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PlasmodiaMesquitoHuman Life Cycle

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Mosquitos. ... Out of the 380 species of Anopheline mosquito, 60 can transmit malaria. ... inside the mosquito they mate and begin to reproduce, ... – PowerPoint PPT presentation

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Title: PlasmodiaMesquitoHuman Life Cycle


1
Genetics in the news.
2
Malaria
  • Malaria has long been recognized as an important
    parasitic disease of humans, having been
    described by the early Egyptians in the third
    millennium B.C., 
  • Despite the introduction of control programs in
    many parts of the world over the past few
    decades, the impact of malaria on human
    populations continues to increase.  Recent
    estimates suggest,
  • (1) that 1.5 billion persons live in areas of the
    world where malaria is an endemic disease,
  • (2) that the number of infected humans exceeds
    500,000,000, and,
  • (3) that 1-3 million persons die each year.

3
Malarias Range(cross hatching)
Science Volume 289, Number 5485 (2000), p. 1763
4
LINKS BETWEEN MALARIA AND POVERTY
  • Poverty affects malaria. Communities with low
    incomes, limited education and poor access to
    health care are least able to engage in malaria
    control activities.
  • Malaria affects poverty. In poor households, a
    greater proportion of income is likely to be
    spent on malaria treatment than in affluent
    households,
  • malaria illness causes absenteeism from work and
    school, poor scholastic performance, lack of
    labor for cultivation, and a decline in child
    care, etc.
  • Hence, a negative spiral can develop with malaria
    causing and deepening poverty which, in turn,
    exacerbates inequalities in societies.

5
General Malaria Pathologymore later.
  • Host (human) inflammatory response resulting in
    severe chills and fever,
  • paroxysms,
  • Anemia due to loss of red blood cells,
  • Recurrence of paroxysms at periodic intervals.

6
Causative Agent Plasmodium sp. (Protozoa
Haemosporina)
  • These single celled eukaryotes have heteroxenous
    life cycles with both vertebrate and invertebrate
    hosts,
  • Humans,
  • Mosquitos.
  • Macro- and micro- gametes develop independently
    and the resultant zygote is motile and is called
    the Ookinete.

7
Malarial Plasmodium
  • There are four important species that infect
    humans, causing malaria
  • P. vivax - benign tertian malaria - accounts
    for 43 of cases
  • P. falciparum - malignant tertian malaria
    accounts for 50 cases
  • P. malariae - quartan malaria - accounts for
    7 of cases
  • P. ovale - mild tertian malaria - accounts for

8
P. vivax - benign tertian malaria
  • The disease gets its name from the time between
    paroxysms, which occur every 48 hours,
  • from the Roman custom to call the first day of
    the event day 1 and therefore 48 hours later
    would be day 3,
  • Tertian malaria accounts for 43 of all cases of
    malaria.
  • This species is mainly found in Asia and relapses
    have been known to occur up to 8 years after the
    first infection due to the presence of the
    dormant exoerythrocytic stage also known as
    hypnozoites.
  • P. vivax are only capable of infecting red blood
    cells via genetically determined receptor sites.
    Only people exhibiting these antigenic sites are
    susceptible to the disease.

P. ovale - mild tertian malaria similar
9
Duffy Blood Group
  • Human populations express two dominant alleles,
    signified as Fya and Fyb,
  • the expressed glycoproteins are recognized by
    their respective antigens on the erythocyte cell
    membranes,
  • A third allele Fy has no antigen associated with
    it.
  • The Fy/Fy genotype appears in about 40 of the
    African population as opposed to less than 0.1
    in European populations.
  • Caucasians have Fya//Fyb genotypes and therefore
    express the antigen on the surface of the red
    cell to which the plasmodium can bind, allowing
    it to infect the cell.

10
Tertian Malaria
  • The paroxysms commence with the patient having a
    feeling of intense cold (15 min - 1 hour),
  • this is due to a rapid rise in body temperature
    to 104-106o F,
  • accompanied with violent shivering,
  • often with nausea and vomiting.
  • Followed by the hot phase, which includes
    headache and often delirium (2 - 6 hours).
  • The fever breaks with the copious production of
    perspiration. The body temperature drops back to
    normal after 2 - 4 hours. The patient may sleep
    for 8-12 hours and feel well until the next
    paroxysm.

11
P. falciparum - malignant tertian malaria
  • The time between the onset of paroxysms is 48
    hours, but the period of fever is prolonged,
    lasting from 24-36 hours,
  • The course of this disease is very rapid and it
    is not uncommon to see more than 60 of a
    patient's red blood cells infected.
  • When the number of infected cells rises above 25
    the disease is usually fatal, in spite of
    treatment.

12
Falciparum Malaria
  • In Falciparum malaria the paroxysms last longer
    20-36 hours and so the patient does not recover
    before the onset of the next bout.
  • In 10 of P. falciparum cases patients develop
    cerebral malaria, 80 of which result in death.
  • characterized by headaches followed by the
    patient falling into a coma, often with
    convulsions.
  • This is particularly common in children.
  • Ultimately death ensues with the patients
    temperature reaching up to 108 F.

13
P. malariae - quartan malaria
  • This species of malaria has a 72 hour cycle of
    paroxysms and is found in both the New and Old
    World.
  • Recrudescence has been reported up to 53 years
    after the first infection.
  • It is the principal cause of malaria episodes as
    a result of blood transfusion.

14
Anopheline Mosquitoes
  • Anopheline mosquito are the only vector (except
    blood transfusions, and unprotected sex)
  • Out of the 380 species of Anopheline mosquito, 60
    can transmit malaria.
  • Only female mosquitos are involved as the males
    do not feed on blood.

15
Malaria Cycle Three Main (Human) Stages
  • Stage I Upon infection by the mosquito, the
    malaria parasites move rapidly into the liver
    (within 30 minutes ),
  • and reproduce rapidly (mitosis) for 5 days or
    more, depending on the species ( P. falciparum or
    P. vivax) ,
  • Stage II The malaria parasite exits the liver,
    enters the bloodstream, and within minutes invade
    red blood cells, where they grow and divide,
  • every 48-72 hours (time differences depend on
    the species) the red blood cells rupture,
  • dispersing more parasites along with waste
    products/toxins into the blood stream,
  • this step causes fever, chills and anaemia in the
    victim,
  • the released parasites then invade other red
    blood cells, beginning the cycle again.
  • Stage III Some parasites invade red blood cells
    and develop into sexual forms,
  • may be ingested by uninfected biting mosquitoes,
  • inside the mosquito they mate and begin to
    reproduce,
  • the zygotes (plasmodia) make their way to the
    salivary glands of the mosquito, ready to move on
    to another victim when the mosquito takes its
    next blood meal.

16
Plasmodia/Mesquito/Human Life Cycle
  • 1. Sporozoite (schizonts)/liver,
  • 2-5. Mitosis, liver cell lysis,
  • 6. Trophozoite (schizonts)/red blood cell,
  • 7-11 (mitosis),
  • 12. Gametocytes (via meiosis),
  • 13,14. Gametes via mitosis (midgut),
  • 15,16. Ookinetes, zygotes via conjugation,
  • cross the midgut epithilium,
  • 17. Oocysts, mitosis,
  • 18-20. Cross salivary epithelium.

17
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18
Fighting Malaria
  • Insecticides (mosquitoes develop resistance
    quickly),
  • most effective (DDT) banned,
  • Bed nets (often coated with insecticides),
  • Post infection drugs, even the best (chloroquine
    and sulfadoxine-pyrimethamine) generally lose
    effectiveness over time.
  • New Chinese remedy Qinghaosu plant, used in
    fever remedies for 2,000 years,
  • Artemisia annua, known as sweet wormwood or
    Chinese wormwood, grows wild, even in the United
    States.
  • Incredibly effective, although long term
    effectiveness and side effects are not known.

19
Plasmodium Mosquito Life Cycle
20
Receptor Mediated Transfer
Ookinete
Ookinete
Ross Cell Mosquito epithelium
Ross Cell Mosquito epithelium
normal/infection
no infection
21
The Experiment
  • Try to find a transgene that produces a peptide
    that will effectively block the receptor mediated
    transfer of ookinetes,
  • a transgene is an introduced gene.

22
Preliminary Work
23
Phage Display
T7 phage
recombinant phage express the sequence as part
of the gIII protein.
24
Recombinatorial Library
Insert DNA coding for dodecapeptides (XCX8CX)
into T7 genome.
Allow recombinant DNAs to replicate and repackage
themselves.
Run T7 phage over the receptor.
25
Procedure
  • 109 different phages in the population,
  • XCX8CX yields 2010 different possible peptides,
  • Mosquitoes were injected with 1011 phages.

26
SM1
  • PCQRAIFQSICN protein sequence bound both
    salivary gland and midgut epithelia,
  • Injection of the peptide into the mosquitos body
    cavity inhibited oocyst formatin by roughly 90.

27
SM1 Injected into Mosquitoes
A mouse model system has been established in
order to provide a clinical vertebrate host.
28
Delivery
  • We cant hope to hand-inject every mosquito in
    the world,
  • or, even feed them all SM1 peptide,
  • How can we deliver the 12 amino acid peptide into
    the correct cells in mosquitoes?

29
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30
Transgenic Construct
31
Transformation
  • Embryos are injected with the transgene
    construct, and a helper plasmid,
  • Surviving embryos are raised and crossed with
    virgin wt mosquitos,
  • Subsequent offspring are scored for GFP eyes.

32
trans (top)
wt (top)
trans (bottom)
wt (front)
trans (front)
33
Southern Blot
till a NotI or BglII site.
Probe 3xP3-EGFP-SV40 and SM1 DNA,
Blot Genomic DNA from different transgenic
individuals
34
Northern Blot
Probes SM1 DNA (mt rRNA for control)
35
Transgenic SM1 Results
36
Other Controls
  • wt Phage infection no anti-ookinete function,
  • GFP transgene only no anti-ookinete function,
  • Transposons ditto,
  • Other peptides ditto.

Further SM1 was inserted into 4 different
genomic regions, all had the same
phenotype, This suggests that the anti-ookinete
function wasnt due to a disrupted gene.
37
Epitope Tagging
  • Fluorescent antibodies are available for a
    portion of a virus protein (HA1)...

38
Trangenic
Wt
Midguts
39
Does SM1 Stop New Infections?
  • Transgenic and wt mosquitoes are fed blood meals
    on malaria infected mice,
  • Then are placed with uninfected mice,
  • These mice are then assayed for malaria.

40
And its Only a Heterozygote
  • The authors conjecture that the possibility
    exists that homozygous SM1 expressing mosquitoes
    might display stronger anti-ookinete function.

41
But(t)
  • SM1 transgenic mosquitoes dont seem to kill all
    plasmodium, thus, the plasmodium may be able to
    mutate and maybe find a way around the
    SM1-blocked receptor,
  • SM1 transgenic mosquitoes
  • healthy?
  • how do you replace natural populations?
  • are there unknown environmental consequences of
    the transgene?
  • how do you test for this?
  • are all of the anopholines similar?
  • maybe the receptor differs between species?

42
Biotechnology in General
Scenario 1
Scenario 2
43
To Know
  • Understand the basic life cycle of Plasmodium,
  • Understand the Figures and Tables in this
    experiment,
  • Understand the general rational of the experiment.

44
Friday
  • Assigned Questions 6.8, 6.13, 6.18, 6.19, 11.6,
    11.7,
  • Chapter 7 (7.1 - 7.5),
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