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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)