Title: PlasmodiaMesquitoHuman Life Cycle
1Genetics in the news.
2Malaria
- 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.
3Malarias Range(cross hatching)
Science Volume 289, Number 5485 (2000), p. 1763
4LINKS 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.
5General 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.
6Causative 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.
7Malarial 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
8P. 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
9Duffy 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.
10Tertian 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.
11P. 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.
12Falciparum 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.
13P. 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.
14Anopheline 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.
15Malaria 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.
16Plasmodia/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.
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18Fighting 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.
19Plasmodium Mosquito Life Cycle
20Receptor Mediated Transfer
Ookinete
Ookinete
Ross Cell Mosquito epithelium
Ross Cell Mosquito epithelium
normal/infection
no infection
21The 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.
22Preliminary Work
23Phage Display
T7 phage
recombinant phage express the sequence as part
of the gIII protein.
24Recombinatorial Library
Insert DNA coding for dodecapeptides (XCX8CX)
into T7 genome.
Allow recombinant DNAs to replicate and repackage
themselves.
Run T7 phage over the receptor.
25Procedure
- 109 different phages in the population,
- XCX8CX yields 2010 different possible peptides,
- Mosquitoes were injected with 1011 phages.
26SM1
- 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.
27SM1 Injected into Mosquitoes
A mouse model system has been established in
order to provide a clinical vertebrate host.
28Delivery
- 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?
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30Transgenic Construct
31Transformation
- 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.
32trans (top)
wt (top)
trans (bottom)
wt (front)
trans (front)
33Southern Blot
till a NotI or BglII site.
Probe 3xP3-EGFP-SV40 and SM1 DNA,
Blot Genomic DNA from different transgenic
individuals
34Northern Blot
Probes SM1 DNA (mt rRNA for control)
35Transgenic SM1 Results
36Other 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.
37Epitope Tagging
- Fluorescent antibodies are available for a
portion of a virus protein (HA1)...
38Trangenic
Wt
Midguts
39Does 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.
40And its Only a Heterozygote
- The authors conjecture that the possibility
exists that homozygous SM1 expressing mosquitoes
might display stronger anti-ookinete function.
41But(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?
42Biotechnology in General
Scenario 1
Scenario 2
43To Know
- Understand the basic life cycle of Plasmodium,
- Understand the Figures and Tables in this
experiment, - Understand the general rational of the experiment.
44Friday
- Assigned Questions 6.8, 6.13, 6.18, 6.19, 11.6,
11.7, - Chapter 7 (7.1 - 7.5),