The Walter and Eliza Hall Institute of Medical Research

Functional Genomics

Identification of exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes

AG Maier, M Rug, MT O’Neill, M Brown, J Chesson, JG Beeson, BS Crabb, AF Cowman in collaboration with S Chakravorty, T Szestak, Y Wu, K Hughes, A Craig (Liverpool School of Tropical Medicine, UK), RL Coppel (Microbiology, Monash University), C Newbold (University of Oxford, Weatherall Institute of Molecular Medicine and John Radclifffe Hospital, Oxford, UK) Pub ref: 94

Plasmodium falciparum causes the most severe form of malaria in humans with over 2 million deaths annually. Once in the blood, multiplication of the parasite inside erythrocytes is responsible for associated morbidity and mortality. Profound structural and morphological changes occur in erythrocytes after parasite invasion, dramatically altering their physical properties and impairing circulation in vivo. In contrast to normal erythrocytes, parasitised cells are rigid and adhere to host endothelium as well as other cell type. The increased rigidity and adhesiveness of P. falciparum-infected erythrocytes result in augmented haemodynamic resistance in the microvasculature and play an important role in the pathogenesis of malaria. Adherence of infected red cells to vascular endothelium is mediated by P. falciparum erythrocyte membrane protein (PfEMP1), an antigenically diverse protein family trafficked to the infected red cell surface. The changes are caused by parasite proteins exported to the erythrocyte using novel trafficking machinery assembled in the host cell. To understand these unique modifications, we used a large-scale gene knockout strategy combined with functional screens to identify proteins exported into parasite-infected erythrocytes and involved in remodeling these cells. Eight genes were identified encoding proteins required for export of the parasite adhesin PfEMP1 and assembly of knobs that function as physical platforms to anchor the adhesin. Additionally, we showed that multiple proteins play a role in generating increased rigidity of infected erythrocytes. Collectively, these proteins function as a pathogen secretion system, similar to bacteria and may provide targets for anti-virulence based therapies against malaria.

A P. falciparum-infected mosquito feeding on a human arm. The capillaries under the skin are represented. The mosquito inserts its proboscis into the human and releases malaria sporozoite forms that migrate into the circulation through the capillary walls. Image from Drew Berry, Biomedical Animator, WEHI-TV.

Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum

TS Voss, CJ Tonkin, AJ Marty, JK Thompson, J Healer, BS Crabb, AF Cowman Pub ref: 146

P. falciparum erythrocyte membrane protein 1, encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We have shown that subtelomeric var gene promoters are silenced by default and that promoter activation is sufficient to silence all other family members. Our findings suggest a common logic underlying transcriptional control of var genes and this has important implications for understanding of epigenetic processes involved in regulation of this major virulence gene family.

An essential blood-stage function for the Plasmodium falciparum protease-like antigen SERA5 and its atypical active site serine

AN Hodder, JE McCoubrie, PGilson, SK Miller, RT Good, TF de Koning-Ward, BS Crabb in collaboration with RL Malby, OB Clarke, WD Fairlie, PM Colman, BJ Smith (Structural Biology), T Sargeant, TP Speed (Bioinformatics) Pub ref: 47, 97

Malaria-causing Plasmodium falciparum parasites infect red blood cells progressively, but before they can invade new cells they need to be released from the old cells in which they have been growing. To aid their release, the parasites are believed to secrete a potent cocktail of proteases that break down the old blood cell. These proteases could be attractive new targets for the future development of anti-malarial drugs, but first we need to understand the structure and functions of these proteases.

SERA5 is one of the essential proteases believed to play a role in host cell rupture. We have recently solved the crystal structure of the protease domain of SERA5. Interestingly, the structure of a recombinant version of the protein revealed that SERA5 might not be a classic protease that digests a broad range of substrates: it may have a more restricted function. To explore this further, we expressed a mutant form of the SERA5 protease in parasites to compete with the native protein and inhibit its normal function. Unexpectedly, the mutant protein caused the host cell to rupture prematurely resulting in the death of the parasite. One of the functions of SERA5 may therefore be to suppress host cell rupture rather than assist it and future studies will define the mechanism by which this occurs.

SERA has a protease-like structure that when mutated caused premature host cell rupture of the schizont stage parasite. (A) Ribbon model of SERA5 protease-like domain. (B) Unlike parasites expressing the wildtype ssGPS5 protein, parasites induced to express the ssGFP(S-A) mutant form of SERA5 disappear from the parasite cultures by premature lysis. (C) Microscopy was used to compare differences between parasites expressing 3D7-ssGFPS5 (normal) or 3D7-ssGFPS5(S-A) SERA5 genes.