Download Plasmodia/Mesquito/Human Life Cycle

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, including about 1 million children
under 5 years old (3,000 per day).
Malaria’s Range
(cross hatching)
Science: Volume 289, Number 5485 (2000), p. 1763
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.
General Malaria Pathology
…more 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.
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.
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 < 1% of cases.
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 a
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
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.
• Individuals with Fya//Fyb genotypes therefore express the antigen on
the surface of the red cell to which the plasmodium can bind, allowing
it to infect the cell.
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.
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.
“knobs”
Knobs promote
cell clumping…
clogging veins
and arteries.
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.
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.
Anopheline Mosquitoes
• Anopheline mosquitoes are
the only vector (except
blood transfusions, and
unprotected sex)
• Out of the 380 species of
Anopheline mosquitoes, 60
can transmit malaria.
• Only female mosquitoes are
involved as the males do not
feed on blood.
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 anemia 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 (ookinetes) 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.
Plasmodia/Mesquito/Human Life Cycle
1. Sporozoite / liver,
2-5. Mitosis, liver cell lysis,
6. Trophozoite / 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.
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.
Plasmodium:
Mosquito Life
Cycle
Sporozoites cross the salivary
gland epithelium,
This is thought to be receptor
mediated.
Ookinetes cross the midgut epithelium,
This is thought to be receptor mediated.
Receptor Mediated Transfer
Ookinete
Ookinete
binds open
receptor.
Ookinete
Molecules bind receptor
sites, block Ookinete
transfer.
enters cell
Ross Cell
Ross Cell
Mosquito epithelium
Mosquito epithelium
normal/infection
no infection
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.
Preliminary Work
Phage Display
T7 phage
…insert a DNA sequence into
phage gIII gene sequence.
…recombinant phage
express the sequence as part
of the gIII protein.
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.
Procedure
• 109 different phages
in the population,
– XCX8CX yields
~2010 different
possible peptides…,
• Mosquitoes were
injected with ~ 1011
phages.
SM1
• PCQRAIFQSICN: protein sequence bound both
salivary gland and midgut epithelia,
• Injection of the peptide into the mosquitos body
cavity inhibited oocyst formation by roughly 90%.
PCQRAIFQSICN
XCXXXXXXXXCX
XCX8CX
SM1 Injected into Mosquitoes
A mouse model system has been established in order to
provide a clinical vertebrate host.
Delivery
• We can’t 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?
Transgenic Construct
pBacR: piggyback vector, transposon derived
3xP3-EGFP-S40: Green fluorescent protein, eye specific promoter
AgCP promoter: mosquito promoter, activated by blood feast.
Signal: peptide sequence that sends protein to the midgut.
SM14: SM1 DNA sequence repeated 4 times, linked
Transformation
• Embryos are injected with the transgene/vector
construct, and a “helper” plasmid,
• Surviving embryos are raised and crossed with
virgin wt mosquitos,
– Subsequent offspring are scored for GFP eyes.
trans (top)
wt (top)
trans (bottom)
wt (front)
trans (front)
Southern Blot
…till a NotI
or BglII site.
Probe
3xP3-EGFP-SV40 and SM1
DNA,
Blot
Genomic DNA from different
transgenic individuals, digested
w/ NotI and BglII.
…establishes that
the inserts were in
different parts of
the genome.
Show that
the insert is
present.
Probes: SM1 DNA (mt rRNA for control)
Northern Blot
Why no male expression?
mt rRNA Control: constant expression gene, shows total RNA level was similar in all lanes.
SM1 expression…after a blood meal.
Transgenic SM1 Results
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 wasn’t due to a disrupted gene.
Epitope Tagging
• Fluorescent antibodies are available for a portion
of a virus protein influenza hemagglutin (HA1)...
When in a transgene, and subsequently translated, the location
of transgene expression in the organism can be assayed.
in situ, Western
Trangenic
Western:
protein target,
antibody probe.
Wt
Midguts
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.
And it’s Only a Heterozygote
• The authors conjecture that the possibility
exists that homozygous SM1 expressing
mosquitoes might display stronger antiookinete function.
But(t)
• SM1 transgenic mosquitoes don’t 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?
Genetics
…in the news.
paper (pdf)
To Know
• Understand the basic life cycle of
Plasmodium,
• Understand the Figures and Tables in this
experiment,
• Understand the general rational of the
experiment.
Monday
• Assigned Questions: 6.8, 6.13, 6.18, 6.19,
10.6, 10.7, 10.9, 10.11,
• Chapter 7: (7.1 - 7.5),
• Start Bacterial Genetics.