Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Genetics. Includes bibliographical references.

Of the four Plasmodium species which cause malaria in humans, P. falciparum is responsible for the majority of the morbidity and mortality associated with this disease. The surface expression of parasite-derived proteins in the middle of the asexual cycle coincides with two important modifications of the host erythrocyte. First, a protective immune response is directed against a family of variant antigens, known as P. falciparum Erythrocyte Membrane Protein-1 (PfEMPl). Second, ligands are detected at the surface which mediate the specific cytoadherence of infected erythrocytes to vascular endothelium, such that infected cells are sequestered away from the peripheral circulation in deep vascular beds. The potentially fatal syndrome known as cerebral malaria can ensue when infected cells sequester at high density in the brain. Indirect studies have shown that the antigenic and adhesive phenotypes at the surface are linked to the expression of PfEMPl. However, there is a paucity of biochemical data which relate to PfEMPl, and this problem is addressed in this thesis. This study has confirmed, at the biochemical level, inferences from serology that clonal antigenic variation occurred rapidly. Variation produced a number of novel antigenic and adhesive phenotypes which were associated with unique forms of PfEMPl. Further insights into the mechanism of sequestration were possible because of the finding that single infected erythrocytes had the capacity to bind to at least three putative endothelial cell receptors; CD36, Intercellular Adhesion Molecule-1 (ICAM1), and Thrombospondin (TSP). It was demonstrated for the first time that PfEMPl was responsible for cytoadherence to CD36 and ICAM1, but was probably not involved in adhesion to TSP. Extensive analysis with sequence-specific proteases proved that adhesive interactions with each receptor were separable properties of the surface, and facilitated the proposal of a domain model for PfEMPl. Detailed analysis of the antigenic and adhesive phenotypes of a series of clonally-derived parasites demonstrated that infected cells expressing all variant antigenic types could adhere to CD36 whereas adhesion to ICAM1 was seen in a restricted subset. This may be clinically relevant if, as current data suggests, adhesion of infected cells to ICAM1 is important in the development of cerebral malaria. Identification of all ICAM1 binding phenotypes could lead to the design of novel therapeutic strategies for this life-threatening condition.

Malaria is still a significant public health problem in the Tropics, with an estimated 200 million cases a year and more than 1 million deaths, mostly in young children in sub-Saharan Africa. Plasmodium falciparum is the parasite responsible for the majority of the morbidity and mortality due to malaria. We know from the historical use of malaria to treat neurosyphilis that there were several different strains of P. falciparum, some of which were more pathogenic and had higher multiplication rates than others. High multiplication rates of P. falciparum isolates have been associated with severe disease in Thailand, but not in Kenya or Mali. In determining what differences exist between fast- and slow-growing malaria parasites, and understanding their relationship with clinical outcome, we may discover a way of targeting those parasites that cause most disease. This thesis describes a genetic analysis of the determinants of growth and pathogenicity in the rodent malaria parasite, Plasmodium chabaudi. The use of rodent malaria parasite strains for genetic analysis has several experimental, ethical and financial advantages over the use of human malaria parasites. In addition, rodent malaria parasite strains also vary significantly in their growth and pathogenicity, making them excellent candidates for a genetic analysis of these characteristics. The first section of this thesis is concerned with the characterisation of the growth, pathogenicity and transmissibility of two strains, DS and DK, of the rodent malaria parasite P. c. adami. The DS strain is fast-growing, pathogenic, non-selective in its invasion of red blood cells and a poor transmitter to mosquitoes. The DK strain is slow-growing, non-pathogenic, selective in its invasion of red blood cells and a good transmitter to mosquitoes. In the second section of this thesis is a detailed study of the growth characteristics of DS and DK in mixed infections, relative to their growth in single infections. Both sections provide information relevant for the main objective of this thesis, but also contribute to the body of work on pathogenicity and transmissibility, and pathogenicity and strain behaviour in mixed infections, which has been carried out in rodent malaria parasites to-date. The third section of the thesis contains the results of a genetic analysis of the difference in growth between P. c. adami strains DS and DK, using the Linkage Group Selection (LGS) technique. On several occasions, DS and DK were crossed in the mosquito vector and, following selection for fast growth in mice, the cross progeny were initially screened with genome-wide, quantitative AFLP markers. Markers specific to the slow-growing parent DK which were greatly reduced in intensity after selection were found on P. chabaudi chromosomes 6, 7 and 9. This result suggests that the difference in growth between the two strains is determined by multiple genetic loci. The selection on chromosomes 7 and 9 was then looked at in greater detail, using SNP-based markers quantified by Pyrosequencing™. It was found, consistently, that a region at one end of DS chromosome 9 was inherited as a single, non-recombining unit in cross progeny selected for fast growth. As this was the region most strongly selected against, it suggests that a gene (or genes) in this region has a major role in the determination of growth characteristics, and therefore pathogenicity, in P. c. adami. Narrowing down this region further, in order to identify the candidate gene(s), remains a key future objective.

Invasion of host erythrocytes is an essential stage in the life cycle of Plasmodium parasites and in development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by the coordinated release of specialised apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and the erythrocyte-binding like (EBL) proteins, that mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin. Indeed, antibodies targeting either PfRH5 or basigin can block parasite invasion with high efficiency in vitro, making PfRH5 an excellent candidate for a vaccine to protect against the most deadly form of malaria. Here I present crystal structures of PfRH5 in complex with basigin and with two distinct inhibitory antibodies. This is the first structure of any RH protein, revealing a novel fold in which two three-helical bundles come together to form a kite-like architecture. The two immunoglobulin domains of basigin and the inhibitory antibodies bind to one tip of the kite. These findings provide the first structural insights into erythrocyte binding by the Plasmodium RH protein family and identify novel inhibitory epitopes to guide the design of a new generation of vaccines against the blood-stage parasite.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant surface antigens, encoded by the var gene family, play a crucial role in malaria pathogenesis through mediating immunomodulation and host cell adhesion. Var genes can be sub-grouped according to genetic or functional features. This thesis examined var gene transcription of conserved groups of var genes in the context of clinical malaria disease manifestation in African field isolates. Analysis of var gene transcription in 26 P. falciparum field isolates from Malian children revealed that field isolates from children with cerebral malaria show significantly higher transcription of group A var genes than the field isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia). These results suggest that group A var genes are important determinants of parasite virulence and strengthen the growing body of evidence associating group A var expression with severe disease in children. Analysis of var gene transcription in six P. falciparum placental malaria field isolates showed that var2csa was transcribed in all placental malaria field isolates, but not in 10 childhood isolates examined. This finding, also reported in other recent and subsequent studies, suggests that var2csa expression is a critical factor in the onset of clinical malaria disease in pregnant women. Examination of type 3 var gene transcription in laboratory and field isolates established that these var genes were commonly transcribed in blood-stage parasites, and sequence analysis of the transcribed domains confirmed a very high level of conservation across this var gene sub-family. Finally, rosetting is a property of some group A PfEMP1 and is associated with disease severity in African childhood malaria. Certain glycoconjugate compounds can disrupt rosetting, possibly due to the functional similarities of interactions between rosetting PfEMP1 and host rosetting ligands. A non-toxic compound (curdlan sulfate) was found to be effective at disrupting rosettes in all 18 rosetting field isolates examined, showing potential for use in treatment of severe malaria due to rosetting P. falciparum isolates. The findings presented in this thesis expand current knowledge of the role and significance of var genes/PfEMP1 in P. falciparum malaria disease pathogenesis. The work demonstrates the importance of continued research on var genes/PfEMP1 in further understanding this complex parasite, and ultimately in combating this severe disease.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 99-100).

Thesis (Ph.D. (Biochemistry))--Mahidol University, 1982.

Thesis (Ph.D. (Tropical Medicine))--Mahidol University, 2007. / LICL has E-Thesis 0043 ; please contact computer services. LIRV has E-Thesis 0043 ; please contact circulation services.

Tübingen, Univ., Diss., 2005.

Human malaria caused by Plasmodium falciparum is a major global burden killing between 700,000 and 2.7 million people every year. Africa bears the greatest portion of this burden, with over three quarters of deaths occurring in African children, accounting for 18% of all child deaths in sub-Saharan Africa. Synthesis of tetrahydrofolate through the folate biosynthetic pathway is vital for the survival of P. falciparum parasites and is lacking in the human host. As such, enzymes of this pathway have long presented attractive targets for drug therapy and although increasingly being compromised by resistance, anti-folates such as pyrimethamine and sulfadoxine are still very valuable drugs in many malaria-endemic regions.In this project, further investigation of the enzymes of the folate biosynthetic pathway has been attempted by developing protocols to quantify these proteins and others through proteomic techniques. Two quantification techniques were pursued. The first was quantification using whole, heterologously expressed, stable-isotope labelled forms of P. falciparum proteins for use as heavy standards in mass spectrometry. Great difficulty was experienced in the effort to express and purify P. falciparum enzymes in E. coli expression systems, with only one enzyme successfully expressed and purified in a 13C-labelled form. This one protein was taken forward into quantification experiments. The second quantification technique used a stable-isotope labelled ‘QConcat’ protein, consisting of a number of peptides from 12 P. falciparum proteins of interest, as a heavy standard in mass spectrometry. This was successfully expressed and purified in a 13C-labelled form from an artificial gene using an E. coli expression system. This too was taken forward into quantification experiments.Quantification experiments using the QConcat-based quantification technique were successfully performed on whole P. falciparum extract. Among the proteins quantified were SHMT and DHFR, two proteins of great interest from the folate biosynthetic pathway. Consistent with results from different expression analysis techniques in the literature, the folate enzymes were found to be of lower abundance than housekeeping enzymes and SHMT was found to be more abundant than DHFR.For deep quantitative analysis of the P. falciparum proteome, it was found that fractionation was necessary. Fractionation in this project was performed using a ZOOM™ IEF fractionator (Invitrogen), an OFFGEL™ IEF fractionator (Agilent) and 1D SDS-PAGE. It was found that by using these fractionation techniques, more proteins could be identified within the P. falciparum proteome, with all but one of the enzymes of the folate biosynthetic pathway being identified. Significant advances in the sensitivity of mass spectrometers during this project have also greatly facilitated the investigation of the proteome. In some cases, this meant that proteins which were only previously accessible by prefractionation of the proteome could be seen in whole P. falciparum extract. Unfortunately, QConcat-based quantification using both fractionation and sensitive mass spectrometry could not be successfully achieved in the time available. However, the promising results obtained suggest that, after careful optimisation, such an approach will be valuable.