Dr. Lilach Sheiner’s accent?
Apicomplexan invasion
 Active, parasite driven process
 Depends on parasite actin/myosin motility (conveyor belt
model)
 Involves secretion of micronemes (attachment, motility),
rhoptries (PV & MJ formation) and dense granules
(makes PV into a suitable home)
 Sets up a parasitophorous vacuole which initially is
derived from the host cell cell-membrane
 A moving junction is formed which screens out host
membrane proteins from the PV, the PV is fusion
incompetent and the parasite protected
Two great movie clips summarizing the
malaria life cycle:
 Development in the human:
 http://www.hhmi.org/biointeractive/disease/malaria_anim/malariahuman.html
 Development in the mosquito:
 http://www.youtube.com/watch?v=7sHB56AjHQ8&feature=related
 (Best done before Thursday)
Malaria II
Malaria the disease
Pathogenesis of
severe falciparum
malaria
Drugs used to treat
malaria and the
development of drug
resistance
Malaria the disease
 Human malaria is primarily a
blood disease, however it
causes pathology in a
variety of organs & tissues
 All disease is due to the
parasites development
within the red blood cell
(merozoite, trophozoite,
schizont).
 Other stages are important
for transmission but they do
not contribute to
pathogenesis
Malaria the disease
9-14 day incubation
period
Fever, chills,
headache, back and
joint pain
Gastrointestinal
symptoms (nausea,
vomiting, etc.)
Malaria the disease
Malaria the disease
 Malaria tertiana: 48h
between fevers (P.
vivax and ovale)
 Malaria quartana: 72h
between fevers (P.
malariae)
 Malaria tropica:
irregular high fever (P.
falciparum)
Malaria the disease
 Symptoms intensify
 Irregular high fever
 Anxiety, delirium and
other mental problems
 Sweating, increased
pulse rate, severe
exhaustion
 Worsening GI symptoms
 Enlarged spleen and liver
Malaria the disease
3 Severe manifestations
Cerebral malaria
Irritability, loss of reflexes,
neurological symptoms
similar to menigitis, coma
20% fatality
Severe anemia
Progressive severe drop
of hematocrit, poor oxygen
Supply for organs and
tissues
Renal failure
Dwindling urine, high urea
Level in serum, hyperventilation
Coma, poor prognosis
Malaria the disease
WHO-TDR
Pathogenesis of malaria
 In highly endemic areas: high
mortality among children due to
severe anemia, children who
survive beyond the first years
show decreasing parasitemia and
disease (this immunity is not
sterile and depends on constant
exposure)
 In areas with less infection
pressure: malaria is an epidemic
disease with varying intensity.
Adults and children are equally
susceptible and death in adults is
mostly due to cerebral malaria
Pathogenesis of malaria
Parasitemia
Anemia
Age
Cerebral malaria
Pathogenesis of cerebral
malaria
 Cerebral malaria is characterized
by multiple brain hemorrhages
(vessel rupture and bleeding)
 Excessive serum and tissue
levels of TNFa and INFg (two
important cytokines driving
inflammation) are associated
with severe malaria
 Some researchers believe this
inflammation is the main cause
for pathology (remember the
immunology introduction: an
overshooting immune response
against a chronic pathogen that
can not be cleared can cause
severe disease)
Sequestration & cytoadherence
Ring stages
 The second model suggests
sequestration to be the main
culprit
 In P. falciparum infections
only early stages (rings) are
found in the peripheral blood
 Trophozoites and schizonts
are sequestered to the postcapilary venules by
attachment to the
endothelium
Pathogenesis of falciparum
malaria
 Parasite infected RBC
become ‘sticky’ and adhere
to endothelial cells
 This phenomenon takes
about 10-12 hours to
develop after parasite
invasion
 Under high flow (here
modeled using a
microfluidic device) this first
results in rolling and then in
attachment
Pathogenesis of falciparum
malaria
 Cytoadherence seems to be
the main culprit for
pathogenesis
 Infected RBCs will adhere to
the endothelium as well as to
each other and cause clogging
and hemorhaging
 Note that high cytokine levels
induce expression of
endothelial adhesins -inflammation makes the
endothelia ‘stickier’
 Adherence and inflammation
reinforce each other in an
unholy circle causing
pathology
Knobs and cytoadherence
 Cytoadhrence correlates with
the presence of “knobs” (left
column) on the surface of the
infected RBC
 The right column shows a
RBC infected with a knobless strain which does not
cause cerebral malaria
 Knobs are made up of
parasite derived proteins
knobs
knob-less
Knobs and cytoadherence
 PfEMP1 (P. falciparum
erythrocyte membrane protein)
is found in knobs and is
responsible for cytoadherence
and rosetting
 PfEMP1 is a large membrane
protein anchored in the RBC
membrane with the bulk
extending into the blood stream
 Various domains of PfEMP1
have been shown to bind to
ligands on the endothelia of the
vasculature and the placenta
 PfEMP1 is an important
pathogenesis factor
Knobs and cytoadherence
 The parasite exports
PfEMP1 and other
proteins (this picture is
showing Knob associated
protein) into the RBC and
its surface to form knobs
 F: in early rings protein is
in the parasite and the
parasitophorous vacuole,
G,H: in trophozoites it is
found first within the RBC
cytoplasm and then at
the RBC membrane (I).
Knobs and cytoadherence
 How precisely the parasite transports
proteins through the RBC is still under study
 However it is clear that the parasite has not
only to provide the cargo but also the
transport machinery as the RBC has
reduced its capability for membrane
transport and secretion
 All these proteins are initially secreted by
the parasite into the parasitophorous
vacuole
 A recently discovered gatekeeper in the
vacuole membrane appears to shuttle
proteins across (PTEX Plasmodium
translocon of exported proteins)
 Maurer’s clefts (parasite induced
membranous structures in the RBC) appear
to be an important bridgehead acting in the
sorting and trafficking of exported proteins
Chinchona the source of
quinine
 Peruvian Indians appear to have been
the first to know about the medicinal
effects of quinine, they chewed
Chinchona bark while working in the
mines as forced laborers for the
Spanish
 Jesuits brought the bark back to
Europe to treat febrile diseases
 In the early 1600s the bark was used
to treat the fever of the Countess of
Chinchon and became well known as
Jesuit’s powder or Peruvian bark
 Initial preparations were often quite
variable in the amount of active
ingredient resulting in varying effects
Chichona the source of
quinine
 High demand had brought the
Chinchona tree almost to
extinction in the wild
 Charles Ledger a trader in Peru
send out Manuel Incra Macrami
to collect seeds from a stand of
special trees they had found
earlier
 After three years Manuel came
back with 15 kg of seeds which
they sold for 100 guilders to the
Dutch consul as the British were
not interested
 C. ledgeriana formed the basis of
a very profitable Dutch quinine
monopoly which lasted until
World War II
Chloroquine the wonder
drug
 Chloroquine, a synthetic
quinine analog developed by
German and American
chemists during WWII, was a
very potent drug that was
cheap to make, stable, and
had no serious side effects
 Chloroquine was a major
component of the 60/70s
malaria eradication campaign
 None of the drugs developed
since come close to
chloroquine
Chloroquine the wonder
drug
 During its development within
the RBC the malaria parasite
ingests the cytoplasma of its
host
 Note that in this schematic
(and in real micrographs) the
red color of the blood cell gets
considerably lighter -- at the
same time malaria pigment
accumulates
 The parasite digests large
ammounts of hemoglobin to
cover part of its amino acid
needs
Chloroquine the wonder
drug
 RBC cytoplasm is taken up by
endocytosis
 The endocytosis vesicle fuse with the
food vacuole (a homolog of the
secondary lysosome) were hemoglobin
digestion occurs
 Digestion frees large ammounts of
heme
 Heme is toxic to the parasite and is
neutralized by polymerization into the
malaria pigment or hemozoin
 Chloroquine accumulates in the food
vacuole (it’s a weak base and like all
lysosomes the FV is an acidic
compartment)
 Chloroquine is thought to interfere with
the polymerization and detoxification of
heme
Resistance to chloroquine
1960
1965
1978
1989
http://www.tigr.org/tdb/edb/pfdb/CQR.html
Mechanisms of drug
resistance
Mechanisms of drug
resistance
Changes in target enzyme (e.g.
decreased affinity to drug)
Overexpression of target (amplification)
Decreased activation of drug
Changes in accessibility (less import, or
more export of drug)
Resistance to chloroquine
PfCRT, resistance mutations highlighted
 Genetic studies have shown that
resistance is linked to the
transporter protein PfCRT
 Studies using parasite cultures
suggests that a series of point
mutations in PfCRT are
responsible for resistance
 This putative transporter localizes
to the membrane of the food
vacuole
 Large field studies have found
strong association of these
mutations with chloroquine
resistance
 Currently it is not known what the
physiological function of PfCRT is
Antifolates as malaria drugs
Nucleotide
synthesis
dUMP
dTMP
Tetrahydrofolate
 The synthesis of certain building
blocks of DNA requires reduced
folate (more specifically the
syntheisis of dTMP)
 No reduced folate -- no DNA
 The malaria drug Fansidar uses a
drug combination to hit the same
target pathway twice
 Combinations that are more
effective than the sum of their
individual activities are called
synergistic
Antifolates as malaria drugs
Parasite
GTP
Folate
synthesis
Dihydrofolate
Folate
‘recharging’
Tetrahydrofolate
Antifolates as malaria drugs
Parasite
Human
GTP
Folate
synthesis
Dihydrofolate
Dihydrofolate
Folate
‘recharging’
Nucleotide
synthesis
Tetrahydrofolate
Tetrahydrofolate
Antifolates as malaria drugs
Parasite
Human
GTP
Folate
synthesis
Sulfonamide
Dihydrofolate
Dihydrofolate
Folate
‘recharging’
Nucleotide
synthesis
Tetrahydrofolate
Tetrahydrofolate
Antifolates as malaria drugs
Parasite
Human
GTP
Folate
synthesis
Sulfonamide
Dihydrofolate
Folate
‘recharging’
Dihydrofolate
Nucleotide
synthesis
Pyrimethamine
Tetrahydrofolate
Tetrahydrofolate
Antifolates as malaria drugs
Parasite
GTP
Folate
synthesis
Sulfonamide
Dihydrofolate
Folate
‘recharging’
Pyrimethamine
Tetrahydrofolate
 First strike: Folate synthesis. We
can’t make folate and take it up
with food as a vitamin. The
parasite makes it and is therefore
susceptible to sulfonamides which
block synthesis
 Second strike: After each use
dihydrofolate has to be reduced
again (think of it as recharging).
The enzyme which does this
(dihydrofolate reductase) is
different in human and parasite
 The drug pyrimethamine inhibits
parasite DHFR but not human
DHFR
 Fansidar combines pyrimethamine
with sulfadoxine
 A very similar drug combination is
used to treat toxoplasmosis
Antifolate resistance
developed very fast
Combinations of Artemisinin and other
antimalarials are promising
 Extracts of Artemisia annua (sweet
wormwood) have long been used in
traditional Chinese medicine to treat fever
 Chinese investigators extracted the active
ingredients and showed that they and there
chemical modifications are powerful
antimalarials
 However monotherapy results in high level of
recrudescence
 Combining Artemisinin with other drugs have
been very successful especially for severe
malaria
 Artemisinin acts very fast which helps to
reduce mortality and get patients out of their
coma quickly
Summary
 Severe forms of malaria include: severe anemia in kids, and
cerebral and renal malaria in adults
 Severe pathogenesis is related to adherence of infected RBC to
entothelia
 Adherence is mediate by knobs in the RBC surface made up by
parasite proteins (PFEMP1)
 PFEMP1 undergoes antigenic variation
 Chloroquine resistance has been a public health catastrophe
 Chloroquine accumulates in the food vacuole and prevents heme
polymerization, resistance is linked to mutations in a transport
protein in the food vacuole membrane
Immunity to malaria
 There is no sterile immunity to malaria
 Patients produce strong antibody responses to PfEMP1
which is exposed to the immune system on the surface
of the infected RBC.
 Why is the immunity to malaria relatively weak?
 PfEMP1 is encoded by a large multigene family (VAR
genes) and parasites switch to new variants (antigenic
variation again)
 The parasite genome encodes 60 VAR genes, only one
is expressed at a time (allelic exclusion)
Immunity to malaria
 Successful vaccination in humans has been achieved
with large doses of irradiated sporozoites, however that
is likely not practical
 Many approaches have been and are explored to
stimulate immunity against sporozoites (infection
blocking), merozoites (disease blocking) or gametocytes
(transmission blocking)
 None has yielded a satisfactory and safe human
vaccine yet
 The most promising new strategies use genetic
manipulation to engineer attenuated parasites strains
(parasites that enter cells and induce immunity yet fail to
develop fully and cause disease)
 For now, control depends heavily on drug therapy