess formation

Amoebapores: protein toxins that perforate target cells

Amoebapores one of the candidate pathogenicity factors

Family of small (77 AA) proteins contained in secretory granules

Similar in structure and function to NK lysins

Used to kill bacteria and host cells

Amoebapores insert into target membranes and form ion channels

Amoeba mutants which make less amoebapores cause less disease in animal model studies

amoeba lecture

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