Genomic and transcripto variation in blood stage Plasmodium falciparum
Author: Mok, Bobo
Date: 2007-09-21
Location: Föreläsningssalen vi institutionen för Mikrobiologi, Tumör- och Cellbiologi, Theorells väg 1
Time: 09.15
Department: Institutionen för mikrobiologi, tumör- och cellbiologi / Department of Microbiology, Tumor and Cell Biology
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thesis.pdf (5.839Mb)
Abstract
Malaria research has entered a postgenomic era since October 2002, when
the complete genomic sequence of Plasmodium falciparum strain 3D7 was
published. A massive amount of information generated by the P. falciparum
genome project has facilitated the development of many novel platforms
for profiling different levels of biological aspects. In this thesis, by
employing various high throughput approaches, 1 aimed at understanding
the genetic basis of phenotype variation in P. falciparum.
Genomic diversity of P. falciparum strains originating from different
geographical areas was studied using microarray-based comparative genomic
hybridization (CGH). Here I show presence of multiple genome widecopy
number polymorphisms (CNPs) covering 82 genes. The genes, amplified in up
to six copies, encode molecules involved in cell-cycle regulation, cell
division, drug resistance, erythrocyte invasion, sexual differentiation
and unknown functions. Our results suggest that P. falciparum employs
gene duplications and deletions as general strategies to enhance its
survival and spread.
Even though the P. falciparum subtelomeres are considerably variable,
segmental duplication were observed within these regions. High-resolution
mapping and CGH data of the subtelomeric regions revealed a block of
genes that had been amplified nine times on multiple subtelomeres of P.
falciparum. These duplicated segments (SD) are of more than 10 kilobases
in size and span six genes, including three known variant gene families:
var, rif and pfmc-2tm and three hypothetical genes (n, o-, q-gene).
Sequencing data revealed that both inter- and intragenic regions are
highly conserved across the species, despite their variation in copy
numbers. One of the hypothetical genes within the SD, the n-gene,
encoding a PEXEL/VTScontaining two transmembrane proteins was found to be
transcribed at an early stage of the asexual erythrocytic cycle and its
transcriptional levels were correlated to the gene dosage. The ubiquity
and uniqueness of these SDs in the P. falciparum subtelomeres, and the
conserved nature of the gene content within, suggests an important role
in plasmodia speciation.
A major part of the parasite virulence attributes to the extensive host
cell modification of blood stages P. falciparum. Two 3D7 isogenic clones
with distinct adhesive and antigenic phenotypes (rosettes formation for
3D7S8.4 and CD36 binding for 3D7AH 1 S2) were isolated by
micropanipulation. To have a global view of gene expression governing
their phenotypes, we have employed a P. falciparum genome array,
supplemented with a panel of in-house oligonucleotides, for comparative
transcriptomal analysis. Fifteen genes were found highly differentially
expressed (greater than a 5-fold change) encoding proteins for apical
organellar (Gbph2, GBPrelated antigen), cell-rescue, defense/virulence
(RESA-2, RIFIN, PfEMP1), DNA/RNA processing (RNA methylase), erythrocyte
invasion (SERA-5) and a number of hypothetical proteins. A number of
short and fulllength var transcripts were differentially expressed
between the clones; yet, only one full-length transcript was dominant in
both rings and trophozoites. In fact, var genes were found at the top of
the list of the highly differentially expressed gene in between the two
clones and its protein product, PfEMP1, is believed to be the most
important determinant of antigenic phenotypes in P. falciparum.
To understand better the mechanisms and dynamics of var gene switching,
we propagated 3D7S8.4 and 3D7AHIS2 under the same conditions for more
than one year without enrichment or panning. We found that, upon
long-term culturing, both parasites switch to express a common var gene
(var2csa) matched by the loss of PfEMP1 surface expression and host
cellbinding. The var2csa gene repositioned in the perinuclear area upon
activation away from the telomeric clusters and heterochromatin to
express spliced, full-length RNA. We suggest switching to var2csa to be
an inherited trait that allows for small populations of P. falciparum to
express new var genes. The process may coordinate the variant-antigen
repertoire and thus protect against its rapid exhaustion.
List of papers:
I. Ribacke U, Mok BW, Wirta V, Normark J, Lundeberg J, Kironde F, Egwang TG, Nilsson P, Wahlgren M (2007). "Genome wide gene amplifications and deletions in Plasmodium falciparum." Mol Biochem Parasitol 155(1): 33-44. Epub 2007 May 18
Pubmed
II. Mok BW, Ribacke U, Sherwood E, Wahlgren M (2007). "Highly conserved segmental duplication of variable copy numbers in the subtelomeres of distinct Plasmodium falciparum." (Submitted)
III. Mok BW, Ribacke U, Winter G, Yip BH, Tan CS, Fernandez V, Chen Q, Nilsson P, Wahlgren M (2007). "Comparative transcriptomal analysis of isogenic Plasmodium falciparum clones of distinct antigenic and adhesive phenotypes." Mol Biochem Parasitol 151(2): 184-92. Epub 2006 Dec 4
Pubmed
IV. Mok BW, Ribacke U, Rasti N, Kironde F, Chen Q, Nilsson P, Wahlgren M (2007). "var gene switching in Plasmodium falciparum as an inherited trait." (Submitted)
I. Ribacke U, Mok BW, Wirta V, Normark J, Lundeberg J, Kironde F, Egwang TG, Nilsson P, Wahlgren M (2007). "Genome wide gene amplifications and deletions in Plasmodium falciparum." Mol Biochem Parasitol 155(1): 33-44. Epub 2007 May 18
Pubmed
II. Mok BW, Ribacke U, Sherwood E, Wahlgren M (2007). "Highly conserved segmental duplication of variable copy numbers in the subtelomeres of distinct Plasmodium falciparum." (Submitted)
III. Mok BW, Ribacke U, Winter G, Yip BH, Tan CS, Fernandez V, Chen Q, Nilsson P, Wahlgren M (2007). "Comparative transcriptomal analysis of isogenic Plasmodium falciparum clones of distinct antigenic and adhesive phenotypes." Mol Biochem Parasitol 151(2): 184-92. Epub 2006 Dec 4
Pubmed
IV. Mok BW, Ribacke U, Rasti N, Kironde F, Chen Q, Nilsson P, Wahlgren M (2007). "var gene switching in Plasmodium falciparum as an inherited trait." (Submitted)
Issue date: 2007-08-31
Rights:
Publication year: 2007
ISBN: 978-91-7357-291-0
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