Malaria pathogenesis

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Malaria pathogenesis
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Parasite 'turns women into sex kittens'By Jane
Bunce, December 26, 2006 0527pm, Article from
AAP
A COMMON parasite can increase a women's
attractiveness to the opposite sex but also make
men more stupid, an Australian researcher
says. About 40 per cent of the world's
population is infected with Toxoplasma gondii,
including about eight million Australians. Until
recently it was thought to be an insignificant
disease in healthy people, Sydney University of
Technology infectious disease researcher Nicky
Boulter said, but new research has revealed its
mind-altering properties. "Interestingly, the
effect of infection is different between men and
women,'' Dr Boulter writes in the latest issue of
Australasian Science magazine. "Infected men
have lower IQs, achieve a lower level of
education and have shorter attention spans. They
are also more likely to break rules and take
risks, be more independent, more anti-social,
suspicious, jealous and morose, and are deemed
less attractive to women. "On the other hand,
infected women tend to be more outgoing,
friendly, more promiscuous, and are considered
more attractive to men compared with non-infected
controls. "In short, it can make men behave like
alley cats and women behave like sex kittens''.
(http//www.news.com.au/story/0,10117,20975555-2
9277,00.html)
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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

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Two great movie clips summarizing the malaria
life cycle

• Development in the human
• http//www.hhmi.org/biointeractive/disease/malaria
  _anim/malaria-human.html
• Development in the mosquito
• http//www.youtube.com/watch?v7sHB56AjHQ8feature
  related
• (make sure to watch these before the next exam)

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Malaria II

• Malaria the disease
• Pathogenesis of severe falciparum malaria
• Drugs used to treat malaria and the development
  of drug resistance

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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

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Malaria the disease

• 9-14 day incubation period
• Fever, chills, headache, back and joint pain
• Gastrointestinal symptoms (nausea, vomiting, etc.)

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Malaria the disease
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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)

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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

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Malaria the disease
3 Severe manifestations
Cerebral malaria
Severe anemia
Renal failure
Irritability, loss of reflexes, neurological
symptoms similar to menigitis, coma 20 fatality
Progressive severe drop of hematocrit, poor
oxygen Supply for organs and tissues
Dwindling urine, high urea Level in serum,
hyperventilation Coma, poor prognosis
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Malaria the disease
WHO-TDR
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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

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Pathogenesis of malaria
Age
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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 immune hormones 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)

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Sequestration cytoadherence

• 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
  post-capilary venules by attachment to the
  endothelium

Ring stages
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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
• (movie courtesy of Dr. Pradip Rathod University
  of Washington)

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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

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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
  knob-less strain which does not cause cerebral
  malaria
• Knobs are made up of parasite derived proteins

knobs
knob-less
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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

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Knobs and cytoadherence
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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).

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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)
• Maurers clefts (parasite induced membranous
  structures in the RBC) appear to be an important
  bridgehead acting in the sorting and trafficking
  of exported proteins

Reiff Striepen, Nature 459918
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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)

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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

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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 Jesuits powder or Peruvian bark
• Initial preparations were often quite variable in
  the amount of active ingredient resulting in
  varying effects

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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

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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

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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

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Chloroquine the wonder drug

• RBC cytoplasm is taken up by endocytosis
• The endocytotic vesicles 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 (its
  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

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Resistance to chloroquine
1965
1960
1978
1989
http//www.tigr.org/tdb/edb/pfdb/CQR.html
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Mechanisms of drug resistance
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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)

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Resistance to chloroquine

• 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
• It is now thought that the natural role of
  PfCRT is to export small peptides from the food
  vacuole

PfCRT, resistance mutations highlighted
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Antifolates as malaria drugs

• 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

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Antifolates as malaria drugs
Parasite
GTP
Folate synthesis
Dihydrofolate
Folate recharging
Tetrahydrofolate
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Antifolates as malaria drugs
Parasite
Human
GTP
Folate synthesis
Dihydrofolate
Dihydrofolate
Nucleotide synthesis
Folate recharging
Tetrahydrofolate
Tetrahydrofolate
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Antifolates as malaria drugs
Parasite
Human
GTP
Folate synthesis
Sulfonamide
Dihydrofolate
Dihydrofolate
Nucleotide synthesis
Folate recharging
Tetrahydrofolate
Tetrahydrofolate
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Antifolates as malaria drugs
Human
Dihydrofolate
Nucleotide synthesis
Tetrahydrofolate
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Antifolates as malaria drugs

• First strike Folate synthesis. We cant 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

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Antifolate resistance developed very fast
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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

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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