Protozoa

Protozoa, the tree of life and the origin of eukaryotes

What makes an ameba an ameba?Entamoeba histolytica infection and

pathogenesis

protozoa

Primary unicellular eukaryotes, often also called protists

Many important human and veterinary pathogens

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

Where do they come from, how do they relate to each other, and how do they relate to us?

From taxonomy to the tree of life (Linneus to Haeckel)

Taxonomy classifies organisms into meaningful groups that help to conquer and understand the massive diversity

The tree of life concept uses evolution as guiding principle of taxonomy

No evolution – no tree. Choosing the tree model makes

several important assumptions All life is related Life diversifies Life has a common origin

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 anatomical characteristics

“Complex” organisms were viewed as derived and highly evolved “simple” organisms (like the ameba) as primitive

This scheme puts protozoa to the bottom of the tree and animals to the top

the tree of life(Ernst Haeckel, 1866)

Monophyletic tree of organisms again by Haeckel

Loss or gain of characters produces branching of the tree

The advent of electron microscopy brought more morphological characters even for the small protists

However, reduction and simplifications (e.g. due to parasitism) pose significant problems for morphology based trees

Furthermore homology is not always discernable from analogy, and characters are not always easily quantifiable

Molecular phylogeny

Uses the sequence of macromolecules (RNA, DNA & proteins) to measure similarity, and to deduce evolutionary relation

Informative molecules are present in all the organisms to be compared

Relatedness is inferred from the simple argument that two molecules from two related organisms are likely to be more similar than from two organisms that diverged a long time ago

30S ribosomal subunit, rRNA pinkSchluenzen et al. Cell 102 (5): 615–23.

Molecular phylogeny

Molecular phylogeny assumes that sequence changes occur over time and that these changes can be modeled and used to infer a process (evolution) out of the current pattern

Molecular phylogeny

A large number of statistical approaches has been developed to compare sequences, build trees that depict the results, and evaluate their statistical significance

A tree of life based on the ribosomal RNA sequence

Three major domains (two prokaryotic one eukaryotic)

The archezoa hypothesis

Eukaryotes have some features of bacteria, some of archaea and some new ones

How did they evolve? A key event was the acquisition

of the mitochondrion (we will discuss endosymbiosis in detail in a later class) but when did that occur?

Who is the “most primitive eukaryote”

Archezoa & amoeba the most primitive eukaryotes?

No mitochondrion and no typical mitochondrial enzymes (Krebs cycle and oxidative phosphorylation is missing)

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

An alternative hypothesis stated that these organisms once had mitochondria and subsequently lost them while adapting to parasitism and life in anaerobic environments

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 and Giardia identified several genes for proteins targeted to the mitochondrion in other organisms

Is the absence of mitochondria a primary of secondary trait?

Localizing 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

Cpn60

DIC

Microbiology 150 (2004), 1245-1250

what is amoeboid about amoebae?

Amoeboid movement

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

what is amoeboid about amoebae?

Uroid

Pseudopodium

Hyaline ectoplasm

Endoplasm (sol)

While the endoplasm is ‘liquid’ and filled with organelles the ectoplasm appears ‘solid’ 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

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)

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

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 and cysts

Trophozoites 20-40 m diameter

Ribosomes arranged in helical patterns

Tissue forms often contain phagocytized red blood cell

trophozoites and cysts

Trophozoites encyst and cysts mature as they travel through the colon

Only mature cysts are infective

trophozoites and cysts

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)

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 fermentative metabolism of E. histolytica (PFOR – Dr. Moreno will explain this in the next lecture)

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

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 causes (Brumpt, 1928)

This theory was discounted Recent molecular data have

revived the two species hypothesis We now know that most people

are infected with the apathogenic E. dispar

How do ameba cause disease?Emile Brumpt 1877-1951

Pathogenic amoeba show contact dependent killing

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

Humans harbor numerous amebae (most are nonpathogenic)