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)
ungulates
whales
fish
crustaceans
man
 The tree of life (who is
related and how did they
carnivores
evolve) was initially
based on anatomical
characteristics
 “Complex” organisms
reptiles
were viewed as derived
and highly evolved
“simple” organisms (like
the ameba) as primitive
molluscs  This scheme puts
protozoa to the bottom of
worms
the tree and animals to
the top
protozoa
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 pink
Schluenzen 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
Acanthamoeba
http://cmgm.stanford.edu/theriot/movies.htm#Hits
what is amoeboid about
amoebae?
Pseudopodium
Hyaline ectoplasm
(gel)
Endoplasm (sol)
Uroid
While the endoplasm is ‘liquid’ and filled with
organelles the ectoplasm appears ‘solid’ and clear.
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
Listeria in Xenopus extract (right panel
Phase contrast, left panel actin-GFP fluorescence)
Listeria in host cell (150x)
http://cmgm.stanford.edu/theriot/movies.htm#Hits
 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 mm) with
small central nucleolus and
characteristic radial spokes
trophozoites and cysts
Trophozoites 20-40 mm
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
 Round (10- 16 mm), 4
nuclei
 150 nm cyst wall with
fibrillar structure
 Chromidial bodies and
bars are semicrystalline
arrays of riobosomes
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 Entamoeba
36,000,000 develop clinical symptoms
40,000 - 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)