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?
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
From Exercitationes de Vena Mediensis et de Vermiculis 
Capilaribus Infantium by G.H. Velschius, published in 
Augsburg, Germany 1674
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)
ungulates man ❚ The tree of life (who is
whales carnivores related and how did they
evolve) was initially
based on morphological
characteristics
fish ❚ “Complex” organisms
reptiles were viewed as derived
and highly evolved
crustaceans “simple” organisms as
primitive
molluscs ❚ This scheme puts
protozoa as a cohesive
worms group to the bottom of
the tree

protozoa
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”
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❚ It was assumed that amoeba
represent the stage of early
eukaryotes before the
endosymbiosis event that let to the
mitochondrion
Is the absence of mitochondria a
primary of secondary trait?
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❚ 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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Acanthamoeba
http://cmgm.stanford.edu/theriot/movies.htm#Hits
what is amoeboid about amoebae?

Pseudopodium

Hyaline ectoplasm
(gel)
QuickTimeª and a
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While the endoplasm (sol) is ‘liquid’ and
Uroid filled with organelles the ectoplasms appears
more solid (gel) 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

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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

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Listeria in Xenopus extract (right panel

Phase contrast, left panel actin-GFP fluorescence)

❚ The actin polymerization model is

Listeria in host cell (150x) based on cell free reconstitution of
the movement of intracellular
bacteria
❚ These studies allowed to identify
the factors involved in the initiation
http://cmgm.stanford.edu/theriot/movies.htm#Hits 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 µm) with
small central nucleolus and
characteristic radial spokes
trophozoites and cysts

❚ Trophozoites 20-40 µm
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 µm), 4 ❚ Chromidial bodies and
nuclei bars are semicrystalline
❚ 150 nm cyst wall with arrays of riobosomes
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)
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
ATP
ADP acetate ❚ Entamoeba uses a pyruvate
ferredoxin oxidoreductase
CO2
(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
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nitroradical
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❚ 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 (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
Emile Brumpt 1877­1951
❚ We now know that most people
are infected with the
apathogenic E. dispar
Genetic evidence for two species

❚ Species specific isoenzyme patterns

❚ Multiple antibodies specific for either the
pathogenic or apathogenic species
❚ Numerous genes sequenced which
show clear differences
❚ Repetitive DNA elements are different
❚ Genomic organization of conserved
gene loci like actin is different
❚ Ribosomal 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