Transcript Isolation of Dynein Light Chains and determination of

Identification of Plasmodium
Falciparum MSP-1 Variants in Malaria
sequential infections
YAA Reuben Mangi (I56/ 81536/ 2012)
Bsc. University of Nairobi (2012)
Supervisors
Dr. Isabella Oyier,
Wellcome- Trust, Kenya Medical Research Institute (KEMRI),
Kilifi
Dr. George Obiero,
Centre for Bioinformatics and Biotechnology (CEBIB)
University of Nairobi
Introduction
 Malaria is an intermittent and
remittent fever disease caused by a
species in the genus Plasmodium.
 Malaria thrives in the tropical areas of
Asia, Africa, and Central & South
America, where it infects millions of
people.
Figure 1. Malaria Victim( National Geographic)
 There are approximately 219 million malaria cases and mortality rates of
approximately 1 million cases a year in the world.
 28 million Kenyans living in malaria risk regions and majority being children.
Introduction Cont..
• The erythrocytic stages of the
parasite life cycle are
responsible for all symptoms
and pathologies of the disease.
• The symptoms are; fever,
headache, malaise, fatigue,
muscular pains, and occasionally
nausea, vomiting and diarrhoea.
Figure 2 Malaria Parasites Amid Red Blood Cells (National
Geographic)
• Sequestered RBCs adhere to the
blood vessels resulting to
destruction and blockage of
vessels leading to hypoxia and
toxemia in vital tissues and
organs.
Life cycle of Plasmodium falciparum
The parasite undergoes a complex life
cycle that requires both a human host
and a female Anopheles mosquito.
Figure 3. Life cycle of P. falciparum (Nature)
Molecular basis of invasion
The surface is comprised of Glycosyl
Phosphatidyl Inositol (GPI) anchored
membrane proteins(Ligands) and their
associated partners.
These include Merozoite Surface
proteins(MSP), Apical Membrane Antigen1(AMA-1), The Reticulocyte Binding
Homologue proteins (RBLs), Erythrocyte
Binding like proteins (EBLs).
Other peripheral proteins involved in primary
contact also exist.
Well characterized EBLs are, EBA-175,
EBA-140 and their receptors are glycophorin A,
Glycophorin C respectively.
Figure 4: Fine structure of
Plasmodium falciparum Merozoite
(Cowman and Crabb 2006)
Invasion of Erythrocytes by Plasmodium falciparum
Merozoite
Figure 5: Diagram of RBC invasion -(Cowman and Crabb 2006)
A process that involves is multi-step sequence of initial recognition, reorientation and entry.
The contact requires interactions of merozoite surface proteins (MSP) and
specific receptors on the host cell.
Immunity to Invasion by Merozoites
Clinical immunity to malaria is slow to develop and short lived. One reason
for this is the extensive diversity found in Plasmodium antigens.
Merozoites are among the parasite stages targeted by immune system.
The merozoite stage represents an attractive target for controlling malaria
due to its uniqueness towards its association with the disease clinical
symptoms.
The merozoite surface proteins(MSP) are the major targets of immune
attack by antibodies.
The well characterized MSP is MSP-1. Other MSP gene families exist.
Plasmodium falciparum MSP-1 gene
• Located at the middle of chromosome 9
• Organized into blocks
• Its polymorphic and genetically diverse
The product of this is MSP-1, a high-molecular-weight precursor which is
then processed into several fragments . At the time of red cell invasion, only
the 19-kDa C-terminal fragment remains on the surface. and is carried into
the invaded erythrocytes.
Since MSP-1, can elicit immune responses in natural malaria infections,
and is genetically diverse containing multiple polymorphisms in P.
falciparum it will be considered in sequential infections in this study.
Research Question
Are there Plasmodium falciparum MSP-1 polymorphic differences in
sequential clinical malaria infections
Hypothesis
There are no Plasmodium falciparum MSP-1 polymorphic variants in
sequential cases of clinical malaria infections.
General Objective
Determine MSP-1 19kDa genetic variants in parasite obtained from individuals
with multiple malaria sequential infections.
Specific Objectives
• Identify MSP-119kDa gene polymorphisms in sequential clinical malaria
infections.
• Determine allelic changes in individuals across the sequential infections.
• Determine the population proportion of allele frequencies between
infections.
Justification
• Malaria parasite resistant to
drugs.
• Vector resistant to insecticides.
• Developing a vaccine towards malaria would be the most practical way to
arrest this quagmire. However, efforts that are underway have been
unsuccessful.
• This work will determine the frequency of MSP-119kDa gene
polymorphisms and allele prevalence in individuals with multiple sequential
infections and contribute to the knowledge of MSP-1 diversity which is
useful towards the development of malaria vaccine merozoite candidates.
Methodology
Study design:
This will be Longitudinal study where MSP-1 variable will be measured in
all subjects in the cohort
Study site
The lab work will be conducted at Centre for Biotechnology and
Bioinformatics (CEBIB) molecular labs in Nairobi and at ILRI.
Study population
The target group will be composed of 33 children aged between 0 and 5
years who had been followed up weekly with over 9 malaria infection
episodes after visiting a dispensary in Junju, Kilifi County.
Ethical clearance
Ethical clearance for this study has already been obtained from the
KEMRI ethics review board for integrated studies of natural immunity to
malaria.
.
Amplification and sequencing of MSP-119 kDa genes
PCR
•
Optimization
•
Gene specific primers for MSP-1 19KDa, High
Fidelity PCR Taq (Roche) and Controls (3D7)
•
Gel
electrophoresis
Samples run in batches
Gel preparation using Cyber Safe® and
view by Gel Doc
BigDye PCR
•
•
•
Sequencing
Amplicons will be purified
using ethanol precipitation.
Cycle sequencing PCR.
Analysis by DNA analyser machine
(Applied Biosystems)
Data
Analysis
•
•
•
Quality checking and contig assembly using BioEdit, DNASTAR, CLC bio softwares.
Alignment to identify Nucleotide polymorphisms
Gene polymorphisms frequencies by R
Timeline
Budget
Acknowledgements:
 Parwos Abraham: CEBIB admin
 Colleagues
Identification of Plasmodium
Falciparum MSP-1 Variants in Malaria
sequential infection
YAA Reuben Mangi (I56/ 81536/ 2012)
Bsc. University of Nairobi (2012)
THANK YOU