Plasmodium falciparum binding interactions with human brain endothelial cells
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Date
29/11/2016Author
Azasi, Yvonne
Metadata
Abstract
Cerebral malaria is the most severe form of malaria and mostly affects children
under 5 years causing impaired consciousness, coma and neurological disorders,
with life-threatening consequences in affected individuals. A pathological feature
of the disease is the sequestration of mature Plasmodium falciparum infected
erythrocytes (IEs) in the microvasculature of the brain. P. falciparum Erythrocyte
Membrane Protein 1 (PfEMP1) expressed on the surface of IEs is thought to enable
the parasites bind to human brain endothelial cells (HBEC) to avoid splenic
clearance. An in vitro model of cytoadherence in cerebral malaria has been
developed using a human brain endothelial cell line called HBEC-5i, which enables
the study of IEs binding to HBEC.
Previous work based on three laboratory parasite lines showed that HBEC-binding IEs express a specific subset of the diverse PfEMP1 family which contain
sets of cysteine-rich domains called domain cassettes (DC) 8 and 13. Parasites from
children diagnosed with cerebral malaria have also been independently shown to
express the DC8 and DC13 PfEMP1 types. The adhesion of IEs to HBEC is suggested
to occur by binding of a domain of the DC8 and DC13 PfEMP1 variants called
CIDRα1, to Endothelial Protein C Receptor (EPCR) on HBEC. However,
investigations of the effect of parasite and host environmental factors on adhesion
to HBEC are lacking, and further studies are needed to confirm the association of
these DC8 and DC13 PfEMP1 to HBEC-binding and the role of EPCR in mediating
the cytoadhesion. Therefore, the aim of this thesis was to examine the hypothesis
that HBEC-binding would be affected by changes in environmental conditions, and
that all IEs that bind to HBEC would express group A-like PfEMP1 containing DC8
and DC13, for binding to EPCR on HBEC.
In this study, the effect of pH, parasitaemia, gas, temperature, and serum on
cytoadhesion, were investigated using four HBEC-binding parasite lines. Adhesion
of IEs to HBEC was found to be pH and parasitaemia -dependent with optimal
binding at pH 7.3 and IE adhesion positively correlated with parasitaemia. There
was no significant effect of increase in temperature to 39°C and no significant
difference between hypoxic and normoxic conditions on adhesion in all parasite
lines. Human serum, however, abolished binding of the DC8-expressing parasite
line but had minimal effects on adhesion of the DC13-expressing parasite lines.
Two Kenyan isolates recently adapted to culture were selected for binding
to HBEC and were found to also predominantly express group A-like PfEMP1
including a DC8 PfEMP1 variant and PfEMP1 (s) that contained DBLα1.2 domains.
Attempts were made to localise the binding domain within the DC8 and DC13
PfEMP1 variants using recombinant proteins and antibodies. However, the CIDRα1
domain appeared to mediate adhesion of the DC8-expressing parasite line but had
no effect on adhesion of the DC13-expressing parasite lines. Only antibodies to the
N-terminal domain, known as NTS.DBLα, significantly inhibited binding of all the
parasites lines.
The role of EPCR and other receptor molecules on endothelial cells
including ICAM-1, CD36, CSA, PECAM-1, HABP-1 and heparin, in mediating
adhesion to HBEC was also investigated. Using EPCR recombinant protein,
monoclonal and polyclonal antibodies, and EPCR-siRNA knockdown in binding
assays, EPCR was shown to be involved in adhesion of only the DC8-expressing
parasite line and did not affect adhesion of the DC13-expressing parasite lines to
HBEC. Binding of DC8-expressing parasite line to HBEC was also inhibited by
soluble recombinant PECAM-1. There was no significant adhesion of both types of
parasite lines to the other receptor molecules, although minimal binding to HABP-1
was observed.
This study expands current knowledge on the parasite binding interactions
with HBEC by elucidating some of the environmental factors that affect the binding
properties, and gives the optimal conditions for the in vitro model of HBEC-adhesion in cerebral malaria. Findings presented here confirm the association of
expression of group A-like PfEMP1 to HBEC-binding and shows that the EPCR-CIDRα1 interaction does not mediate adhesion of all DC8 and DC13- expressing
parasite lines to HBEC. Additional receptors, other than EPCR, are therefore
required for HBEC-binding in cerebral malaria. The ability of normal human serum
to abolish binding of the DC8-expressing parasite line also raises the question of
whether IE binding to EPCR is physiologically relevant and suggest that the DC8-
expressing parasites associated with cerebral malaria may contribute to the disease
in a mechanism other than binding to brain endothelial cells