Wrap up antimalarial treatment

1

• Wrap up anti-malarial treatment drug resistance
• Babesia Theileria, tick transmitted
  apicomplexan parasites
• Theileria hijacks several host cell functions to
  spread within the host and cause disease

2
Chloroquine the wonder drug

• During its development within the RBC the malaria
  parasite ingests the cytoplasm of its host cell
• 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

3
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 (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

4
Resistance to chloroquine
1965
1960
1978
1989
http//www.tigr.org/tdb/edb/pfdb/CQR.html
5
Mechanisms of drug resistance

• Changes in target enzyme (e.g. decreased affinity
  to drug)
• Over-expression of target (amplification)
• Decreased activation of drug
• Changes in accessibility (less import, or more
  export of drug)

6
Resistance to chloroquine

• Chloroquine resistance is associated with
  decreased accumulation of the drug in the food
  vacuole
• Genetic studies have shown that resistance is
  linked to the integral membrane protein PfCRT
• This putative transporter localizes to the
  membrane of the food vacuole
• Studies using parasite cultures suggests that a
  series of point mutations in PfCRT are
  responsible for resistance
• Large field studies have found strong association
  of these mutations with chloroquine resistance
• Currently the exact physiological function of
  PfCRT is unknown

PfCRT, resistance mutations highlighted
7
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

8
Antifolates as malaria drugs
Parasite
Folate synthesis
Dihydrofolate
Folate recharging
Tetrahydrofolate
9
Antifolates as malaria drugs
Parasite
Human
Folate synthesis
Dihydrofolate
Dihydrofolate
Nucleotide synthesis
Folate recharging
Tetrahydrofolate
Tetrahydrofolate
10
Antifolates as malaria drugs
Parasite
Human
Folate synthesis
Sulfonamide
Dihydrofolate
Dihydrofolate
Nucleotide synthesis
Folate recharging
Tetrahydrofolate
Tetrahydrofolate
11
Antifolates as malaria drugs
Human
Dihydrofolate
Nucleotide synthesis
Tetrahydrofolate
12
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

13
Antifolate resistance developed very fast (5
years)
14
Combinations of Artemisinin and other
antimalarial 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
  anti-malarials
• However mono-therapy 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

15
Piroplasms Babesia Theileria
16
Piroplasms

• Piroplasms or Piroplasmida are an order of the
  Apicomplexa
• They are very small parasites of mammals and
  ticks
• There are two genera which cause import disease
  in livestock (and occasionally in humans)
  Babesia Theileria

17
Babesia

• Numerous species which cause malaria like disease
  in wide variety of animals
• We will only discuss B. bigemina a parasite of
  cattle (however note that there are occasional
  infections in humans by other Babesia species)

18
Devastating outbreaks follow the big cattle drives

• In the 1860s and 1870s Texas longhorns were
  driven in huge numbers to the railheads in Kansas
  (from there they went by train to the
  slaughterhouses of Chicago and other big nothern
  cities)
• Farmers in Kansas and Missouri were plagued by
  outbreaks of Texas fever in their herds which
  they linked to the cattle drives
• Several standoffs ensued as local farmers tried
  to block drives
• Theobald Smith and Frederick Kilbourne show
  (1889-1893) that the disease is caused by a
  protozoan parasite transmitted by ticks (first
  disease shown to be transmitted by an arthropode)

19
Babesia bigemina causes Texas cattle fever

• Mortality in acute untreated cattle 50-90
• Rapid rise in temperature (105-108 F)
• Fever persists for a week or more
• Loss of appetite, dull, listless
• Severe anemia due to rapid loss of red blood
  cells
• Hemoglobinuria (red colored urine) due to massive
  RBC lysis
• Infected RBCs adhere to vasculature of organs
  (likely similar in mechanisms to Plasmodium)
• Evidence for comparable clonal antigenic
  variation
• Cattle may die within 3-8 days

B. bigemina
20
Babesia bigemina causes Texas cattle fever

• Older cattle is much more likely to develop
  severe disease than calves
• In endemic areas calves get infected and
  mortality is low
• In the case of epidemics adults without previous
  exposure get infected resulting in massive loss
• This explains the massive loss of cattle in
  Kansas despite the fact that the longhorns coming
  from Texas (where the disease was endemic) seemed
  perfectly healthy
• What makes the difference between Texas Kansas?

21
Distribution of Tick fever caused by B. bigemina
Babesiosis coincides with the distribution of the
main vector ticks Boophilus annulatus microplus
(Winter temperatures limit distribution)
22
Babesia

• Merozoites (piroplasms) multiply in RBCs of the
  mamalian host
• Tick takes up sexual stages with blood meal,
  gamete formation, fertilization
• Kinetes infect various organs of the tick
  including ovary (transovarial infection of next
  generation of ticks)
• In the larvae the kinetes invade salivary gland
  cells, massive replication results in the
  production of ten thousands of sporozoites which
  are injected upon feeding

23
Boophilus is a single host tick

• In the U.S. Mexico Babesia bigemina is
  transmitted by Boophilus annulatus
• Boophilus are one host ticks larvae hatch from
  the eggs on the ground and attach to a host
• Ticks stay on host and feed and molt several
  times until they are adults
• Engorged and fertilized female drops of to lay
  eggs and dies
• Transovarial infection is very important for
  effective transmission in one host ticks

24
Disease control is mostly through vector control

• Disease can be treated with drugs
• A partially effective attenuated vaccine is
  available
• Tick control mostly through pesticide application
  remains the most important counter measure

25
Vaccination of cows with an antigen from the tick
midgut
Antigen Bm86

• Proteins found on the surface of the gut
  epithelium of Boophilus ticks have be
  characterized and used to make a recombinant
  vaccine (against the tick not the parasite)
• Ticks fed on vaccinated cows are exposed to
  antibody/complement mediated attack of their
  epithelium
• These tick grow poorly and have low fecundity

gut
Salivary gland
Host
vaccinated
normal
26
Border Cowboys patrol the U.S. Mexican border for
ticks

• Boophilus ticks and with them the Texas tick
  fever have been eradicated from the Southern U.S.
  but they are still present in Central America
• USDA employs 60 cowboys which patrol the Southern
  border to find and check stray-cattle for ticks
  to prevent the reintroduction
• See short New York Times feature on border
  cowboys posted on the class web site

Eddie Dillard, left, and Jack Gilpin are tick
riders (NYT 7/03)
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Theileria

• Life cycle and transmission of Theileria
• Host cell invasion by Theileria -- what are the
  differences to Toxoplasma
• East coast fever and tropical theileriosis
• Theileria manipulates its host cells

28
Theileria

• Infects mainly ruminants (cattle, goats, sheep,
  deer)
• Several different species causing both pathogenic
  and benign disease
• Infection in wild animals is mostly asymptomatic

cattle disease
Cape buffalo reservoir
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T. parva T. annulata
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Distribution of theileriosis
red T. annulata 250 million cattle at
risk orange T. parva 50 million cattle
at risk grey T. buffeli/orientalis/sergenti
relatively benign
31
Life cycle of Theileria spec.

• Life cycle in tick similar to that of Babesia
• However, no transovarial transmission (vectors
  are multi-host ticks)
• Two different cell types are infected in the
  mammalian blood stream (initially leukocytes
  later on RBCs)
• Infection of RBCs is important for transmission
  and infection of lymphocytes is important for
  pathology
• T. parva (mostly T-cells), T. annulata (B-cells,
  macrophages)

32
Two stages are found in the bovine host Kochs
bodies and piroplasms

• Kochs bodies, infected lymphocytes
• Piroplasms, infected red blood cells

33
Theileria (sporozoite) invasion differs from
Toxoplasma invasion
34
Theileria invasion

• Zipper mechanism of entry into lymphocyte
• Escape from vacuole into cytoplas coincides with
  rhopthry microneme discharge
• Parasites free in the cytoplasm associate with
  host MT

• Animated version

35
The Theileria paradox

• Although Theileria replicates in lymphocytes
  these cells seem to proliferate enormously in
  infected animals (most of these proliferating
  lymphocytes are infected) -- this is in contrast
  to other infections like malaria or babesiosis
  where parasite replication is associated with the
  decline of the host cell population causing
  anemia
• Also, the sporozoite (injected by the tick)
  appears to be the only stage capable of invading
  lymphocytes
• How can the parasite spread to new lymphocytes?
• The trick Theileria hijacks and exploits two key
  features of the lymphocytes cell biology cell
  division and growth control

36
Divide conquer
37
Divide conquer

• Parasites do not egress from (and in the process
  destroy) their host cells and infect new
  lymphocytes but proliferate along with them
• The tight association of parasites with host cell
  microtubules ensures that they are segregated by
  the host cell mitotic spindle between the two
  daughter cells
• A recently divided infected lymphocyte (the arrow
  indicates the cleavage furrow at which
  cytokinesis occurred. Blue (DNA), red (host cell
  centrioles), green (parasite surface membrane),
  HN (host nucleus)

38
Theileriosis is a lympho-proliferative disease

• Recall the immunology lecture -- lymphocytes are
  usually arrested and only expand upon antigen
  presentation
• If parasite replication requires host cell
  replication the parasite has to somehow induce
  proliferation of its host cells
• Indeed theileriosis is a lympho-proliferative
  disease
• Swelling and proliferation of the lymph node
  draining the bite site is the first sign of
  disease

39
Pathology is mainly due to lymphoproliferation

• Lymphocytes proliferate heavily invading multiple
  organs causing disease similar to a lymphoma
  (cancer of lymphocytes)
• (Top) Infiltration of kidney by Theileria parva
  infected lymphocytes
• (Bottom) Abdominal ulcers due to transformed
  lymphocytes
• Death is in most cases due to infiltration of the
  lung resulting in lung edema (the abnormal build
  up of fluid within the lung)

40
Theileria infected cells show characteristics of
transformation

• Theileria infection seems to share many of the
  features seen in the transformation of normal
  cells into cancer cells
• Uncontrolled growth
• Loss of differentiation
• Immortalization (infected cells taken into
  culture will grow indefinitely)
• Growth in the absence of external growth factors
• Enhanced ability to migrate and to infiltrate
  organs
• When cells are cured from parasite infection they
  die (by apoptosis -- this suicide response is
  usually suppressed in cancer cells)

41
How does Theileria interfere with lymphocyte
growth and cause cancer?
42
NF-kB -- a major regulator of lymphocyte growth

• NFkB (nuclear factor, p50 p65) is an important
  and very well studied transcription factor (a
  protein that interacts with the promoter of genes
  and stimulates gene expression)
• It is a major player in the stimulation and
  clonal expansion of lymphcytes (among other
  functions)
• NFkB is bound by IkB (its inhibitor) which
  sequesters it in the cytoplasm and keeps it
  inactive
• Phosphorylation followed by ubiquitinylation and
  degradation of IkB leads to import into the
  nucleus and transcriptional activity
• Theileria interferes with this pathway by causing
  the destruction of IkB

43
The IKK complex

• IkB is tagged for destruction by phosphorylation
  through the IKK complex
• In the lymphocytes this provides a way to relay
  the reception of signals from the surface of the
  cell to gene expression
• Theileria hijacks and activates the IKK signaling
  complex independent of the usually required
  external stimulation

44
Hijacking and activation of IKK transforms
infected cells

• Theileria parasites (green) interact with and
  activate IKK (red) of their host lymphocytes
• IKK tags IkB for destruction
• NfKb free of its inhibitor enters the nucleus and
  cells start dividing rapidly

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summary

• Theileria sporozoites invade using a zippering
  mechanism
• The PV is lysed upon rhoptry secretion and the
  parasites resides in the cytoplasm and associates
  with the host cells microtubuli centrosomes
• When the host cell divides the parasite divides
  and segregates alongside using the host cells
  mitotic machinery
• Theileria schizonts transform their hosts
  lyphocytes (induce uncontrolled cancer-like
  growth)
• Transformation is parasite dependent and
  reversible
• Parasites interfere with NFkB growth control by
  activating the IKK signalling pathway