Investigating the role of PfRab5 in Plasmodium falciparum

1
Investigating the role of PfRab5 in Plasmodium
falciparum
L. Isabella Ochola, Steve A Ward Michael L
Chance
Molecular and Biochemical Parasitology Group,
Liverpool School of Tropical Medicine, Pembroke
Place, L3 5BA
AIM P. falciparum actively phagocytoses
haemoglobin (Hb) through the cytostome. Vesicles
containing Hb bud off and are trafficked to the
digestive vacuole where Hb is broken down into
haemozoin. This process is being investigated at
the molecular level.
INTRODUCTION We have identified and characterised
3 homologues of PfRab5 in Plasmodium falciparum
PfRab5A, PfRab5B and PfRab5C. PfRab5B will be
looked at in detail.
1 2 3
4
A
B
RING
0h 4h 8h 16h 20h 24h
28h 32h 40h 44h 48h
PfRab5b
23130bp
PfRab5B
1490bp
9417bp
C
6557bp
EARLY TROPHOZOITE
Fig. 1. A- Southern Blot of 5?g of Plasmodium
falciparum 3D7 genomic DNA digested with
restriction enzymes. Lane 1- single digest, lane
2- double digest and lane 3-digest that cuts
outside the gene. B- Northern blot of 4?g of RNA
isolated from 3D7 showing the expression pattern
of PfRab5B over 48hrs. C- Western Blot of 50ng
of 3D7 protein using a polyclonal antibody
raised in a rabbit against recombinant PfRab5B
protein
66.2kDa
45kDa
4361bp
31kDa
PfRab5B
21.5kDa
1
2
3
2322bp
14.4kDa
LATE TROPHOZOITE
2
3
1
A
B
1 2
SCHIZONT
1 2 3
4
EARLY RING
EARLY RING
EARLY TROPHOZOITE
Fig 2. Indirect Immunofluorescence of the
different stages of the 3D7 parasite using
polyclonal rabbit anti-serum to recombinant
PfRab5B, followed by fluorescein-isothiocyanate
conjugated anti-rabbit IgG (green) column 1,
column 2- bright field image, column 3- staining
of the nucleus with propidium iodide (10?g/ml),
column 4- combined image of A, B and C
LATE RING
CONCLUSIONS PfRab5B is a single copy gene,
expressed throughout the erythrocytic cycle, that
peaks mainly in the trophozoite stage and it is a
protein of approximately 30kDa. Punctuate
staining is seen using immunofluorescence whereby
PfRab5B is localised in the erythrocyte cytoplasm
at the ring stage, and mainly in the parasite
cytoplasm around the food vacuole in the
trophozoite and schizont stages. GFP
tagged-PfRab5B localises to the parasite
cytoplasm with varying intensities of protein
around the food vacuole.
MID TROPHOZOITE
EARLY TROPHOZOITE
LATE TROPHOZOITE
LATE TROPHOZOITE
SCHIZONT
SCHIZONT
Fig 3. A- Confocal microscopy images of
transfected 3D7 strain parasite expressing
C-terminally GFP-tagged PfRab5B. PfRab5B was
cloned into the pARL vector and electroporated
(0.31kV, 950?F) into 3D7 and grown under
continuous WR99210 drug pressure. Column 1-
image of fluorescence from GFP tagged to PfRab5B
and column 2- bright field image. B- Images of
erythrocytes hypotonically loaded with
rhodamine-dextran and infected with 3D7
transfectant expressing C-terminally GFP-tagged
PfRab5B, column 1- image of fluorescence from GFP
tagged to PfRab5B, column 2- bright field image,
column 3- red fluorescence from
rhodamine-dextran, column 4- combined image of
columns 1, 2 and 3