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Use of recombinant complement regulatory proteins to enhance killing of Plasmodium falciparum

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posted on 01.02.2019, 10:48 by Mohenned H. Alsaadawi
Plasmodium falciparum, the causative agent of malaria, evades complement mediated killing by recruiting host complement regulatory proteins. All of the known complement evasion mechanisms act on the alternative pathway C3 convertase. Asexual blood stages of Plasmodium falciparum can sequester factor H, which helps to destabilise and inactivate the C3 convertase. The action of factor H could be reduced by several fluid-phase complement regulators, including properdin and factor H-related protein 1 (CFHR1), which are competitive inhibitors of factor H and enhance complement activation. The aim of this work was to explore the possibility of enhancing the complement-mediated killing of P. falciparum by reducing the efficacy of factor H bound to the parasite surface, either by interfering with its binding to the parasite or its ability to inactivate the C3 convertase. Recombinant properdin and CFHR1 were used as model inhibitors of factor H. Recombinant properdin is particularly effective at augmenting complement activation, as it forms very large oligomers that are much more active than native serum properdin. Recombinant human, mouse and chimeric properdin (mouse properdin humanised at one or both termini) were expressed in mammalian cells. CFHR1 was expressed in a bacterial expression system, together with a C-terminal fragment of CFHR1 (SCR3-5), which retains the factor H inhibitory activity, but lacks a C5 convertase inhibition activity that resides in the N-terminus of the protein. The recombinant proteins were purified to near homogeneity and shown to effectively increase C3b deposition on zymosan, a model activator of the alternative pathway. Moreover, the proteins could enhance formation of the membrane attack complex (MAC) via the terminal pathway, leading to haemolysis of erythrocytes. Likewise, serum supplemented with the recombinant proteins had an enhanced ability to opsonize P. falciparum schizonts with C3b. The effect of the proteins on live parasites was tested using an ex vivo model of parasitaemia in infected erythrocytes. When the infected erythrocyte cultures were supplemented with rCFHR1, parasitaemia was significantly reduced. Controls using heat-inactivated serum showed that this effect was complement dependent. Immunofluorescence microscopy showed increased C3b and C5b-9 deposition on infected RBC when the recombinant proteins were added to the cultures. In conclusion, the results indicate that blocking the activity of factor H in serum, or preventing its attachment to P. falciparum, is sufficient to reduce the infectivity of the parasite in blood, opening up the possibility of new therapies that exploit this mechanism.



Lynch, Nicholas

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Department of Infection, Immunity and Inflammation

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University of Leicester

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